Nucleoporin 62-like protein activates canonical Wnt signaling through facilitating the nuclear import of β-catenin in zebrafish.
Journal: 2015/May - Molecular and Cellular Biology
ISSN: 1098-5549
Abstract:
Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/β-catenin pathway positively modulates nup62l transcription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulate nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitation of nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish.
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Mol Cell Biol 35(7): 1110-1124

Nucleoporin 62-Like Protein Activates Canonical Wnt Signaling through Facilitating the Nuclear Import of β-Catenin in Zebrafish

INTRODUCTION

The nuclear pore complex (NPC) is embedded in the nuclear envelope of eukaryotic cells and is a multimeric structure of 60 to 125 MDa in vertebrates (1). NPCs selectively mediate the bidirectional trafficking of various macromolecules between the nucleus and the cytoplasm (2). Despite some organism-specific differences, the principal properties of NPCs, including their composition, architecture, and transport mechanism, appear to be evolutionarily conserved (3, 4). Nucleoporins (Nups) are the critical components of NPCs and have over 400 members belonging to nearly 30 different types (5).

Some Nups are known to function in microtubule regulation (6), mitochondrial transport (7), gene transcription (8, 9), and DNA repair (10), as well as in tissue-specific development and several human pathologies. Mouse Nup133 is essential for differentiation of the neural lineage (11). Genetic mutation of zebrafish elys, encoding a component of the Nup107-160 complex, perturbs proper development and proliferation of the retina and intestine, with massive apoptosis in these affected tissues (12, 13). Zebrafish nup107 mutants exhibit defects in the pharyngeal arches, eyes, and intestine due to the disruption of nuclear pores and mRNA transportation (14). Loss of Nup155 functions in both humans and mice leads to atrial fibrillation, the most common form of clinical arrhythmia that can cause sudden cardiac death (15). Thus, a tight regulation of nucleocytoplasmic transport by some of the Nups is essential for cell homeostasis and proper functioning of tissues and organs.

Nup62 localizes in the central channel of NPCs and usually exists in the Nup62-Nup58-Nup54-Nup45 complex (16, 17). Nup62 is crucial to the control of nuclear pore permeability and selective nucleocytoplasmic shuttling via the direct interaction of its N-terminal phenylalanine-glycine (FG)-rich domain with nuclear transport receptors, and occasionally with cargos (18, 19), such as nuclear transport factor 2 (NTF2), a transport receptor for Ran (20,22). The C-terminal coiled-coil structure functions in anchorage of Nup62 to the NPC (23, 24) and offers targeting information (25, 26). The oncoprotein MUC1-C, which is associated with human carcinomas, enters the nucleus via a pathway involving binding to Nup62 (27). Nup62 is essential for the export of HIV Rev viral RNA particles in human cells (28). Nuclear mRNA export is disrupted in a temperature-sensitive mutant of Nsp1p, the yeast homologue of mammalian Nup62 (29). Moreover, a missense mutation of Nup62 in humans leads to infantile bilateral striatal necrosis (30). Nevertheless, physiological, pathological, and developmental roles of Nup62 in vertebrates remain to be well defined.

The β-catenin protein serves as a crucial coactivator of the canonical Wnt pathway (31) and participates in embryonic development and maintenance of organ and tissue functions in adults (32,34). Certain gene mutations can initiate the aberrant nuclear accumulation of β-catenin, resulting in cell transformation and a variety of human cancers, including melanoma, colon cancer, and hepatocellular carcinomas (35,37). Thus, β-catenin is widely considered a potential drug target for basic research and anticancer therapeutics (38, 39). In zebrafish, β-catenin is required for dorsoventral patterning (40,43), convergence and extension (CE) movements through multifarious signaling molecules (44), and cardiac differentiation before gastrulation (45).

Macromolecules generally shuttle between the nucleus and the cytoplasm via the classical Ran/transport receptor mechanism, but β-catenin is able to shuttle in and out of the nucleus even though it possesses neither a classical nuclear localization signal (NLS) nor a nuclear export signal (NES). β-Catenin is reported to shuttle out of the nucleus via the classical CRM1 pathway after interaction with specific partners, such as APC, Kank, Lzts2, and Axin (46,49), but the major route of its export appears to be independent of CRM1, exogenous soluble factors, and Ran-GTPase (50, 51). Moreover, β-catenin migrates into the nucleus through direct interaction with nuclear pore components (52, 53). Recently, β-catenin was found to behave similarly to transport receptors and to shuttle through direct binding to multiple Nups, such as Nup62, Nup358, Nup98, and Nup153, in the absence of receptors (54). Therefore, precise molecular mechanisms underlying the nucleocytoplasmic transfer of β-catenin remain to be well defined.

In this study, we investigated functions of zebrafish Nup62-like protein (Nup62l) in the control of early dorsoventral patterning, gastrula CE movements, and specification of midline organs. We further dissected molecular mechanisms underlying the nuclear import of β-catenin-1/2 by Nup62l in zebrafish.

MATERIALS AND METHODS

Fish lines and maintenance.

The AB strain of zebrafish was maintained in a recirculating water system, and embryos were raised under standard procedures. Naturally fertilized embryos were staged by morphological features or by hours postfertilization (hpf) (55). The animal studies were approved by the Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (approval ID Keshuizhuan 0829).

Plasmid construction.

Total RNAs were extracted from 24-hpf embryos by use of TRIzol reagent (Invitrogen), and first-strand cDNAs were synthesized using a RevertAid first-strand cDNA synthesis kit (Fermentas). Full-length cDNA of zebrafish nup62l (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001113596","term_id":"165972452","term_text":"NM_001113596"}}NM_001113596) was obtained by PCR amplification with primers Nup62l-SalI-F (5′-CTAGTCGACATGGCATTTAACTTTGGACAAACAGG-3′) and Nup62l-EcoRI-R (5′-TGCGAATTCTCACTGGAAGTTTAAACGAAAGCTC-3′) and subcloned into vectors for in vitro synthesis of capped mRNA and overexpression. The primer pair Nup62l-EcoRI-F (5′-CAGGAATTCGAGCCACCATGGCATTTAACTTTGGACAAAC-3′) and Nup62l-HindIII-R (5′-GATAAGCTTGTCAGTATATGGTTCCACTTTGCTCC-3′) was used for the generation of a nup62l mutant (nup62l-DN) in which amino acids 408 to 507 were deleted from the coiled-coil domain of the encoded protein. Nup62l mutants D1 to D14 were generated by deleting amino acids 1 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 507, 1 to 25, 26 to 50, 51 to 75, 76 to 100, 76 to 80, 81 to 85, 86 to 90, 91 to 95, and 96 to 100, respectively, from the potential β-catenin binding motif.

MO and mRNA injection.

A nup62l morpholino oligonucleotide (nup62l-MO) against the 5′ untranslated region (5′ UTR) (5′-TGAACTACTTCACAGCAGCTCCCGC-3′), nup54-ATG-MO (5′-ACGCGCCGCCGAAGTTGAACGCCAT-3′), and a standard control MO (STD-MO) were purchased from Gene Tools. MOs for β-catenin-1 (5′-CTGGGTAGCCATGATTTTCTCACAG-3′) and β-catenin-2 (5′-CCTTTAGCCTGAGCGACTTCCAAAC-3′) were gifts from Wuhan Xiao. MOs were injected into one-cell-stage embryos at the indicated doses. Capped mRNAs were synthesized using an mMESSAGE mMACHINE kit (Ambion, Austin, TX). Constructs for in vitro synthesis of capped mRNAs included zebrafish nup62l, mutants of zebrafish nup62l, EGFP, kaede, zebrafish β-catenin-1 and β-catenin-2, frzb, bmp4, and a truncated Bmp receptor (56). Capped mRNAs and MOs were prepared as described previously (57). One-cell-stage embryos were injected with 150 or 300 pg capped mRNA/embryo, unless otherwise stated. The synthetic zebrafish nup62l mRNA was diluted to different concentrations and coinjected with nup62l-MO to determine the optimal dose for the rescue of morpholino-injected embryos.

Real-time qPCR.

Total RNAs were treated with RNase-free DNase I, and cDNAs were synthesized using a First Strand cDNA synthesis kit (Thermo Scientific). Real-time quantitative PCR (qPCR) was performed as described previously (58). SYBR green real-time PCR master mix (QPK-212; Toyobo) was used for PCR in a real-time detection system (Bio-Rad). The primer pairs for nup62l, ved, and vent were nup62l-qPCRF (5′-CTCTTGGTGCTCCTACTG-3′) and nup62l-qPCRR (5′-CAATGTGAATCCTGTGACTG-3′), ved-qPCRF (5′-GTCTGGAGCGAGTCTTTA-3′) and ved-qPCRR (5′-GGTGAGTTTGAGTGTTCTG-3′), and vent-qPCRF (5′-AGTGATGACAGTGAAGTAGAA-3′) and vent-qPCRR (5′-CGCTATCTTCCTCCTCTG-3′), respectively. The primer pair for β-actin was actin-qPCRF (5′-CGGAATATCATCTGCTTGTAA-3′) and actin-qPCRR (5′-CATCATCTCCAGCGAATC-3′).

WISH.

Antisense and sense RNA probes were generated by in vitro transcription using T7 or T3 RNA polymerase and then labeled with digoxigenin (DIG; Roche). Embryos were fixed overnight at 4°C in 4% paraformaldehyde–phosphate-buffered saline (PFA-PBS). After dechorionation, embryos were dehydrated in 100% methanol and stored at −20°C for use. Before whole-mount in situ hybridization (WISH), embryos were rinsed in 75%, 50%, and 25% methanol-PBS and PBS with Tween 20 (PBST). All embryos at 14 to 24 hpf should be permeabilized by proteinase K treatment. After treatment with 4% PFA for 20 min and with PBST four times, embryos were incubated in prehybridization Hyb mix for 4 to 6 h at 70°C and then in Hyb mix containing a probe overnight at 70°C. Embryos were treated with posthybridization washes at 68°C and room temperature, blocked with 1× blocking solution containing 2 mg/ml bovine serum albumin (BSA) and 2% sheep serum for 4 h at room temperature, and incubated with anti-DIG antibody (Roche) at 4°C overnight. Washed embryos were incubated with nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) solution (Amresco) for the development of a color reaction, and signals were detected using a SteReo Lumar V12 microscope from Carl Zeiss.

In situ hybridization on cryostat sections.

Zebrafish larvae were fixed in 4% PFA overnight, washed in PBS, and equilibrated in 30% sucrose in PBS overnight at 4°C. The embryos were then embedded in OCT compound (Sakura), frozen, and cut serially in 6-μm-thick sections in the sagittal, coronal, and transverse planes. Sections were collected on microscope slides treated with polylysine and stored at −80°C in sealed boxes. Sections were air dried at room temperature for 20 min to 3 h and postfixed with 4% PFA in PBS for 20 min. After three washes in PBS, sections were acetylated and incubated in 50% formamide-3× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Riboprobes were diluted to 100 ng/ml in warm (65°C) hybridization solution (50% formamide, 5× SSC, 10 mM β-mercaptoethanol, 10% dextran sulfate, 2× Denhardt's solution, 250 μg/ml yeast tRNA, 500 μg/ml heat-inactivated salmon sperm DNA). Hybridization was carried out in a humid chamber at 65°C for 16 h. Slides were rinsed in 50% formamide, 2× SSC at 65°C, treated with RNase A for 30 min at 37°C, rinsed twice with 0.2× SSC at 65°C and with PBST at room temperature, and blocked with 10% sheep serum in PBST at room temperature for 1 to 3 h. Blocking solution with anti-DIG–alkaline phosphatase (AP) antibody at 1:2,000 was added and incubated overnight at 4°C. Slides were checked under an LSM710 microscope from Carl Zeiss.

