Bone morphogenetic proteins (BMPs) are implicated in cell-fate determination of embryonic stem (ES) cells and cancer cells. GREM1 (CKTSF1B1 or DAND2) and CER1 (Cerberus 1 or DAND4) are cysteine knot superfamily proteins, functioning as secreted-type BMP antagonists. BMP4 is preferentially expressed in diffuse-type gastric cancer cells. Here, vertebrate BMP4 orthologs were identified and characterized by using bioinformatics for comparative proteomics and comparative genomics analyses. Baboon BMP4 gene within AC153751.2 genome sequence encoded a 408-aa protein, showing A152V and S298P amino-acid substitutions compared with human BMP4. Cow Bmp4, bat Bmp4 and zebrafish bmp4 genes were located within AC149774.2, AC156788.2 and CR391996.2 genome sequences, respectively. Human BMP4 showed 99.5%, 98.0%, 97.8%, 97.1%, 96.3%, 83.3% and 71.1% total-amino-acid identity with baboon BMP4, cow Bmp4, bat Bmp4, mouse Bmp4, rat Bmp4, chicken bmp4 and zebrafish bmp4, respectively. Human BMP4 gene was found consisting of six exons, including novel exon 1C, and known exons 1 (1A or I), 1B (II), 2 (III), 3 (IV) and 4 (V). Forty human BMP4 ESTs started from exon 1, seven from intron 1 (5'-flanking region of exon 2), and two from exon 1C. Fourteen mouse Bmp4 ESTs started from exon 1, and one from intron 1. The 5'-flanking region of exon 1 and exon 1 itself, but not exons 1C and 1B, were well conserved between human BMP4 and rodent Bmp4 genes. The major promoter region of human BMP4 and rodent Bmp4 genes were located within the 5'-flanking region of exon 1. FOXA2, OLF1, and MYC-binding sites were conserved among the major promoter region of human, baboon, cow, bat, mouse and rat BMP4 orthologs.

We have previously identified an approximately 7 cM long common eliminated region (CER), involving the 3p21.3 markers AP20R, D3S966, D3S3559, D3S1029, WI-7947, D3S2354, AFMb362wb9, and D3S32, in human chromosome 3/A9 mouse fibrosarcoma microcell hybrid (MCH) derived SCID mouse tumors. We now report the results of our more detailed analysis on 24 SCID mouse tumors derived from two MCH lines that originally carried intact human chromosomes 3. They were analyzed by fluorescence in situ hybridization (FISH) painting and PCR, using 24 markers covering the region between D3S1611 and D3S13235 at 3p22-p21.2. D3S32 and D3S2354 were regularly eliminated during in vivo tumor growth, whereas the other 22 markers, D3S1611, ACAA, D3S1260, WI-692, AP20R, D3S3521, D3S966, D3S1029, D3S643, WI-2420, MSTI. GNAI2, D3S1235, D3S1298, GLBI, WI-4193, D3S3658, D3S3559, D3S3678, WI-6400, WI-7947, and WI-10865, were regularly retained. We have defined a common eliminated region of approximately 1.6 cM (designated as CER1) inside the 7 cM CER described earlier. CER1 is flanked distally by D3S1029 and proximally by D3S643.

We have previously shown that inoculation of human chromosome 3 (chr3)/A9 mouse fibrosarcoma microcell hybrids (MCHs) into severely combined immunodeficient (SCID) mice was followed by the regular elimination of certain 3p regions, whereas a 3q region was retained even after prolonged mouse passage. Using this approach, referred to as the elimination test (Et), we identified a common eliminated region (CER) of about 7 cM at 3p22-p21.3 that was absent in all tumors generated from five MCHs. A second frequently eliminated region (FER, originally called ER2) was found at 3p21.1-p14.2. These segments have been reported to be frequently deleted in a variety of carcinomas. In the following experiments, we have identified at the centromeric border of CER a common eliminated region 1 (CER1) of about 1.6 cM. We now report the results of more detailed analyses of the original tumor panel that contained 30 SCID mouse tumors. Using polymerase chain reaction and chromosome reverse painting, we have identified at the telomeric border of CER a second common eliminated region (designated as CER2). CER2 is flanked distally by RH94338 and proximally by SHGC-154057. The size of CER2 is about 1 Mb, according to the Homo Sapiens Complete Genome databases at EMBL, and is located about 0.5 Mb centromeric to the known homozygous deletion region, identified in lung cancer. Remarkably, two chemokine-receptor genes (CCRs), CCR8 and CX3CR1, are located within CER2, whereas seven CCRs were found within CER1.