Cell tracing experiments.

Cell tracing experiments were performed as described previously (59), with minor modifications. The full-length kaede cDNA was subcloned into the pCS2+ vector. Two hundred picograms of capped kaede mRNA was coinjected with 10 ng nup62l-MO, 300 pg nup62l capped mRNA, 300 pg nup62l-DN capped mRNA, or 10 ng nup62l-MO plus 300 pg nup62l capped mRNA into one-cell-stage embryos. UV light was directed at the dorsal or lateral blastoderm margin of embryos for 45 s.

Cell culture, transfection, and luciferase reporter assays.

The culture and transfection of ZF4 cells were performed as described previously (58). Luciferase reporter assays were performed with the vectors Top-flash, Bre, and (CAGA)12-Luc (58). Luciferase activity in transfected ZF4 cells or microinjected embryos was determined using a dual-luciferase assay system from Promega and a Sirius luminometer from Berthold by following the manufacturers' instructions.

Immunofluorescence staining.

Immunofluorescence staining was performed in transfected ZF4 cells as described previously (58). Antibodies included rabbit antihemagglutinin (anti-HA) (Sigma-Aldrich) and goat anti-rabbit antibody–fluorescein isothiocyanate (FITC) (Santa Cruz). DAPI (4′,6-diamidino-2-phenylindole) was used to label nuclei. Fluorescence images of fixed cells were acquired with a Zeiss LSM 710 confocal microscope.

Coimmunoprecipitation (co-IP) and WB.

Cells were lysed in RIPA buffer. Cell lysates were incubated on ice for 10 min and then centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was incubated with anti-HA or anti-Flag antibody for 1 h at 4°C. The supernatant was then incubated with 10 μl protein A/G Plus-agarose immunoprecipitation reagent (Santa Cruz) at 4°C overnight. After centrifugation at 12,000 × g for another 10 min at 4°C, precipitates were rinsed four times with 1× PBS and boiled in 1× loading buffer. Precipitated proteins were separated in 10% polyacrylamide gels and electrotransferred to Immobilon-P transfer membranes (Millipore). The membranes were blocked with 5% (wt/vol) dried milk in 1× Tris-buffered saline–Tween (TBST) buffer overnight at 4°C, incubated with the desired primary antibody (anti-HA or anti-Flag), and then probed with horseradish peroxidase (HRP)-conjugated secondary antibody. Immobilon Western blot (WB) chemiluminescence HRP (Millipore) acted as the substrate, and signals were observed using a Fujifilm LAS-4000 imaging system.

Lentiviral shRNA assays.

Lentiviral short hairpin RNA (shRNA) assays were performed as previously described (60). The target sequence of zebrafish nup62l (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001113596","term_id":"165972452","term_text":"NM_001113596"}}NM_001113596) was 5′-GCAGCCTATGGGAGGATTTAG-3′. The pLKO.1-puro empty vector was used as a negative control. Lentiviral shRNA plasmids or control vectors were cotransfected with packaging plasmids into HEK293T cells by use of X-tremeGENE HP DNA transfection reagent (Roche) to produce lentivirus. Medium containing lentivirus was collected after 72 h, centrifuged at 2,000 × g, and filtered through 0.22-μm filters. ZF4 cells were infected with lentivirus in the presence of 8 μg/ml Polybrene for 24 h and then selected with 0.5 μg/ml puromycin for 3 weeks.

ChIP assay.

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (61), with a few modifications. ZF4 cells were fixed in 4% formaldehyde for 10 min at room temperature. After swelling and lysis, 20 μg chromatin from a cell extract (average DNA length of 500 bp) was used in each ChIP mixture. Tcf4 antibody (Santa Cruz) was added to the cleared chromatin extract and incubated with rotation at 4°C overnight. Beta-actin antibody (Santa Cruz) was used on a parallel negative group. Purified DNA was used in a PCR to monitor the ChIP results. Primers 1054F (5′-CGTAGCAACGGTAACTAAGGAG-3′) and 1054R (5′-CGAAACGAGCAGTTTTTACTGTC-3′) were used for detection of the potential binding site of Tcf4 in the regulatory sequence of the nup62l gene; primers Neg-F (5′-CAGACACAGATGAGAGGTTTGC-3′) and Neg-R (5′-CTAGCTTTCCTATGAACATGATTTG-3′) were used as a negative control.

Fish lines and maintenance.

The AB strain of zebrafish was maintained in a recirculating water system, and embryos were raised under standard procedures. Naturally fertilized embryos were staged by morphological features or by hours postfertilization (hpf) (55). The animal studies were approved by the Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (approval ID Keshuizhuan 0829).

Plasmid construction.

Total RNAs were extracted from 24-hpf embryos by use of TRIzol reagent (Invitrogen), and first-strand cDNAs were synthesized using a RevertAid first-strand cDNA synthesis kit (Fermentas). Full-length cDNA of zebrafish nup62l (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001113596","term_id":"165972452","term_text":"NM_001113596"}}NM_001113596) was obtained by PCR amplification with primers Nup62l-SalI-F (5′-CTAGTCGACATGGCATTTAACTTTGGACAAACAGG-3′) and Nup62l-EcoRI-R (5′-TGCGAATTCTCACTGGAAGTTTAAACGAAAGCTC-3′) and subcloned into vectors for in vitro synthesis of capped mRNA and overexpression. The primer pair Nup62l-EcoRI-F (5′-CAGGAATTCGAGCCACCATGGCATTTAACTTTGGACAAAC-3′) and Nup62l-HindIII-R (5′-GATAAGCTTGTCAGTATATGGTTCCACTTTGCTCC-3′) was used for the generation of a nup62l mutant (nup62l-DN) in which amino acids 408 to 507 were deleted from the coiled-coil domain of the encoded protein. Nup62l mutants D1 to D14 were generated by deleting amino acids 1 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 507, 1 to 25, 26 to 50, 51 to 75, 76 to 100, 76 to 80, 81 to 85, 86 to 90, 91 to 95, and 96 to 100, respectively, from the potential β-catenin binding motif.

MO and mRNA injection.

A nup62l morpholino oligonucleotide (nup62l-MO) against the 5′ untranslated region (5′ UTR) (5′-TGAACTACTTCACAGCAGCTCCCGC-3′), nup54-ATG-MO (5′-ACGCGCCGCCGAAGTTGAACGCCAT-3′), and a standard control MO (STD-MO) were purchased from Gene Tools. MOs for β-catenin-1 (5′-CTGGGTAGCCATGATTTTCTCACAG-3′) and β-catenin-2 (5′-CCTTTAGCCTGAGCGACTTCCAAAC-3′) were gifts from Wuhan Xiao. MOs were injected into one-cell-stage embryos at the indicated doses. Capped mRNAs were synthesized using an mMESSAGE mMACHINE kit (Ambion, Austin, TX). Constructs for in vitro synthesis of capped mRNAs included zebrafish nup62l, mutants of zebrafish nup62l, EGFP, kaede, zebrafish β-catenin-1 and β-catenin-2, frzb, bmp4, and a truncated Bmp receptor (56). Capped mRNAs and MOs were prepared as described previously (57). One-cell-stage embryos were injected with 150 or 300 pg capped mRNA/embryo, unless otherwise stated. The synthetic zebrafish nup62l mRNA was diluted to different concentrations and coinjected with nup62l-MO to determine the optimal dose for the rescue of morpholino-injected embryos.

Real-time qPCR.

Total RNAs were treated with RNase-free DNase I, and cDNAs were synthesized using a First Strand cDNA synthesis kit (Thermo Scientific). Real-time quantitative PCR (qPCR) was performed as described previously (58). SYBR green real-time PCR master mix (QPK-212; Toyobo) was used for PCR in a real-time detection system (Bio-Rad). The primer pairs for nup62l, ved, and vent were nup62l-qPCRF (5′-CTCTTGGTGCTCCTACTG-3′) and nup62l-qPCRR (5′-CAATGTGAATCCTGTGACTG-3′), ved-qPCRF (5′-GTCTGGAGCGAGTCTTTA-3′) and ved-qPCRR (5′-GGTGAGTTTGAGTGTTCTG-3′), and vent-qPCRF (5′-AGTGATGACAGTGAAGTAGAA-3′) and vent-qPCRR (5′-CGCTATCTTCCTCCTCTG-3′), respectively. The primer pair for β-actin was actin-qPCRF (5′-CGGAATATCATCTGCTTGTAA-3′) and actin-qPCRR (5′-CATCATCTCCAGCGAATC-3′).

WISH.

Antisense and sense RNA probes were generated by in vitro transcription using T7 or T3 RNA polymerase and then labeled with digoxigenin (DIG; Roche). Embryos were fixed overnight at 4°C in 4% paraformaldehyde–phosphate-buffered saline (PFA-PBS). After dechorionation, embryos were dehydrated in 100% methanol and stored at −20°C for use. Before whole-mount in situ hybridization (WISH), embryos were rinsed in 75%, 50%, and 25% methanol-PBS and PBS with Tween 20 (PBST). All embryos at 14 to 24 hpf should be permeabilized by proteinase K treatment. After treatment with 4% PFA for 20 min and with PBST four times, embryos were incubated in prehybridization Hyb mix for 4 to 6 h at 70°C and then in Hyb mix containing a probe overnight at 70°C. Embryos were treated with posthybridization washes at 68°C and room temperature, blocked with 1× blocking solution containing 2 mg/ml bovine serum albumin (BSA) and 2% sheep serum for 4 h at room temperature, and incubated with anti-DIG antibody (Roche) at 4°C overnight. Washed embryos were incubated with nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) solution (Amresco) for the development of a color reaction, and signals were detected using a SteReo Lumar V12 microscope from Carl Zeiss.

In situ hybridization on cryostat sections.

Zebrafish larvae were fixed in 4% PFA overnight, washed in PBS, and equilibrated in 30% sucrose in PBS overnight at 4°C. The embryos were then embedded in OCT compound (Sakura), frozen, and cut serially in 6-μm-thick sections in the sagittal, coronal, and transverse planes. Sections were collected on microscope slides treated with polylysine and stored at −80°C in sealed boxes. Sections were air dried at room temperature for 20 min to 3 h and postfixed with 4% PFA in PBS for 20 min. After three washes in PBS, sections were acetylated and incubated in 50% formamide-3× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Riboprobes were diluted to 100 ng/ml in warm (65°C) hybridization solution (50% formamide, 5× SSC, 10 mM β-mercaptoethanol, 10% dextran sulfate, 2× Denhardt's solution, 250 μg/ml yeast tRNA, 500 μg/ml heat-inactivated salmon sperm DNA). Hybridization was carried out in a humid chamber at 65°C for 16 h. Slides were rinsed in 50% formamide, 2× SSC at 65°C, treated with RNase A for 30 min at 37°C, rinsed twice with 0.2× SSC at 65°C and with PBST at room temperature, and blocked with 10% sheep serum in PBST at room temperature for 1 to 3 h. Blocking solution with anti-DIG–alkaline phosphatase (AP) antibody at 1:2,000 was added and incubated overnight at 4°C. Slides were checked under an LSM710 microscope from Carl Zeiss.