The initial landmark of anterior-posterior (A-P) axis formation in mouse embryos is the distal visceral endoderm, DVE, which expresses a series of anterior genes at embryonic day 5.5 (E5.5). Subsequently, DVE cells move to the future anterior region, generating anterior visceral endoderm (AVE). Questions remain regarding how the DVE is formed and how the direction of the movement is determined. This study compares the detailed expression patterns of OTX2, HHEX, CER1, LEFTY1 and DKK1 by immunohistology and live imaging at E4.5-E6.5. At E6.5, the AVE is subdivided into four domains: most anterior (OTX2, HHEX, CER1-low/DKK1-high), anterior (OTX2, HHEX, CER1-high/DKK1-low), main (OTX2, HHEX, CER1, LEFTY1-high) and antero-lateral and posterior (OTX2, HHEX-low). The study demonstrates how this pattern is established. AVE protein expression in the DVE occurs de novo at E5.25-E5.5. Neither HHEX, LEFTY1 nor CER1 expression is asymmetric. In contrast, OTX2 expression is tilted on the future posterior side with the DKK1 expression at its proximal domain; the DVE cells move in the opposite direction of the tilt.

Plant surface waxes form an outer barrier that protects the plant from many forms of environmental stress. The deposition of cuticular waxes on the plant surface is regulated by external environmental changes, including light and dark cycles. However, the underlying molecular mechanisms controlling light regulation of wax production is are still poorly understood, especially at the post-transcriptional level. In this paper, we report the regulation of cuticular wax production by the miR156-SPL9 module in Arabidopsis. Compared with wild-type plants, miR156 and SPL9 mutants showed significantly altered cuticular wax amounts in both stems and leaves. Furthermore, it was found that SPL9 positively regulates gene expression of the alkane-forming enzyme CER1, as well as the primary (1-) alcohol-forming enzyme CER4, to enhance alkane and 1-alcohol synthesis respectively. More importantly, our results indicate that complex formation of SPL9 with a negative regulator of wax synthesis, DEWAX, will hamper SPL9 DNA binding ability, possibly by interfering with SPL9 homodimerization. Combined with their diurnal gene and protein expressions, this dynamic repression-activation transcriptional module defines a dynamic mechanism that may allow plants to optimize wax synthesis during daily cycles. These findings provide a regulatory framework for environmental signal integration in the regulation of wax synthesis.

Drought events are a major challenge for many horticultural crops, including grapes, which are often cultivated in dry and warm climates. It is not understood how the cuticle contributes to the grape berry response to water deficit (WD), furthermore the cuticular waxes and the related biosynthetic pathways are poorly characterized in this fruit. In this study, we identified candidate wax-related genes from the grapevine genome by phylogenetic and transcriptomic analyses. Developmental and stress-response expression patterns of these candidates were characterized across pre-existing RNA-seq datasets and confirmed a high responsiveness of the pathway to environmental stresses. We then characterized the developmental and WD induced changes in berry cuticular wax composition, and quantified differences in berry transpiration. Cuticular aliphatic wax content was modulated across development and an increase was observed under WD, with wax esters being strongly upregulated. These compositional changes were related to upregulated candidate genes of the aliphatic wax biosynthetic pathway, including CER1CER1, CER4, and WSD1. The effect of WD on berry transpiration was not significant. This study indicates that changes in cuticular wax amount and composition are part of the metabolic response of the grape berry to WD, but these changes do not reduce berry transpiration.

WNT family proteins bind to transmembrane proteins FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, ROR1, ROR2, RYK, and also secreted proteins SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, and DAND4 (CER1). DKK family members antagonize WNT binding to LRPs, while SFRP family members antagonize WNT binding to FZDs. Here, we identified and characterized rat Dkk1 gene and cow Dkk1 gene by using bioinformatics. Rat and cow Dkk1 genes, consisting of four exons, were located within AC095296.4 and AC157142.1 genome sequences, respectively. Dkk1 gene at rat chromosome 1q52 was found to encode a 270-aa protein, showing 94.1, 81.5, 78.9, 53.7 and 48.1% total-amino-acid identity with mouse Dkk1, human DKK1, cow Dkk1, Xenopus dkk1 and zebrafish dkk1, respectively. Vertebrate Dkk1 orthologs were secreted proteins with two Cys rich regions, each containing ten conserved Cys residues. The C-terminal Cys rich region was well conserved among vertebrate Dkk1 orthologs. Nucleotide position 148898-147860 of AC009986.10 human genome sequence was identified as evolutionarily conserved human DKK1 promoter, and nucleotide position 55266-56301 of AC095296.4 rat genome sequence as evolutionarily conserved rat Dkk1 promoter. Human DKK1 promoter and rat Dkk1 promoter, showing 66.2% total nucleotide identity, were well conserved. TCF/LEF, CP2, POU2F1 (OCT1), HNF1 and FOXJ2-binding sites and TATA-box were conserved among human DKK1, rat Dkk1, mouse Dkk1, and cow Dkk1 promoters. Double TCF/LEF-binding sites within the proximal promoter region of mammalian Dkk1 orthologs are implicated in the negative feed back mechanism of WNT/beta-catenin signaling pathway.

BACKGROUND

A back curvature defect similar to kyphosis in humans has been observed in swine herds. The defect ranges from mild to severe curvature of the thoracic vertebrate in split carcasses and has an estimated heritability of 0.3. The objective of this study was to identify genomic regions that affect this trait.

RESULTS

Single nucleotide polymorphism (SNP) associations performed with 198 SNPs and microsatellite markers in a Duroc-Landrace-Yorkshire resource population (U.S. Meat Animal Research Center, USMARC resource population) of swine provided regions of association with this trait on 15 chromosomes. Positional candidate genes, especially those involved in human skeletal development pathways, were selected for SNP identification. SNPs in 16 candidate genes were genotyped in an F2 population (n = 371) and the USMARC resource herd (n = 1,257) with kyphosis scores. SNPs in KCNN2 on SSC2, RYR1 and PLOD1 on SSC6 and MYST4 on SSC14 were significantly associated with kyphosis in the resource population of swine (P ≤ 0.05). SNPs in CER1 and CDH7 on SSC1, PSMA5 on SSC4, HOXC6 and HOXC8 on SSC5, ADAMTS18 on SSC6 and SOX9 on SSC12 were significantly associated with the kyphosis trait in the F2 population of swine (P ≤ 0.05).

CONCLUSIONS

These data suggest that this kyphosis trait may be affected by several loci and that these may differ by population. Carcass value could be improved by effectively removing this undesirable trait from pig populations.

The mouse Tyrp1 deletion complex is a valuable resource for high-resolution mapping of genes and phenotypes to the central region of Chromosome (Chr) 4. The distal part of the complex is homologous to human Chr 9p21-23, and we have used the available radiation hybrid maps to identify human transcripts in the region. We localize seven genes to a human YAC contig that spans the full extent of the distal deletion complex and show that the mouse homologs of four of these, including Cer1, map within the complex. On the basis of location and/or expression, we exclude genes as candidates for several known phenotypes in the region and identify a candidate transcript for the neonatal lethal phenotype l(4)Rn2.

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21.1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.

The great majority of terrestrial plants produce epicuticular wax that is used to protect plants from a variety of biotic and abiotic stresses. Cabbage epicuticular wax is a white crystalline compound of various lipids. Wax-less cabbage has the characteristics of lustrous green leaves and beautiful exterior, which facilitates the brilliant green cabbage breeding. CGL-3 is a spontaneous wax-less mutant identified from cabbage. Genetic analysis indicated that the waxy deficiency of the mutant was controlled by a single dominant gene. To clarify the mechanism of the waxy deficiency, fine-mapping and transcriptome analysis of the wax-less gene, BoGL-3, were carried out in this study. The result of fine mapping showed that the wax-less gene, BoGL-3, was delimited in a 33.5-kb interval which is between the flanking marker C08-98 and the end of chromosome 8. Two cDNA libraries, constructed with wax-less cabbage CGL-3 and the wild-type cabbage WT, were sequenced for screening of the target gene BoGL-3. A total of 8340 genes were identified with significant differential expression between CGL-3 and WT. Among these genes, 3187 were up-regulated and 5153 were down-regulated in CGL-3. With homologous analysis, four differential expressed genes related to wax metabolism were obtained. Among these four genes, only Bol018504 is located within the region of fine-mapping. Bol08504 is homologous to CER1, which encodes fatty acid hydroxylase and plays an important role in wax synthesis in Arabidopsis. However, there was no difference of Bol08504 sequence between CGL-3 and WT. We suggested that Bol018504 was regulated by BoGL-3. The suppression of Bol018504 leads to the reduction of wax. These findings will be helpful to reveal the mechanism of the wax metabolism in cabbage and develop lustrous green cabbage germplasm material.