Cell tracing experiments.

Cell tracing experiments were performed as described previously (59), with minor modifications. The full-length kaede cDNA was subcloned into the pCS2+ vector. Two hundred picograms of capped kaede mRNA was coinjected with 10 ng nup62l-MO, 300 pg nup62l capped mRNA, 300 pg nup62l-DN capped mRNA, or 10 ng nup62l-MO plus 300 pg nup62l capped mRNA into one-cell-stage embryos. UV light was directed at the dorsal or lateral blastoderm margin of embryos for 45 s.

Cell culture, transfection, and luciferase reporter assays.

The culture and transfection of ZF4 cells were performed as described previously (58). Luciferase reporter assays were performed with the vectors Top-flash, Bre, and (CAGA)12-Luc (58). Luciferase activity in transfected ZF4 cells or microinjected embryos was determined using a dual-luciferase assay system from Promega and a Sirius luminometer from Berthold by following the manufacturers' instructions.

Immunofluorescence staining.

Immunofluorescence staining was performed in transfected ZF4 cells as described previously (58). Antibodies included rabbit antihemagglutinin (anti-HA) (Sigma-Aldrich) and goat anti-rabbit antibody–fluorescein isothiocyanate (FITC) (Santa Cruz). DAPI (4′,6-diamidino-2-phenylindole) was used to label nuclei. Fluorescence images of fixed cells were acquired with a Zeiss LSM 710 confocal microscope.

Coimmunoprecipitation (co-IP) and WB.

Cells were lysed in RIPA buffer. Cell lysates were incubated on ice for 10 min and then centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was incubated with anti-HA or anti-Flag antibody for 1 h at 4°C. The supernatant was then incubated with 10 μl protein A/G Plus-agarose immunoprecipitation reagent (Santa Cruz) at 4°C overnight. After centrifugation at 12,000 × g for another 10 min at 4°C, precipitates were rinsed four times with 1× PBS and boiled in 1× loading buffer. Precipitated proteins were separated in 10% polyacrylamide gels and electrotransferred to Immobilon-P transfer membranes (Millipore). The membranes were blocked with 5% (wt/vol) dried milk in 1× Tris-buffered saline–Tween (TBST) buffer overnight at 4°C, incubated with the desired primary antibody (anti-HA or anti-Flag), and then probed with horseradish peroxidase (HRP)-conjugated secondary antibody. Immobilon Western blot (WB) chemiluminescence HRP (Millipore) acted as the substrate, and signals were observed using a Fujifilm LAS-4000 imaging system.

Lentiviral shRNA assays.

Lentiviral short hairpin RNA (shRNA) assays were performed as previously described (60). The target sequence of zebrafish nup62l (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_001113596","term_id":"165972452","term_text":"NM_001113596"}}NM_001113596) was 5′-GCAGCCTATGGGAGGATTTAG-3′. The pLKO.1-puro empty vector was used as a negative control. Lentiviral shRNA plasmids or control vectors were cotransfected with packaging plasmids into HEK293T cells by use of X-tremeGENE HP DNA transfection reagent (Roche) to produce lentivirus. Medium containing lentivirus was collected after 72 h, centrifuged at 2,000 × g, and filtered through 0.22-μm filters. ZF4 cells were infected with lentivirus in the presence of 8 μg/ml Polybrene for 24 h and then selected with 0.5 μg/ml puromycin for 3 weeks.

ChIP assay.

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (61), with a few modifications. ZF4 cells were fixed in 4% formaldehyde for 10 min at room temperature. After swelling and lysis, 20 μg chromatin from a cell extract (average DNA length of 500 bp) was used in each ChIP mixture. Tcf4 antibody (Santa Cruz) was added to the cleared chromatin extract and incubated with rotation at 4°C overnight. Beta-actin antibody (Santa Cruz) was used on a parallel negative group. Purified DNA was used in a PCR to monitor the ChIP results. Primers 1054F (5′-CGTAGCAACGGTAACTAAGGAG-3′) and 1054R (5′-CGAAACGAGCAGTTTTTACTGTC-3′) were used for detection of the potential binding site of Tcf4 in the regulatory sequence of the nup62l gene; primers Neg-F (5′-CAGACACAGATGAGAGGTTTGC-3′) and Neg-R (5′-CTAGCTTTCCTATGAACATGATTTG-3′) were used as a negative control.

RESULTS

Expression and transcriptional regulation of nup62l during zebrafish embryogenesis.

Zebrafish nup62l, a homolog of mammalian Nup62 genes, encodes a protein consisting of 507 amino acids. Amino acid sequence alignment indicates that zebrafish Nup62l has a high degree of similarity to Nup62 proteins from mice (47%) and humans (47%) (see Fig. S1A in the supplemental material). Like human Nup62, Nup62l is predicted to be composed of three different segments: an unstructured FG-repeat region in the N terminus (residues 1 to 164), a central serine/threonine-rich linker (residues 165 to 312), and a highly conserved C terminus (residues 313 to 507) that adopts an α-helical coiled-coil conformation (see Fig. S1B).

The spatiotemporal expression of zebrafish nup62l during embryogenesis was first examined by whole-mount in situ hybridization (WISH). Maternally derived nup62l transcripts were weakly detected in unfertilized and one-cell-stage embryos (Fig. 1A1 and andA2).A2). Ubiquitous expression of nup62l was observed at the shield and 12-hpf stages (Fig. 1A3 and andA4).A4). By 24 hpf, nup62l RNA was detected predominantly in the tectum, hindbrain, and somites (Fig. 1A5 and andA6).A6). High levels of nup62l transcripts were found in branchial arches, pectoral fin buds, and posterior head regions at 48 hpf (Fig. 1A7) and in the forebrain ventricular zone, posterior tectum, hindbrain rhombomere, hyoid, telencephalon, forebrain-midbrain boundaries, cerebellum, branchial arches, pectoral fin buds, intestine, bipolar cells, and ganglion cells at 72 and 96 hpf (Fig. 1A8 to A10). These results imply that Nup62l plays certain roles in early embryonic development and organogenesis of zebrafish.

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Expression patterns and transcriptional regulation of zebrafish nup62l. (A) Spatiotemporal expression of nup62l during embryogenesis was detected by WISH. (A1 and A2) Unfertilized eggs and one-cell-stage embryos. The images show lateral views with the dorsal toward the right. (A3 and A4) Embryos at 6 hpf and 12 hpf. The images show lateral views with the dorsal toward the right (A3) or the anterior region toward the top (A4). (A5 and A6) Embryos at 24 hpf. The images show a dorsal view with the anterior region toward the top (A5) and a lateral view with the tail toward the right (A6). (A7 and A9) Embryos at 48 hpf and 72 hpf. The images show dorsal views with the anterior region toward the top (A7) or toward the left (A9). (A8 and A10) Embryos at 72 hpf and 96 hpf. The images show lateral views with the anterior region toward the left. (A9-a, A10-a, and A10-b) Positions of cryosections. t, tectum; hb, hind brain; bs, somites; ba, branchial arches; pf, pectoral fin buds; fvz, forebrain ventricular zone; pt, posterior tectum; te, telencephalon; bfm, boundaries of forebrain-midbrain; i, intestine; hr, hindbrain rhombomere; hy, hyoid; bc, bipolar cells; gc, ganglion cells; ce, cerebellum. (B) Transcriptional regulation of nup62l. Embryos at the one-cell stage were injected with 300 pg constitutively active β-catenin-1 (β-cat) mRNA, 300 pg frzb mRNA, 300 pg bmp4 mRNA, 300 pg truncated bmpRI mRNA, 150 pg β-cat mRNA plus 150 pg bmp4 mRNA, or 150 pg frzb mRNA plus 150 pg truncated bmpRI mRNA. WT embryos were used as the control. Embryos are shown at the shield stage: for lateral views, the animal pole is toward the top; and for animal pole views, the dorsal is toward the right. (C) Total mRNAs extracted from pooled embryos in panel B were detected by qPCR assays. Data represent means and standard deviations (SD) for five independent experiments. *, P < 0.05; **, P < 0.01 (versus WT).

We next tested how transcriptional expression of nup62l was modulated by key signaling pathways implicated in early embryogenesis. As shown in Fig. 1B, expression of nup62l at 6 hpf was strongly induced on the dorsal side by ectopic expression of a constitutively active β-catenin-1 gene (β-cat) and on the ventral side by overexpression of a truncated Bmp receptor I gene (bmpRI), whereas it was repressed by the Wnt inhibitor gene frzb or bmp4. Additionally, reduced nup62l expression was observed in embryos injected with equal amounts of mRNAs for β-cat and bmp4, and its induction was detected in the ventral region of embryos injected with equal amounts of frzb and bmpRI. However, stimulation of the Nodal signal by injection of zebrafish Cyclops mRNA or suppression of the Fgf signal by injection of zebrafish sef mRNA resulted in undetectable effects on nup62l transcripts (data not shown). Similar results were obtained by detection of nup62l expression in pooled embryos by real-time PCR (qPCR) (Fig. 1C). In addition, WISH and qPCR analyses indicated that overexpressed β-catenin-1/2 can cause a significant decline in the expression of the Bmp-dependent genes ved and vent (see Fig. S2A and B in the supplemental material). These findings suggest that Wnt signaling acts upstream of Bmp signaling to upregulate nup62l expression and that activation of Bmp signaling negatively regulates transcriptional expression of nup62l during embryogenesis.

Previous studies have proposed that the β-catenin/Tcf complex regulates gene transcription via binding to TCF/LEF-binding sites (5′-A/T A/T CAAAG-3′) within the promoter regions of target genes (62, 63). Thus, we further checked whether nup62l transcription activity was directly regulated by the β-catenin/Tcf complex. Seven core binding sites within the predicted nup62l promoter were identified (see Fig. S2C in the supplemental material). ChIP assays were performed with Tcf4 antibody and ZF4 cells, and Tcf4 could bind specifically to the nup62l promoter region (see Fig. S2D). These data reflect that nup62l transcription is directly modulated by the β-catenin/Tcf4 complex.

Alterations in nup62l expression lead to severe defects in dorsoventral patterning of developing embryos.

To investigate functions of endogenous Nup62l during zebrafish embryogenesis, an antisense morpholino oligonucleotide (MO) against the 5′ UTR sequence of nup62l was microinjected into one-cell-stage embryos to suppress nup62l expression. The efficiency of nup62l suppression by nup62l-MO was examined by use of a construct called nup62l/EGFP that can produce an in-frame transcript of enhanced green fluorescent protein (EGFP) with the nup62l-MO target at its N terminus. EGFP was markedly expressed in more than 92% of embryos injected with STD-MO and nup62l/EGFP plasmid DNA; however, EGFP was faintly found in fewer than 4% of embryos injected with nup62l-MO and nup62l/EGFP (see Fig. S3A and B in the supplemental material). These data indicate that nup62l-MO can effectively block the translation of nup62l transcripts.

Compared to wild-type (WT) embryos, nup62l morphants exhibited abnormal phenotypes, such as a thickened ventral at the shield stage, an expanded anterior-posterior (AP) axis, and a laterally spreading notochord at 12 hpf. In contrast, injection of capped nup62l mRNA led to a thickened organizer and an elongated AP axis. In addition, the majority of morphological changes obtained for nup62l-MO were neutralized back to the WT by coinjection with nup62l mRNA (Fig. 2A). Total numbers of embryos examined and ratios of embryos with morphological defects are summarized in Fig. 2B.