The lipid organization in the upper layer of the skin, the stratum corneum (SC), is important for the skin barrier function. This lipid organization, including the characteristic 13 nm lamellar phase, can be reproduced in vitro with mixtures based on cholesterol, free fatty acids and natural as well as synthetic ceramides (CER). In human SC, nine CER classes have been identified (CER1-CER9). Detailed studies on the effect of molecular structure of individual ceramides on the SC lipid organization are only possible with synthetic lipid mixtures, as their composition can be accurately chosen and systematically modified. In the present study, small-angle X-ray diffraction was used to examine the organization in synthetic lipid mixtures of which the synthetic ceramide fraction was prepared with sphingosine-based CER1 or phytosphingosine-based CER9. The latter acylceramide contains an additional hydroxyl group at the sphingoid backbone. The results show that a gradual increase in CER1 level consistently promotes the formation of the 13 nm lamellar phase and that partial replacement of CER1 by CER9 does not affect the phase behavior. Interestingly, complete substitution of CER1 with CER9 reduces the formation of the long periodicity phase and results in phase separation of CER9.

Plant aerial organs are coated with cuticular waxes, a hydrophobic layer that primarily serves as a waterproofing barrier. Cuticular wax is a mixture of aliphatic very-long-chain molecules, ranging from 22 to 48 carbons, produced in the endoplasmic reticulum of epidermal cells. Among all wax components, alkanes represent up to 80% of total wax in Arabidopsis (Arabidopsis thaliana) leaves. Odd-numbered alkanes and their derivatives are produced through the alkane-forming pathway. Although the chemical reactions of this pathway have been well described, the enzymatic mechanisms catalyzing these reactions remain unclear. We previously showed that a complex made of Arabidopsis ECERIFERUM1 (CER1) and CER3 catalyzes the conversion of acyl-Coenzyme A's to alkanes with strict substrate specificity for compounds containing more than 29 carbons. To learn more about alkane biosynthesis in Arabidopsis, we characterized the biochemical specificity and physiological functions of a CER1 homolog, CER1-LIKE1. In a yeast strain engineered to produce very-long-chain fatty acids, CER1-LIKE1 interacted with CER3 and cytochrome B5 to form a functional complex leading to the production of alkanes that are of different chain lengths compared to that produced by CER1-containing complexes. Gene expression analysis showed that both CER1 and CER1-LIKE1 are differentially expressed in an organ- and tissue-specific manner. Moreover, the inactivation or overexpression of CER1-LIKE1 in Arabidopsis transgenic lines specifically impacted alkane biosynthesis and wax crystallization. Collectively, our study reports on the identification of a further plant alkane synthesis enzymatic component and supports a model in which several alkane-forming complexes with distinct chain-length specificities coexist in plants.

To protect above-ground plant organs from excessive water loss, their surfaces are coated by waxes. The genes involved in wax formation have been investigated in detail in Arabidopsis but scarcely in crop species. Here, we aimed to isolate and characterize a CER1 enzyme responsible for formation of the very long-chain alkanes present in high concentrations especially during late stages of wheat development. On the basis of comparative wax and transcriptome analyses of various wheat organs, we selected TaCER1-1A as a primary candidate and demonstrated that it was located to the endoplasmic reticulum, the subcellular compartment for wax biosynthesis. A wheat nullisomic-tetrasomic substitution line lacking TaCER1-1A had significantly reduced amounts of C₃₃ alkane, whereas rice plants overexpressing TaCER1-1A showed substantial increases of C₂₅ -C₃₃ alkanes relative to wild type control. Similarly, heterologous expression of TaCER1-1A in Arabidopsis wild type and the cer1 mutant resulted in increased levels of unbranched alkanes, iso-branched alkanes and alkenes. Finally, the expression of TaCER1-1A was found activated by abiotic stresses and abscisic acid treatment, resulting in increased production of alkanes in wheat. Taken together, our results demonstrate that TaCER1-1A plays an important role in wheat wax alkane biosynthesis and involved in responding to drought and other environmental stresses.

Cuticle waxes, which are primarily comprised of very-long-chain (VLC) alkanes, play an important role in plant reproductive development. ECERIFERUM1 (CER1) is recognized as the core element for VLC alkane biosynthesis in Arabidopsis (Arabidopsis thaliana). However, genes involved in the VLC alkane biosynthesis in rice remain unclear, and the alkane-form pathway in rice has still to be further explored. Here, we show that OsCER1, a homology of CER1, functions in VLC alkanes biosynthesis, which also could regulate anther development and plastids differentiation in rice. OsCER1 was highly expressed in the tapetum (stage 10) and bicellular pollen cells (stage 11). The decreased content of VLC alkanes (C25 and C27) in the OsCER1 knocked down plants as well as the increased content of C27 alkanes in the OsCER1 overexpression plants indicates that OsCER1 participates in VLC alkane biosynthesis. Downregulation of OsCER1 in rice led to sterility, and fewer amyloplasts within the mature pollen grains. In addition, the downregulation of OsCER1 in rice caused delayed tapetal programmed cell death and abnormal development of plastids in the tapetal cells. Furthermore, significantly altered levels of expression of genes involved in the pollen development were exhibited in the OsCER1 knocked down plants. These results indicate that OsCER1 is critical for VLC alkanes biosynthesis, plastids differentiation, and pollen development. This work provides insights into the VLC alkanes biosynthesis in anther development in rice.

Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4(Co/Co);TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4(Co/Co);TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction.

DAND1 (NBL1), DAND2 (CKTSF1B1 or GREM1 or GREMLIN), DAND3 (CKTSF1B2 or GREM2 or PRDC), DAND4 (CER1), DAND5 (CKTSF1B3 or GREM3 or DANTE), MUC2, MUC5AC, MUC5B, MUC6, MUC19, WISP1, WISP2, WISP3, VWF, NOV and Norrie disease (NDP or NORRIN) genes encode proteins with cysteine knot domain. Cysteine-knot superfamily proteins regulate ligand-receptor interactions for a variety of signaling pathways implicated in embryogenesis, homeostasis, and carcinogenesis. Although Ndp is unrelated to Wnt family members, Ndp is claimed to function as a ligand for Fzd4. Here, we identified and characterized rat Ndp, cow Ndp, chicken ndp and zebrafish ndp genes by using bioinformatics. Rat Ndp gene, consisting of three exons, was located within AC105563.4 genome sequence. Cow Ndp and chicken ndp complete CDS were derived from CB467544.1 EST and BX932859.2 cDNA, respectively. Zebrafish ndp gene was located within BX572627.5 genome sequence. Rat Ndp (131 aa) was a secreted protein with C-terminal cysteine knot-like (CTCK) domain. Rat Ndp showed 100, 96.9, 95.4, 87.8 and 66.4 total-amino-acid identity with mouse Ndp, cow Ndp, human NDP, chicken ndp and zebrafish ndp, respectively. Exon-intron structure of mammalian Ndp orthologs was well conserved. FOXA2, CUTL1 (CCAAT displacement protein), LMO2, CEBPA (C/EBPalpha)-binding sites and triple POU2F1 (OCT1)-binding sites were conserved among promoters of mammalian Ndp orthologs.

The aerial parts of plants are covered with a cuticular wax layer, which is the first barrier between a plant and its environment. Although cuticular wax deposition increases more in the light than in the dark, little is known about the molecular mechanisms underlying the regulation of cuticular wax biosynthesis. Recently DEWAX (Decrease Wax Biosynthesis) encoding an AP2/ERF transcription factor was found to be preferentially expressed in the epidermis and induced by darkness. Wax analysis of the dewax knockout mutant, wild type, and DEWAX overexpression lines (OX) indicates that DEWAX is a negative regulator of cuticular wax biosynthesis. DEWAX represses the expression of wax biosynthetic genes CER1, LACS2, ACLA2, and ECR via direct interaction with their promoters. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX; throughout the night and another for stomata closing. Taken together, it is evident that DEWAX-mediated negative regulation of the wax biosynthetic genes plays role in determining the total wax loads produced in Arabidopsis during daily dark and light cycles. In addition, significantly higher levels of DEWAX transcripts in leaves than stems suggest that DEWAX-mediated transcriptional repression might be involved in the organ-specific regulation of total wax amounts on plant surfaces.