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Overexpression and knockdown of nup62l cause defects in dorsoventral patterning and CE movements. (A) Morphologies of embryos injected with 300 pg nup62l mRNA, 10 ng nup62l-MO, or 300 pg nup62l mRNA plus 10 ng nup62l-MO at the one-cell stage. WT embryos were used as the control. Lateral views are shown for 6 hpf, with the animal pole toward the top and the dorsal toward the right. Lateral and dorsal views are shown for 12 hpf, with the anterior region toward the top. (B) Ratios of defective embryos, as shown in panel A, and the numbers of embryos examined (indicated below the bar). (C) Lateral views of embryos injected with 300 pg nup62l mRNA or 10 ng nup62l-MO. Abnormal embryos were divided into three groups: those with mild, intermediate, and severe defects.

At later stages, MO-induced Nup62l deficiency caused ventralized phenotypes, including a shrunken dorsal, loss of head and notochord structure, enlarged somites and tail, and broadened blood islands, at and after 24 hpf (Fig. 2C). These defects were not altered by coinjection of 1 to 5 ng p53 MO (data not shown), suggesting that these abnormal phenotypes were not an artifact of MO toxicity. In contrast, aberrant phenotypes of nup62l-overexpressing embryos were characterized as dorsalization: loss of the caudal ventral fin, a shorter and twisted tail, and deformed somites (Fig. 2C). Most of defects caused by nup62l-MO were rescued by coinjection of nup62l capped mRNA. Ratios of abnormal embryos after injection of nup62l-MO and/or nup62l mRNA are summarized in Table S1 in the supplemental material.

To further characterize the roles of Nup62l in dorsalization of early developing embryos, several marker genes for dorsoventral patterning were detected by WISH assay of embryos injected with nup62l-MO or capped nup62l mRNA. As shown in Fig. 3A and andB,B, expression of the dorsal genes boz, chordin, and gsc decreased, while expression of the ventral genes bmp2b, bmp4, eve1, and gata1 expanded, in 41% to 62% of nup62l morphants. In contrast, a reduced expression of ventral genes and increased expression of dorsal genes were found in 38% to 64% of embryos injected with nup62l mRNA.

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Effects of nup62l overexpression and knockdown on expression of dorsoventral and CE-related marker genes. (A) Expression of genes in WT embryos and embryos injected with 10 ng nup62l-MO or 300 pg nup62l mRNA at the indicated stages. Embryos with expression of dorsal genes (boz, gsc, and chordin) and ventral genes (bmp2b, bmp4, and eve1) are shown as animal pole views with the dorsal toward the right. Dorsal views of embryos with the anterior region toward the top are shown for the ventral gene gata1. The margins of expressed eve1 are indicated by arrows. (B) Percentages of embryos displaying the indicated patterns of expression of dorsoventral genes (shown in panel A) and the numbers of embryos detected (provided at the bottom). The expression patterns of these markers in the remaining embryos resembled those in WT embryos. (C) WISH assays of CE movement markers in zebrafish. WT embryos or embryos injected with 10 ng nup62l-MO or 300 pg nup62l mRNA were stained for markers of the endoderm (foxd3), mesendoderm (hgg1), endomesoderm (chordin), mesoderm (shh, ntl, myoD, and papc), and ectoderm (dlx3). Embryos are shown as dorsal views with the animal pole or anterior region toward the top. (D) The numbers of embryos detected (provided at the bottom) and percentages of embryos with the expression patterns shown in panel C.

Taken together, these observations imply the involvement of Nup62l in early dorsoventral patterning of zebrafish.

Nup62l regulates gastrula CE movements and formation of midline organs.

We assessed the expression patterns of marker genes for specification of the endoderm (foxd3), endomesoderm (chordin), mesendoderm (hgg1), mesoderm (shh, ntl, myoD, and papc), and ectoderm (dlx3) (57). Expression levels of these markers were not severely affected by nup62l-MO or capped nup62l mRNA, but their distribution patterns were remarkably altered (Fig. 3C). In comparison with those of WT embryos, transcripts of these genes expanded along the dorsoventral axis and shortened along the AP axis of nup62l morphants, as shown by a slightly more posteriorly positioned prechordal plate (hgg1), a restrained convergence of the neural plate (dlx3), a shorter but wider notochord (ntl), an increased distance of bilateral adaxial cells (myoD), and a mediolaterally broadened and shortened presomitic mesoderm (papc). In contrast, transcript distributions of these genes in nup62l-overexpressing embryos narrowed along the dorsoventral axis and elongated along the AP axis. Total numbers of embryos and ratios of embryos with the different expression patterns are shown in Fig. 3D. These observations suggest that Nup62l is essential for normal migration but not for the specification of cells in both the mesendoderm and ectoderm.

We next performed cell tracing assays to monitor the migration of different cell populations that were irradiated with a beam of UV light at the shield stage. As shown in Fig. 4A, the dorsal-ward movement of red fluorescence-labeled cell populations in embryos injected with nup62l-MO or nup62l-dominant negative (nup62l-DN) mRNA was significantly delayed at 8 to 9.5 hpf. nup62l-DN mRNA was generated by deletion of 100 residues of the putative coiled-coil domain of the encoded protein, and injection of capped nup62l-DN mRNA led to embryonic ventralization and altered expression of a series of markers, all of which resembled the results seen with nup62l-MO injection. Coinjection of nup62l mRNA effectively rescued these alterations caused by nup62l-DN mRNA (see Fig. S4A and B and Table S1 in the supplemental material). Moreover, nup62l-DN overexpression significantly restrained the β-catenin-induced activity of the Wnt reporter Top-flash (see Fig. S4C). Thus, nup62l-DN can be used as a dominant negative inhibitor of the activity of Wnt/β-catenin signaling. In contrast, the migration of lateral cells to the dorsal was apparently promoted in embryos overexpressing nup62l mRNA. Coinjection of nup62l mRNA with nup62l-MO restored the delayed movement of lateral cells. We then examined the extension movement of axial mesoderm cell populations in the dorsal organizer. The elongation ability of labeled cells along the AP axis was severely inhibited in embryos injected with nup62l-MO or nup62l-DN mRNA; however, injection of nup62l mRNA enhanced the migration of dorsal organizer cells along the AP axis, and the delayed extension movement by nup62l-MO was rescued to normal by coinjecting an appropriate dose of nup62l mRNA. These findings suggest that Nup62l is indispensable for precise CE movements during gastrulation of zebrafish embryos.

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Nup62l regulates CE movements and specification of midline organ precursors in zebrafish. (A) Effects of Nup62l on CE movements were examined by cell tracing experiments. WT embryos at the one-cell stage were injected with kaede mRNA (200 pg/embryo), and 200 pg kaede mRNA was coinjected with 10 ng nup62l-MO, 300 pg nup62l mRNA, 300 pg nup62l-DN mRNA, or 10 ng nup62l-MO plus 300 pg nup62l mRNA into individual embryos in other groups. Cells were labeled by photoconversion of the Kaede protein at the shield stage. Images were taken directly after labeling. The images show lateral views with the animal pole toward the top. Each experiment was repeated five times. (B) The influence of Nup62l in formation of midline organ precursors was determined by WISH at the indicated stages. WT embryos and embryos injected with 10 ng nup62l-MO or coinjected with 10 ng nup62l-MO and 300 pg nup62l mRNA were stained for the heart marker cmlc (dorsal views with anterior region toward the top), the liver markers cp and lfabp (dorsal views with anterior region toward the top), a the exocrine pancreas marker trypsin (dorsal views with anterior region toward the top), and the intestinal marker ifabp (dorsal views with anterior region to the left). (C) Ratios of embryos with expression patterns shown in panel B and total numbers of embryos detected (indicated at the bottom).

Since nup62l transcripts were detected in anterior and midline structures of later-stage larvae (Fig. 1A), we addressed whether Nup62l is required for the formation of midline organs. The expression levels of marker genes for the liver, heart, pancreas, and intestine in embryos injected with nup62l-MO were examined by WISH assays. Knockdown of Nup62l interrupted the expression of markers, such as heart cmlc at 24 hpf, liver cp at 48 hpf, liver lfabp at 72 hpf, and exocrine pancreas trypsin and intestinal ifabp at 72 hpf. The decreased expression of these markers in nup62l morphants was restored by coinjection of nup62l mRNA (Fig. 4B). Total numbers of embryos examined and ratios of embryos with altered expression of markers are summarized in Fig. 4C. Thus, our data clearly reveal that Nup62l is essential for the specification of midline organ precursors.

Nup62l specifically modulates Wnt/β-catenin and Bmp signaling in zebrafish.

To further investigate mechanisms underlying the developmental roles of Nup62l in zebrafish, WISH assays were performed to assess the effects of Nup62l on the expression of marker genes for canonical Wnt, Bmp, Nodal, and Fgf signaling pathways. These genes include dkk1, boz, otx2, wnt8a, and tbx6 for Wnt/β-catenin, ved and vent for Bmp, spry4 and sef for Fgf, and flh and xbp1 for Nodal signaling (57). As shown in Fig. 5A, the expression of dkk1, boz, tbx6, otx2, and wnt8a was attenuated in >76% of nup62l morphants and increased in >85% of embryos injected with capped nup62l mRNA. The expression of ved and vent was induced by nup62l-MO and inhibited by nup62l mRNA. Importantly, enhanced expression of Wnt target genes and decreased expression of Bmp targets by nup62l mRNA were neutralized by coinjection with β-catenin-1-MO or β-catenin-2-MO in >90% of embryos, suggesting that Nup62l activates classical Wnt and restrains Bmp in a β-catenin-mediated manner. However, the expression of sef, spry4, xbp1, and flh was unperturbed by knockdown or ectopic expression of nup62l during zebrafish embryogenesis (data not shown).

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Effects of Nup62l on activities of Wnt/β-catenin and Bmp signaling in zebrafish. (A) Expression patterns of markers (indicated at the top) for Wnt and Bmp pathways in WT embryos or embryos injected with 300 pg nup62l mRNA, 10 ng nup62l-MO, or 300 pg nup62l mRNA plus 8 ng β-catenin-1/2-MO at the indicated stages. Images show dorsal views with the animal pole toward the top for boz, animal pole views with the dorsal toward the right for ved, vent, and dkk1, dorsoanterior views with the anterior region toward the top for otx2, vegetal views with the dorsal oriented toward the right for wnt8a, and dorsal views with the anterior region toward the top for tbx6. The arrows indicated the margins of ved and vent expression. (B) ZF4 cells were cotransfected with 250 ng of plasmids, including 84 ng Top-flash reporter, 16 ng pRL-TK as an internal control, 50 ng constitutively active β-catenin-1 (HA–β-cat) plasmid as a positive control, and/or 50 to 100 ng Flag–nup62l-DN. Empty vectors were used to balance the total plasmid amount. (C) ZF4 cells were transfected with a mixture of plasmids and analyzed as indicated for panel B. The HA-lef1-VP16 construct (positive control) was able to stimulate Top-flash. (D) ZF4 cells were cotransfected with 250 ng plasmids, including 84 ng Bre reporter, 16 ng pRL-TK as an internal control, 50 ng HA-bmp4 as a positive control, and/or 50 to 100 ng Flag-nup62l. Empty vectors were used to balance the total plasmid amount. Luciferase activity was measured using cellular lysates after transfection for 24 h. WB was carried out to find the proper expression of HA–β-cat, HA-lef1-VP16, HA-bmp4, and Flag–nup62l-DN/Flag-nup62l. Data in panels B and D represent means and SD for three wells. **, P < 0.01 versus Top-flash- or Bre-transfected cells.

Next, reporter systems for Wnt/β-catenin, Bmp, and Nodal/TGF-β signaling were utilized to verify these observations. Luciferase assays were performed with transfected ZF4 cell lysates. Top-flash-luciferase expression of the Wnt/β-catenin reporter was significantly induced by coexpression of β-catenin-1 (HA–β-cat) (P < 0.01), while the induction was antagonized by coexpression of Flag–nup62l-DN, in a dose-dependent manner (P < 0.01 in all cases) (Fig. 5B). In addition, the activity of Top-flash was markedly activated by overexpression of a dominant positive Lef1-VP16 fusion (>100-fold), but the activation was unperturbed by coexpression of Flag–nup62l-DN (Fig. 5C). These data mirror the observation that activation of canonical Wnt signaling by Nup62l is mediated by β-catenin but not by Lef1.

Bre-luciferase expression of the Bmp reporter was evidently elevated by HA-bmp4, but coexpression of Flag-nup62l attenuated this activity in a dose-dependent manner (P < 0.01 in all cases) (Fig. 5D). Western blot (WB) assays indicated that HA–β-cat, HA-lef1-VP16, HA-bmp4, Flag–nup62l-DN, and Flag-nup62l were expressed normally in corresponding groups of transfected cells (Fig. 5B to toD).D). However, expression of the (CAGA)12-Luc reporter for Nodal/transforming growth factor beta (TGF-β) signaling was not affected by overexpression or knockdown of nup62l (data not shown).

These findings suggest that developmental functions of Nup62l in zebrafish are closely associated with its regulation of Wnt and Bmp signaling.

Nup62l is required for nuclear import of β-catenin in zebrafish.

A recent study demonstrated that β-catenin is able to traverse the NPC by direct binding to Nup62, Nup153, and RanBP2/Nup358 (54). Thus, the physical association of Nup62l and β-catenin-1/2 was first examined by co-IP and WB in ZF4 cells. As shown in Fig. 6A and andB,B, overexpressed Flag-Nup62l and HA–β-catenin-1/2 (HA–β-cat-1 or HA–β-cat-2) were coimmunoprecipitated with each other, reflecting that β-catenin-1/2 proteins are binding partners of Nup62l in zebrafish.

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Nup62l facilitates nuclear import of β-catenins in zebrafish. (A and B) The physical interaction between Flag-nup62l and HA–β-cat-1/2 in embryos was analyzed via co-IP and WB. Embryos at the one-cell stage were injected with 150 pg HA–β-cat-1/2, 150 pg Flag-nup62l, or 150 pg HA–β-cat-1/2 plus 150 pg Flag-nup62l. Total cell lysates (TCL) were used as the control. (C and D) Nup62l facilitates nuclear accumulation of β-catenin-1/2. Immunofluorescence staining was performed with ZF4 cells growing on 35-mm dishes after transfection with 800 ng HA–β-cat-1/2 plus 800 ng empty vector DNA, 800 ng HA–β-cat-1/2 plus 800 ng nup62l DNA, or 800 ng HA–β-cat-1/2 plus 800 ng nup62l-D9 DNA. ZF4 cells infected with a lentivirus expressing Nup62l siRNA (siNup62l) or a control siRNA (siCtrl) were analyzed by immunofluorescence staining after transfection with 800 ng HA–β-cat-1/2 plus 800 ng empty vector DNA. Cellular distributions of HA–β-cat proteins are displayed by the locations of FITC signals. (E and F) Ratios of β-catenin-1/2 proteins localized to the nucleus, the cytoplasm, or both the nucleus and the cytoplasm in differently transfected cells.

To investigate whether Nup62l affects the cellular localization of β-catenins, immunofluorescence staining assays were conducted by cotransfection of a plasmid expressing HA–β-cat-1/2 with another plasmid expressing Flag-Nup62l or Flag−Nup62l-D9 into ZF4 cells. Flag–Nup62l-D9 was a deletion mutant of Nup62l without the capability of interaction with β-catenins. As shown in Fig. 6C and andD,D, HA signals were positioned in both the cytoplasm and nuclei of HA–β-cat-1/2-transfected cells and mainly distributed in the nuclei of cells cotransfected with Nup62l, indicating that Nup62l promotes the nuclear accumulation of HA–β-catenins. The nuclear accumulation of HA–β-catenins was stimulated by Nup62l but not by Nup62l-D9. Moreover, Nup62l small interfering RNA (siRNA) (siNup62l) was used to effectively suppress the expression of endogenous Nup62l in ZF4 cells (see Fig. S4D in the supplemental material). As expected, the nuclear import of β-cat-1/2 was markedly impeded in siNup62l-infected cells but not in siCtrl-infected cells (Fig. 6C and andD).D). The numbers of cells showing HA signals in three visual fields are summarized in Fig. 6E and andF.F. These data suggest that Nup62l can facilitate the nuclear import of active β-catenins in zebrafish.

Identification of Nup62l domain for β-catenin binding.

To further elucidate the molecular mechanisms underlying the Nup62l-mediated translocation of β-catenins, a series of Flag-nup62l deletion mutants (D1 to D14) was generated to identify the binding motifs of β-catenin-1 in Nup62l (Fig. 7A). The results of co-IP and WB assays indicated that nup62l mutants D1 and D9 to D14 failed to associate with HA–β-catenin-1/2 in transfected ZF4 cells; however, other deletion mutants retained the ability to bind to HA–β-catenin-1 (Fig. 7B). Overexpressed mutants D1 and D9 to D14 were not able to activate canonical Wnt signaling in transfected cells (Fig. 7C). Similarly, Nup62l bound to β-catenin-2 via the same functional domain (see Fig. S5 in the supplemental material). Moreover, injection of capped D9 mRNA exhibited few effects on morphology and the expression of markers for dorsoventral patterning, CE movements, and midline organ precursors (see Fig. S4A and B and Table S1). Thus, residues 76 to 100 of Nup62l, which contain two FG sequences, are responsible for the interaction and nuclear translocation of β-catenin-1/2 in zebrafish.

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Biochemical analyses of β-catenin-1 binding motif in Nup62l. (A) Schematic diagrams representing a series of Nup62l deletion constructs. +, interaction; −, no interaction. (B) Interactions between a series of Flag-tagged Nup62l mutants and HA–β-catenin-1 were detected by co-IP and WB. (C) Effects of Nup62l mutants on expression of the Wnt signal-responsive reporter Top-flash, which can be activated by overexpression of β-catenin-1. Mutants D1 and D9 to D14, harboring deletions within residues 76 to 100, failed to induce reporter activation. Data from the luciferase reporter assays are presented as means and SD for samples in three wells. **, P < 0.01 versus Top-flash-transfected cells.

Since Nup62-Nup58-Nup54-Nup45 is a crucial subcomplex of the NPC, we addressed the consequences of zebrafish nup54 knockdown during embryogenesis by injection of nup54-ATG-MO. The results indicated that silencing of nup54 led to ventralized embryos and altered expression of marker genes (see Fig. S4A and B and Table S1 in the supplemental material), similar to the effects exhibited in nup62l morphants. These data indicate that the Nup62l subcomplex, in addition to the interaction of Nup62l and β-catenin, is indispensable for the precise nuclear import of β-catenins that is tightly correlated with the phenotypes shown and with gene expression.

Mapping of β-catenin domain for Nup62l binding.

The β-catenin protein is a transport carrier that can effectively compete with importin-β for shuttling of the NPC due to its transport armadillo (arm) repeats, which resemble the transport-active HEAT repeats of importin-β (64, 65). Thus, six mutants with a deletion in HA–β-catenin-1 were generated (DN1 to DN6) to map the binding region of Nup62l in β-catenin-1 (Fig. 8A). Results from co-IP and WB assays indicated that DN3 and DN5 (lacking arm repeats R3 to R8 and R10 to R12, respectively) had lost the ability to interact with Flag-Nup62l (Fig. 8B). Next, we examined the subcellular localization of two β-catenin-1 mutants and found that the nuclear accumulation of HA–β-cat-1-DN3 and HA–β-cat-1-DN5 was not affected by Nup62l (Fig. 8C). The numbers of cells showing HA signals are summarized in Fig. 8D. Collectively, arm repeats R3 to R8 and R10 to R12 of β-catenin-1 are essential for both Nup62l binding and nuclear import. Moreover, the same motifs of β-catenin-2 are required for interaction and nuclear translocation (see Fig. S6 in the supplemental material).

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Mapping of β-catenin-1 domain required for binding to Nup62l and for nuclear transfer. (A) Cartoon illustration of β-catenin-1 domains and deletion mutants. +, interaction; −, no interaction. (B) Co-IP and WB assays were conducted to assess the reciprocal interaction between Flag-tagged Nup62l and β-catenin-1 mutants, and arm repeats R3 to R8 and R10 to R12 in β-catenin-1 were determined to be the Nup62l binding domains. (C) Immunofluorescence staining assays of ZF4 cells transfected with 800 ng HA–β-cat-1-DN3 plus 800 ng nup62l-expressing plasmid or with 800 ng HA–β-cat-1-DN5 plus 800 ng nup62l-expressing plasmid. (D) Ratios of transfected cells in which two β-catenin-1 mutants were localized to the cytoplasm. (E) Schematic model for the feedback network that is closely associated with the functions of Nup62l during early zebrafish development.

Expression and transcriptional regulation of nup62l during zebrafish embryogenesis.

Zebrafish nup62l, a homolog of mammalian Nup62 genes, encodes a protein consisting of 507 amino acids. Amino acid sequence alignment indicates that zebrafish Nup62l has a high degree of similarity to Nup62 proteins from mice (47%) and humans (47%) (see Fig. S1A in the supplemental material). Like human Nup62, Nup62l is predicted to be composed of three different segments: an unstructured FG-repeat region in the N terminus (residues 1 to 164), a central serine/threonine-rich linker (residues 165 to 312), and a highly conserved C terminus (residues 313 to 507) that adopts an α-helical coiled-coil conformation (see Fig. S1B).

The spatiotemporal expression of zebrafish nup62l during embryogenesis was first examined by whole-mount in situ hybridization (WISH). Maternally derived nup62l transcripts were weakly detected in unfertilized and one-cell-stage embryos (Fig. 1A1 and andA2).A2). Ubiquitous expression of nup62l was observed at the shield and 12-hpf stages (Fig. 1A3 and andA4).A4). By 24 hpf, nup62l RNA was detected predominantly in the tectum, hindbrain, and somites (Fig. 1A5 and andA6).A6). High levels of nup62l transcripts were found in branchial arches, pectoral fin buds, and posterior head regions at 48 hpf (Fig. 1A7) and in the forebrain ventricular zone, posterior tectum, hindbrain rhombomere, hyoid, telencephalon, forebrain-midbrain boundaries, cerebellum, branchial arches, pectoral fin buds, intestine, bipolar cells, and ganglion cells at 72 and 96 hpf (Fig. 1A8 to A10). These results imply that Nup62l plays certain roles in early embryonic development and organogenesis of zebrafish.

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Expression patterns and transcriptional regulation of zebrafish nup62l. (A) Spatiotemporal expression of nup62l during embryogenesis was detected by WISH. (A1 and A2) Unfertilized eggs and one-cell-stage embryos. The images show lateral views with the dorsal toward the right. (A3 and A4) Embryos at 6 hpf and 12 hpf. The images show lateral views with the dorsal toward the right (A3) or the anterior region toward the top (A4). (A5 and A6) Embryos at 24 hpf. The images show a dorsal view with the anterior region toward the top (A5) and a lateral view with the tail toward the right (A6). (A7 and A9) Embryos at 48 hpf and 72 hpf. The images show dorsal views with the anterior region toward the top (A7) or toward the left (A9). (A8 and A10) Embryos at 72 hpf and 96 hpf. The images show lateral views with the anterior region toward the left. (A9-a, A10-a, and A10-b) Positions of cryosections. t, tectum; hb, hind brain; bs, somites; ba, branchial arches; pf, pectoral fin buds; fvz, forebrain ventricular zone; pt, posterior tectum; te, telencephalon; bfm, boundaries of forebrain-midbrain; i, intestine; hr, hindbrain rhombomere; hy, hyoid; bc, bipolar cells; gc, ganglion cells; ce, cerebellum. (B) Transcriptional regulation of nup62l. Embryos at the one-cell stage were injected with 300 pg constitutively active β-catenin-1 (β-cat) mRNA, 300 pg frzb mRNA, 300 pg bmp4 mRNA, 300 pg truncated bmpRI mRNA, 150 pg β-cat mRNA plus 150 pg bmp4 mRNA, or 150 pg frzb mRNA plus 150 pg truncated bmpRI mRNA. WT embryos were used as the control. Embryos are shown at the shield stage: for lateral views, the animal pole is toward the top; and for animal pole views, the dorsal is toward the right. (C) Total mRNAs extracted from pooled embryos in panel B were detected by qPCR assays. Data represent means and standard deviations (SD) for five independent experiments. *, P < 0.05; **, P < 0.01 (versus WT).

We next tested how transcriptional expression of nup62l was modulated by key signaling pathways implicated in early embryogenesis. As shown in Fig. 1B, expression of nup62l at 6 hpf was strongly induced on the dorsal side by ectopic expression of a constitutively active β-catenin-1 gene (β-cat) and on the ventral side by overexpression of a truncated Bmp receptor I gene (bmpRI), whereas it was repressed by the Wnt inhibitor gene frzb or bmp4. Additionally, reduced nup62l expression was observed in embryos injected with equal amounts of mRNAs for β-cat and bmp4, and its induction was detected in the ventral region of embryos injected with equal amounts of frzb and bmpRI. However, stimulation of the Nodal signal by injection of zebrafish Cyclops mRNA or suppression of the Fgf signal by injection of zebrafish sef mRNA resulted in undetectable effects on nup62l transcripts (data not shown). Similar results were obtained by detection of nup62l expression in pooled embryos by real-time PCR (qPCR) (Fig. 1C). In addition, WISH and qPCR analyses indicated that overexpressed β-catenin-1/2 can cause a significant decline in the expression of the Bmp-dependent genes ved and vent (see Fig. S2A and B in the supplemental material). These findings suggest that Wnt signaling acts upstream of Bmp signaling to upregulate nup62l expression and that activation of Bmp signaling negatively regulates transcriptional expression of nup62l during embryogenesis.

Previous studies have proposed that the β-catenin/Tcf complex regulates gene transcription via binding to TCF/LEF-binding sites (5′-A/T A/T CAAAG-3′) within the promoter regions of target genes (62, 63). Thus, we further checked whether nup62l transcription activity was directly regulated by the β-catenin/Tcf complex. Seven core binding sites within the predicted nup62l promoter were identified (see Fig. S2C in the supplemental material). ChIP assays were performed with Tcf4 antibody and ZF4 cells, and Tcf4 could bind specifically to the nup62l promoter region (see Fig. S2D). These data reflect that nup62l transcription is directly modulated by the β-catenin/Tcf4 complex.

Alterations in nup62l expression lead to severe defects in dorsoventral patterning of developing embryos.

To investigate functions of endogenous Nup62l during zebrafish embryogenesis, an antisense morpholino oligonucleotide (MO) against the 5′ UTR sequence of nup62l was microinjected into one-cell-stage embryos to suppress nup62l expression. The efficiency of nup62l suppression by nup62l-MO was examined by use of a construct called nup62l/EGFP that can produce an in-frame transcript of enhanced green fluorescent protein (EGFP) with the nup62l-MO target at its N terminus. EGFP was markedly expressed in more than 92% of embryos injected with STD-MO and nup62l/EGFP plasmid DNA; however, EGFP was faintly found in fewer than 4% of embryos injected with nup62l-MO and nup62l/EGFP (see Fig. S3A and B in the supplemental material). These data indicate that nup62l-MO can effectively block the translation of nup62l transcripts.

Compared to wild-type (WT) embryos, nup62l morphants exhibited abnormal phenotypes, such as a thickened ventral at the shield stage, an expanded anterior-posterior (AP) axis, and a laterally spreading notochord at 12 hpf. In contrast, injection of capped nup62l mRNA led to a thickened organizer and an elongated AP axis. In addition, the majority of morphological changes obtained for nup62l-MO were neutralized back to the WT by coinjection with nup62l mRNA (Fig. 2A). Total numbers of embryos examined and ratios of embryos with morphological defects are summarized in Fig. 2B.

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Overexpression and knockdown of nup62l cause defects in dorsoventral patterning and CE movements. (A) Morphologies of embryos injected with 300 pg nup62l mRNA, 10 ng nup62l-MO, or 300 pg nup62l mRNA plus 10 ng nup62l-MO at the one-cell stage. WT embryos were used as the control. Lateral views are shown for 6 hpf, with the animal pole toward the top and the dorsal toward the right. Lateral and dorsal views are shown for 12 hpf, with the anterior region toward the top. (B) Ratios of defective embryos, as shown in panel A, and the numbers of embryos examined (indicated below the bar). (C) Lateral views of embryos injected with 300 pg nup62l mRNA or 10 ng nup62l-MO. Abnormal embryos were divided into three groups: those with mild, intermediate, and severe defects.

At later stages, MO-induced Nup62l deficiency caused ventralized phenotypes, including a shrunken dorsal, loss of head and notochord structure, enlarged somites and tail, and broadened blood islands, at and after 24 hpf (Fig. 2C). These defects were not altered by coinjection of 1 to 5 ng p53 MO (data not shown), suggesting that these abnormal phenotypes were not an artifact of MO toxicity. In contrast, aberrant phenotypes of nup62l-overexpressing embryos were characterized as dorsalization: loss of the caudal ventral fin, a shorter and twisted tail, and deformed somites (Fig. 2C). Most of defects caused by nup62l-MO were rescued by coinjection of nup62l capped mRNA. Ratios of abnormal embryos after injection of nup62l-MO and/or nup62l mRNA are summarized in Table S1 in the supplemental material.

To further characterize the roles of Nup62l in dorsalization of early developing embryos, several marker genes for dorsoventral patterning were detected by WISH assay of embryos injected with nup62l-MO or capped nup62l mRNA. As shown in Fig. 3A and andB,B, expression of the dorsal genes boz, chordin, and gsc decreased, while expression of the ventral genes bmp2b, bmp4, eve1, and gata1 expanded, in 41% to 62% of nup62l morphants. In contrast, a reduced expression of ventral genes and increased expression of dorsal genes were found in 38% to 64% of embryos injected with nup62l mRNA.

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Effects of nup62l overexpression and knockdown on expression of dorsoventral and CE-related marker genes. (A) Expression of genes in WT embryos and embryos injected with 10 ng nup62l-MO or 300 pg nup62l mRNA at the indicated stages. Embryos with expression of dorsal genes (boz, gsc, and chordin) and ventral genes (bmp2b, bmp4, and eve1) are shown as animal pole views with the dorsal toward the right. Dorsal views of embryos with the anterior region toward the top are shown for the ventral gene gata1. The margins of expressed eve1 are indicated by arrows. (B) Percentages of embryos displaying the indicated patterns of expression of dorsoventral genes (shown in panel A) and the numbers of embryos detected (provided at the bottom). The expression patterns of these markers in the remaining embryos resembled those in WT embryos. (C) WISH assays of CE movement markers in zebrafish. WT embryos or embryos injected with 10 ng nup62l-MO or 300 pg nup62l mRNA were stained for markers of the endoderm (foxd3), mesendoderm (hgg1), endomesoderm (chordin), mesoderm (shh, ntl, myoD, and papc), and ectoderm (dlx3). Embryos are shown as dorsal views with the animal pole or anterior region toward the top. (D) The numbers of embryos detected (provided at the bottom) and percentages of embryos with the expression patterns shown in panel C.

Taken together, these observations imply the involvement of Nup62l in early dorsoventral patterning of zebrafish.

Nup62l regulates gastrula CE movements and formation of midline organs.

We assessed the expression patterns of marker genes for specification of the endoderm (foxd3), endomesoderm (chordin), mesendoderm (hgg1), mesoderm (shh, ntl, myoD, and papc), and ectoderm (dlx3) (57). Expression levels of these markers were not severely affected by nup62l-MO or capped nup62l mRNA, but their distribution patterns were remarkably altered (Fig. 3C). In comparison with those of WT embryos, transcripts of these genes expanded along the dorsoventral axis and shortened along the AP axis of nup62l morphants, as shown by a slightly more posteriorly positioned prechordal plate (hgg1), a restrained convergence of the neural plate (dlx3), a shorter but wider notochord (ntl), an increased distance of bilateral adaxial cells (myoD), and a mediolaterally broadened and shortened presomitic mesoderm (papc). In contrast, transcript distributions of these genes in nup62l-overexpressing embryos narrowed along the dorsoventral axis and elongated along the AP axis. Total numbers of embryos and ratios of embryos with the different expression patterns are shown in Fig. 3D. These observations suggest that Nup62l is essential for normal migration but not for the specification of cells in both the mesendoderm and ectoderm.

We next performed cell tracing assays to monitor the migration of different cell populations that were irradiated with a beam of UV light at the shield stage. As shown in Fig. 4A, the dorsal-ward movement of red fluorescence-labeled cell populations in embryos injected with nup62l-MO or nup62l-dominant negative (nup62l-DN) mRNA was significantly delayed at 8 to 9.5 hpf. nup62l-DN mRNA was generated by deletion of 100 residues of the putative coiled-coil domain of the encoded protein, and injection of capped nup62l-DN mRNA led to embryonic ventralization and altered expression of a series of markers, all of which resembled the results seen with nup62l-MO injection. Coinjection of nup62l mRNA effectively rescued these alterations caused by nup62l-DN mRNA (see Fig. S4A and B and Table S1 in the supplemental material). Moreover, nup62l-DN overexpression significantly restrained the β-catenin-induced activity of the Wnt reporter Top-flash (see Fig. S4C). Thus, nup62l-DN can be used as a dominant negative inhibitor of the activity of Wnt/β-catenin signaling. In contrast, the migration of lateral cells to the dorsal was apparently promoted in embryos overexpressing nup62l mRNA. Coinjection of nup62l mRNA with nup62l-MO restored the delayed movement of lateral cells. We then examined the extension movement of axial mesoderm cell populations in the dorsal organizer. The elongation ability of labeled cells along the AP axis was severely inhibited in embryos injected with nup62l-MO or nup62l-DN mRNA; however, injection of nup62l mRNA enhanced the migration of dorsal organizer cells along the AP axis, and the delayed extension movement by nup62l-MO was rescued to normal by coinjecting an appropriate dose of nup62l mRNA. These findings suggest that Nup62l is indispensable for precise CE movements during gastrulation of zebrafish embryos.

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Nup62l regulates CE movements and specification of midline organ precursors in zebrafish. (A) Effects of Nup62l on CE movements were examined by cell tracing experiments. WT embryos at the one-cell stage were injected with kaede mRNA (200 pg/embryo), and 200 pg kaede mRNA was coinjected with 10 ng nup62l-MO, 300 pg nup62l mRNA, 300 pg nup62l-DN mRNA, or 10 ng nup62l-MO plus 300 pg nup62l mRNA into individual embryos in other groups. Cells were labeled by photoconversion of the Kaede protein at the shield stage. Images were taken directly after labeling. The images show lateral views with the animal pole toward the top. Each experiment was repeated five times. (B) The influence of Nup62l in formation of midline organ precursors was determined by WISH at the indicated stages. WT embryos and embryos injected with 10 ng nup62l-MO or coinjected with 10 ng nup62l-MO and 300 pg nup62l mRNA were stained for the heart marker cmlc (dorsal views with anterior region toward the top), the liver markers cp and lfabp (dorsal views with anterior region toward the top), a the exocrine pancreas marker trypsin (dorsal views with anterior region toward the top), and the intestinal marker ifabp (dorsal views with anterior region to the left). (C) Ratios of embryos with expression patterns shown in panel B and total numbers of embryos detected (indicated at the bottom).

Since nup62l transcripts were detected in anterior and midline structures of later-stage larvae (Fig. 1A), we addressed whether Nup62l is required for the formation of midline organs. The expression levels of marker genes for the liver, heart, pancreas, and intestine in embryos injected with nup62l-MO were examined by WISH assays. Knockdown of Nup62l interrupted the expression of markers, such as heart cmlc at 24 hpf, liver cp at 48 hpf, liver lfabp at 72 hpf, and exocrine pancreas trypsin and intestinal ifabp at 72 hpf. The decreased expression of these markers in nup62l morphants was restored by coinjection of nup62l mRNA (Fig. 4B). Total numbers of embryos examined and ratios of embryos with altered expression of markers are summarized in Fig. 4C. Thus, our data clearly reveal that Nup62l is essential for the specification of midline organ precursors.

Nup62l specifically modulates Wnt/β-catenin and Bmp signaling in zebrafish.

To further investigate mechanisms underlying the developmental roles of Nup62l in zebrafish, WISH assays were performed to assess the effects of Nup62l on the expression of marker genes for canonical Wnt, Bmp, Nodal, and Fgf signaling pathways. These genes include dkk1, boz, otx2, wnt8a, and tbx6 for Wnt/β-catenin, ved and vent for Bmp, spry4 and sef for Fgf, and flh and xbp1 for Nodal signaling (57). As shown in Fig. 5A, the expression of dkk1, boz, tbx6, otx2, and wnt8a was attenuated in >76% of nup62l morphants and increased in >85% of embryos injected with capped nup62l mRNA. The expression of ved and vent was induced by nup62l-MO and inhibited by nup62l mRNA. Importantly, enhanced expression of Wnt target genes and decreased expression of Bmp targets by nup62l mRNA were neutralized by coinjection with β-catenin-1-MO or β-catenin-2-MO in >90% of embryos, suggesting that Nup62l activates classical Wnt and restrains Bmp in a β-catenin-mediated manner. However, the expression of sef, spry4, xbp1, and flh was unperturbed by knockdown or ectopic expression of nup62l during zebrafish embryogenesis (data not shown).

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Effects of Nup62l on activities of Wnt/β-catenin and Bmp signaling in zebrafish. (A) Expression patterns of markers (indicated at the top) for Wnt and Bmp pathways in WT embryos or embryos injected with 300 pg nup62l mRNA, 10 ng nup62l-MO, or 300 pg nup62l mRNA plus 8 ng β-catenin-1/2-MO at the indicated stages. Images show dorsal views with the animal pole toward the top for boz, animal pole views with the dorsal toward the right for ved, vent, and dkk1, dorsoanterior views with the anterior region toward the top for otx2, vegetal views with the dorsal oriented toward the right for wnt8a, and dorsal views with the anterior region toward the top for tbx6. The arrows indicated the margins of ved and vent expression. (B) ZF4 cells were cotransfected with 250 ng of plasmids, including 84 ng Top-flash reporter, 16 ng pRL-TK as an internal control, 50 ng constitutively active β-catenin-1 (HA–β-cat) plasmid as a positive control, and/or 50 to 100 ng Flag–nup62l-DN. Empty vectors were used to balance the total plasmid amount. (C) ZF4 cells were transfected with a mixture of plasmids and analyzed as indicated for panel B. The HA-lef1-VP16 construct (positive control) was able to stimulate Top-flash. (D) ZF4 cells were cotransfected with 250 ng plasmids, including 84 ng Bre reporter, 16 ng pRL-TK as an internal control, 50 ng HA-bmp4 as a positive control, and/or 50 to 100 ng Flag-nup62l. Empty vectors were used to balance the total plasmid amount. Luciferase activity was measured using cellular lysates after transfection for 24 h. WB was carried out to find the proper expression of HA–β-cat, HA-lef1-VP16, HA-bmp4, and Flag–nup62l-DN/Flag-nup62l. Data in panels B and D represent means and SD for three wells. **, P < 0.01 versus Top-flash- or Bre-transfected cells.

Next, reporter systems for Wnt/β-catenin, Bmp, and Nodal/TGF-β signaling were utilized to verify these observations. Luciferase assays were performed with transfected ZF4 cell lysates. Top-flash-luciferase expression of the Wnt/β-catenin reporter was significantly induced by coexpression of β-catenin-1 (HA–β-cat) (P < 0.01), while the induction was antagonized by coexpression of Flag–nup62l-DN, in a dose-dependent manner (P < 0.01 in all cases) (Fig. 5B). In addition, the activity of Top-flash was markedly activated by overexpression of a dominant positive Lef1-VP16 fusion (>100-fold), but the activation was unperturbed by coexpression of Flag–nup62l-DN (Fig. 5C). These data mirror the observation that activation of canonical Wnt signaling by Nup62l is mediated by β-catenin but not by Lef1.

Bre-luciferase expression of the Bmp reporter was evidently elevated by HA-bmp4, but coexpression of Flag-nup62l attenuated this activity in a dose-dependent manner (P < 0.01 in all cases) (Fig. 5D). Western blot (WB) assays indicated that HA–β-cat, HA-lef1-VP16, HA-bmp4, Flag–nup62l-DN, and Flag-nup62l were expressed normally in corresponding groups of transfected cells (Fig. 5B to toD).D). However, expression of the (CAGA)12-Luc reporter for Nodal/transforming growth factor beta (TGF-β) signaling was not affected by overexpression or knockdown of nup62l (data not shown).

These findings suggest that developmental functions of Nup62l in zebrafish are closely associated with its regulation of Wnt and Bmp signaling.

Nup62l is required for nuclear import of β-catenin in zebrafish.

A recent study demonstrated that β-catenin is able to traverse the NPC by direct binding to Nup62, Nup153, and RanBP2/Nup358 (54). Thus, the physical association of Nup62l and β-catenin-1/2 was first examined by co-IP and WB in ZF4 cells. As shown in Fig. 6A and andB,B, overexpressed Flag-Nup62l and HA–β-catenin-1/2 (HA–β-cat-1 or HA–β-cat-2) were coimmunoprecipitated with each other, reflecting that β-catenin-1/2 proteins are binding partners of Nup62l in zebrafish.

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Nup62l facilitates nuclear import of β-catenins in zebrafish. (A and B) The physical interaction between Flag-nup62l and HA–β-cat-1/2 in embryos was analyzed via co-IP and WB. Embryos at the one-cell stage were injected with 150 pg HA–β-cat-1/2, 150 pg Flag-nup62l, or 150 pg HA–β-cat-1/2 plus 150 pg Flag-nup62l. Total cell lysates (TCL) were used as the control. (C and D) Nup62l facilitates nuclear accumulation of β-catenin-1/2. Immunofluorescence staining was performed with ZF4 cells growing on 35-mm dishes after transfection with 800 ng HA–β-cat-1/2 plus 800 ng empty vector DNA, 800 ng HA–β-cat-1/2 plus 800 ng nup62l DNA, or 800 ng HA–β-cat-1/2 plus 800 ng nup62l-D9 DNA. ZF4 cells infected with a lentivirus expressing Nup62l siRNA (siNup62l) or a control siRNA (siCtrl) were analyzed by immunofluorescence staining after transfection with 800 ng HA–β-cat-1/2 plus 800 ng empty vector DNA. Cellular distributions of HA–β-cat proteins are displayed by the locations of FITC signals. (E and F) Ratios of β-catenin-1/2 proteins localized to the nucleus, the cytoplasm, or both the nucleus and the cytoplasm in differently transfected cells.

To investigate whether Nup62l affects the cellular localization of β-catenins, immunofluorescence staining assays were conducted by cotransfection of a plasmid expressing HA–β-cat-1/2 with another plasmid expressing Flag-Nup62l or Flag−Nup62l-D9 into ZF4 cells. Flag–Nup62l-D9 was a deletion mutant of Nup62l without the capability of interaction with β-catenins. As shown in Fig. 6C and andD,D, HA signals were positioned in both the cytoplasm and nuclei of HA–β-cat-1/2-transfected cells and mainly distributed in the nuclei of cells cotransfected with Nup62l, indicating that Nup62l promotes the nuclear accumulation of HA–β-catenins. The nuclear accumulation of HA–β-catenins was stimulated by Nup62l but not by Nup62l-D9. Moreover, Nup62l small interfering RNA (siRNA) (siNup62l) was used to effectively suppress the expression of endogenous Nup62l in ZF4 cells (see Fig. S4D in the supplemental material). As expected, the nuclear import of β-cat-1/2 was markedly impeded in siNup62l-infected cells but not in siCtrl-infected cells (Fig. 6C and andD).D). The numbers of cells showing HA signals in three visual fields are summarized in Fig. 6E and andF.F. These data suggest that Nup62l can facilitate the nuclear import of active β-catenins in zebrafish.

Identification of Nup62l domain for β-catenin binding.

To further elucidate the molecular mechanisms underlying the Nup62l-mediated translocation of β-catenins, a series of Flag-nup62l deletion mutants (D1 to D14) was generated to identify the binding motifs of β-catenin-1 in Nup62l (Fig. 7A). The results of co-IP and WB assays indicated that nup62l mutants D1 and D9 to D14 failed to associate with HA–β-catenin-1/2 in transfected ZF4 cells; however, other deletion mutants retained the ability to bind to HA–β-catenin-1 (Fig. 7B). Overexpressed mutants D1 and D9 to D14 were not able to activate canonical Wnt signaling in transfected cells (Fig. 7C). Similarly, Nup62l bound to β-catenin-2 via the same functional domain (see Fig. S5 in the supplemental material). Moreover, injection of capped D9 mRNA exhibited few effects on morphology and the expression of markers for dorsoventral patterning, CE movements, and midline organ precursors (see Fig. S4A and B and Table S1). Thus, residues 76 to 100 of Nup62l, which contain two FG sequences, are responsible for the interaction and nuclear translocation of β-catenin-1/2 in zebrafish.

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Biochemical analyses of β-catenin-1 binding motif in Nup62l. (A) Schematic diagrams representing a series of Nup62l deletion constructs. +, interaction; −, no interaction. (B) Interactions between a series of Flag-tagged Nup62l mutants and HA–β-catenin-1 were detected by co-IP and WB. (C) Effects of Nup62l mutants on expression of the Wnt signal-responsive reporter Top-flash, which can be activated by overexpression of β-catenin-1. Mutants D1 and D9 to D14, harboring deletions within residues 76 to 100, failed to induce reporter activation. Data from the luciferase reporter assays are presented as means and SD for samples in three wells. **, P < 0.01 versus Top-flash-transfected cells.

Since Nup62-Nup58-Nup54-Nup45 is a crucial subcomplex of the NPC, we addressed the consequences of zebrafish nup54 knockdown during embryogenesis by injection of nup54-ATG-MO. The results indicated that silencing of nup54 led to ventralized embryos and altered expression of marker genes (see Fig. S4A and B and Table S1 in the supplemental material), similar to the effects exhibited in nup62l morphants. These data indicate that the Nup62l subcomplex, in addition to the interaction of Nup62l and β-catenin, is indispensable for the precise nuclear import of β-catenins that is tightly correlated with the phenotypes shown and with gene expression.

Mapping of β-catenin domain for Nup62l binding.

The β-catenin protein is a transport carrier that can effectively compete with importin-β for shuttling of the NPC due to its transport armadillo (arm) repeats, which resemble the transport-active HEAT repeats of importin-β (64, 65). Thus, six mutants with a deletion in HA–β-catenin-1 were generated (DN1 to DN6) to map the binding region of Nup62l in β-catenin-1 (Fig. 8A). Results from co-IP and WB assays indicated that DN3 and DN5 (lacking arm repeats R3 to R8 and R10 to R12, respectively) had lost the ability to interact with Flag-Nup62l (Fig. 8B). Next, we examined the subcellular localization of two β-catenin-1 mutants and found that the nuclear accumulation of HA–β-cat-1-DN3 and HA–β-cat-1-DN5 was not affected by Nup62l (Fig. 8C). The numbers of cells showing HA signals are summarized in Fig. 8D. Collectively, arm repeats R3 to R8 and R10 to R12 of β-catenin-1 are essential for both Nup62l binding and nuclear import. Moreover, the same motifs of β-catenin-2 are required for interaction and nuclear translocation (see Fig. S6 in the supplemental material).

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Mapping of β-catenin-1 domain required for binding to Nup62l and for nuclear transfer. (A) Cartoon illustration of β-catenin-1 domains and deletion mutants. +, interaction; −, no interaction. (B) Co-IP and WB assays were conducted to assess the reciprocal interaction between Flag-tagged Nup62l and β-catenin-1 mutants, and arm repeats R3 to R8 and R10 to R12 in β-catenin-1 were determined to be the Nup62l binding domains. (C) Immunofluorescence staining assays of ZF4 cells transfected with 800 ng HA–β-cat-1-DN3 plus 800 ng nup62l-expressing plasmid or with 800 ng HA–β-cat-1-DN5 plus 800 ng nup62l-expressing plasmid. (D) Ratios of transfected cells in which two β-catenin-1 mutants were localized to the cytoplasm. (E) Schematic model for the feedback network that is closely associated with the functions of Nup62l during early zebrafish development.

DISCUSSION

In this study, we characterized the developmental roles of zebrafish Nup62l in dorsoventral patterning, CE movements, and formation of midline organs. During gastrulation, a deficiency of Nup62l leads to abnormal embryos with a thickened ventral and a shortened and expanded AP axis, while its ectopic expression results in a thickened dorsal organizer and elongated AP axis. The morphological abnormalities are closely associated with CE movement defects. Ectopic expression of Nup62l generates a certain ratio of dorsalized embryos that resemble those overexpressing Boz (66) or β-cateninΔN (67, 68) or treated with LiCl (69). In contrast, depletion of Nup62l causes ventralized embryos similar to ichabod mutant embryos (40) and those overexpressing caveolin-1 or Lzts2 (57, 58). In addition, knockdown of Nup62l inhibits CE movements during gastrulation and midline assembly of organ precursors and results in embryos with phenotypes like those seen in slb/wnt11 and ppt/wnt5 loss-of-function mutants (70, 71). Regulatory functions of Nup62l in dorsoventral patterning, CE movements, and midline organs specification are further supported by altered expression of corresponding marker genes.

Several lines of evidence from this study strongly suggest that developmental functions of Nup62l are closely associated with Wnt/β-catenin activity in zebrafish. First, Nup62l and β-catenin have a maternal origin and physically interact with each other. Second, the activity of Wnt/β-catenin during early embryogenesis can be activated by Nup62l in a Lef1-independent manner. Third, ectopic expression of Nup62l leads to dorsalized embryos similar to those expressing active β-catenin (68). Fourth, overexpression of Nup62l results in elevated expression of the dorsal marker genes gsc, chordin, and boz and decreased expression of the ventral markers bmp2b, bmp4, eve1, and gata1. Fifth, a Nup62l mutant with deletion of the β-catenin binding domain fails to regulate Top-flash activity and generate morphologically defective embryos. Moreover, altered expression of marker genes for Wnt and Bmp signaling by ectopic expression of Nup62l is able to be rescued by coinjection with β-catenin-1-MO or β-catenin-2-MO.

In addition to extracellular factors, the activity of Wnt/β-catenin signaling is modulated by multiple intracellular molecules, such as E-cadherin (72,74) and caveolin-1 (58) on the plasma membrane, components in the APC-axin-containing protein complex (75,77) in the cytoplasm, and TCF-type transcription factors (78) and BCL9 (58, 79) in the nucleus. The nuclear export of β-catenin seems to be mediated by the CRM1 route (47, 80) and is closely related to that of other partners, including APC and Lzts2. Nevertheless, major factors and precise pathways for nuclear import of β-catenin remain to be explored, although nuclear import is tightly related to the binding of armadillo repeats to the NPC (81). In this study, we demonstrated that Nup62l is able to activate canonical Wnt signaling through facilitating the nuclear import of active β-catenins in zebrafish. Residues 76 to 100, containing two FG sequences of Nup62l, are necessary for tight binding to β-catenins. These findings are consistent with previous observations that FG-repeat structures of Nup62 are associated with nucleocytoplasmic trafficking through its direct binding to receptors or cargo (18, 19). Furthermore, we have identified arm repeats R3 to R8 and R10 to R12 of β-catenins as being essential for Nup62l binding and nuclear import.

The zebrafish genome contains two distinct genes that encode β-catenin-1 and β-catenin-2. A previous study demonstrated that organizer formation is attributed mainly to the activity of β-catenin-2 (40); however, findings from this and our previous studies have shown that both β-catenin-1 and β-catenin-2 are required for early dorsal formation, CE movements, and specification of organ precursors (57, 58). In this study, depletion of solely β-catenin-1 or β-catenin-2 led to similar phenotypes. Since β-catenin-1/2 proteins contain a putative Nup62l-binding motif with the same amino acid sequence, it is likely that Nup62l exerts its physiological functions in early embryonic patterning through the same interacting interface within β-catenin-1/2. However, zebrafish β-catenin-1/2 proteins exhibit a significant difference in their C-terminal 92 amino acids, corresponding to the major region of a transactivation domain known to interact with CBP and p300 (82, 83), and this domain is also involved in the regulation of selective binding to cadherin or Tcf proteins (84). Thus, we cannot exclude the possibility that β-catenin-1/2 proteins have distinct activities, since the difference in transactivation domains may require the involvement of different cofactors and control the differential expression of downstream genes. Thus, it is reasonable to conclude that zebrafish β-catenin-1/2 proteins exert overlapping but not redundant functions in early developmental processes.

Wnt/β-catenin signaling is known to play a key role in the regulation of embryogenesis and oncogenesis (85,87). In zebrafish, activation of the Wnt/β-catenin pathway facilitates cardiac differentiation before gastrulation but perturbs it at later stages (45). Alteration in Wnt/β-catenin signaling is able to disorganize the fate of the liver and pancreas during early endoderm patterning but facilitates hepatic growth in the bud stage of mouse liver (81, 88). Moreover, β-catenin is involved in liver development, regeneration, and cancer pathogenesis (89,91). In addition to functions in modulating various biological processes, such as cell proliferation, differentiation, and apoptosis (92), BMP signaling plays pleiotropic roles in developmental processes of several organisms, including embryonic dorsoventral patterning (93), induction of neural tissue (94), formation of joints in the skeletal system (95), and neurogenesis in the adult brain (96). The BMP signaling pathway is also closely relevant to the pathophysiology of several diseases, including osteoporosis (97), arthritis (95), kidney diseases (98, 99), pulmonary hypertension (100, 101), cerebrovascular diseases (102), and diverse cancers (103,106). Moreover, both Wnt and BMP signaling pathways are implicated in intestinal development and tumor genesis (107,109).

Furthermore, inappropriate expression of Nup62 is linked to developmental defects and several human diseases, such as primary biliary cirrhosis (110) and autosomal recessive infantile bilateral striatal necrosis (30). Findings from this study have revealed a regulatory network among Nup62l, Wnt/β-catenin, and Bmp signaling in zebrafish (Fig. 8E). Therefore, further investigations of the regulatory feedback network are significant not only to address its essential roles in asymmetric activation of Wnt/β-catenin and Bmp signaling along the dorsoventral axis and specification of tissues and organs during embryonic development but also to provide a well-defined target for therapeutic intervention in relevant human diseases. Additionally, a precise dissection of the positive-feedback loop between nup62l and β-catenin may resolve the discrepant functions of Wnt/β-catenin signaling in organogenesis and oncogenesis and lead to new drug targets for therapeutics of various cancers.

The Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
University of Chinese Academy of Sciences, Beijing, China
School of Basic Medical Sciences, Wuhan University, Wuhan, China
Corresponding author.
Address correspondence to Qing Li, nc.ca.bhi@ilq, or Zongbin Cui, nc.ca.bhi@iucbz.
Citation Yang X, Gu Q, Lin L, Li S, Zhong S, Li Q, Cui Z. 2015. Nucleoporin 62-like protein activates canonical Wnt signaling through facilitating the nuclear import of β-catenin in zebrafish. Mol Cell Biol 35:1110–1124. doi:10.1128/MCB.01181-14.
Citation Yang X, Gu Q, Lin L, Li S, Zhong S, Li Q, Cui Z. 2015. Nucleoporin 62-like protein activates canonical Wnt signaling through facilitating the nuclear import of β-catenin in zebrafish. Mol Cell Biol 35:1110–1124. doi:10.1128/MCB.01181-14.
Received 2014 Oct 31; Revisions requested 2014 Nov 23; Accepted 2014 Dec 31.

Abstract

Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/β-catenin pathway positively modulates nup62l transcription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulate nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitation of nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish.

Abstract
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ACKNOWLEDGMENTS

We thank Wuhan Xiao of the Institute of Hydrobiology, Chinese Academy of Sciences, for his kindly provision of β-catenin-1 and β-catenin-2 antisense MOs.

This work was supported by grants from the National Natural Science Foundation of China (grant 31171390 to Z.C.), the National Basic Research Program of China (grant 2012CB944500), and the Chinese Academy of Sciences (grant KJZD-EW-L08).

ACKNOWLEDGMENTS

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01181-14.

Footnotes

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