Aside from its role in cell membrane integrity, cholesterol is a key component in steroid hormone production. The vital functions of steroid hormones such as estrogen, testosterone, glucocorticoids (Gcrts) and mineralocorticoids (Mnrts) in perinatal and adult life are well understood; however, their role during early embryonic development remains largely unexplored. Here we show that siRNA-mediated perturbation of steroid hormone production during mesoderm formation has important consequences on cardiac differentiation in mouse embryonic stem cells (mESC). Both Gcrts and Mnrts are capable of driving cardiac differentiation in mESC. Interestingly, the Gcrt receptor is widely expressed during gastrulation in the mouse, and is exclusively localized in the nuclei-and thus active-in visceral endoderm cells, suggesting that it functions much earlier than previously anticipated. We therefore studied Gcrt signaling in mESC as a model of the gastrulating embryo, and found that Gcrt signaling regulates expression of the transcription factor Hnf4a and the secreted Nodal and BMP inhibitor Cer1 in the early visceral endoderm. RNAi-mediated knockdown of Gcrt function blocked cardiomyocyte differentiation, with limited effects on other cardiovascular cell types including vascular endothelial cells and smooth muscle. Furthermore, the cardiogenic effect of Gcrts required Hnf4a and paracrine Cer1. These results establish a novel function for cholesterol-derived steroid hormones and identify Gcrt signaling in visceral endoderm cells as a regulator of Cer1 and cardiac fate.

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

Cuticular waxes coat the primary aerial tissues of land plants and serve as a protective barrier against non-stomatal water loss and various environmental stresses. Alkanes are the most prominent cuticular wax components and are thought to have an important role in controlling permeability of the cuticle. However, alkane biosynthesis in plants is not well understood. Arabidopsis eceriferum1 (cer1) and cer22 mutants show dramatic reductions in alkane, secondary alcohol, and ketone content, and concomitant increases in aldehyde content, suggesting that one or both of these genes encode an alkane-forming enzyme. To determine the biochemical identity of CER22, and to investigate the relationship between CER1 and CER22 in alkane formation, we mapped the cer22 mutation as a first step to positional cloning. Unexpectedly, mapping revealed linkage of cer22 to markers on chromosome 1 in the vicinity of CER1, and not to markers on chromosome 3 as previously reported. Failure of the cer1-1 and cer22 mutants to complement each other, and the presence of an allele specific mutation in the CER1 gene amplified from cer22 genomic DNA demonstrated that CER22 is identical to CER1. The cer22 mutant was therefore renamed cer1-6. Analyses of CER1 transcript levels, and stem cuticular wax load and composition in the cer1-6 (cer22) line indicated that cer1-6 is a weak mutant allele of CER1. This represents an important step forward in our understanding of alkane synthesis in plants, and will direct future research in the field to focus on the role of CER1 in this process.

Cuticular wax accumulation and composition affects drought resistance in plants. Brachypodium distachyon plants subjected to water deficit and polyethylene glycol treatments resulted in a significant increase in total wax load, in which very-long-chain (VLC) alkanes were more sensitive to these treatments than other wax compounds, implying that VLC alkanes biosynthesis plays a more important role in drought resistance in B. distachyon. ECERIFERUM1 (CER1) has been reported to encode a core enzyme involved in VLC alkanes biosynthesis in Arabidopsis (Arabidopsis thaliana), but few corresponding genes are investigated in B. distachyon. Here, we identified eight CER1 homologous genes in B. distachyon, namely BdCER1-1 to BdCER1-8, and then analyzed their sequences feature, expression patterns, stress induction, and biochemical activities. These genes had similar protein structure to other reported CER1 and CER1-like genes, but displayed closer phylogenetic relationship to the rice OsGL1 genes. They were further found to exhibit various tissue expression patterns after being induced by abiotic stresses. Among them, BdCER1-8 gene showed extremely high expression in leaves. Heterologous introduction of BdCER1-8 into the Arabidopsis cer1 mutant rescued VLC alkanes biosynthesis. These results indicate that BdCER1 genes are likely to be involved in VLC alkanes biosynthesis of B. distachyon. Taken together, BdCER1-8 seems to play an explicit and predominant role in VLC alkanes biosynthesis in leaf. Our work provides important clues for further characterizing function of CER1 homologous genes in B. distachyon and also an option to improve drought resistance of cereal crops.

The cuticle is a waxy composite that protects the aerial organs of land plans from non-stomatal water loss. The chemical make-up of the cuticular wax mixture plays a central role in defining the water barrier, but structure-function relationships have not been established so far, in part due to gaps in our understanding of wax structures and biosynthesis. While wax compounds with saturated, linear hydrocarbon tails have been investigated in detail, very little is known about compounds with modified aliphatic tails, which comprise substantial portions of some plant wax mixtures. This study aimed to investigate the structures, abundances and biosynthesis of branched compounds on the species for which wax biosynthesis is best understood: Arabidopsis thaliana. Microscale derivatization, mass spectral interpretation and organic synthesis identified homologous series of iso-alkanes and iso-alcohols on flowers and leaves, respectively. These comprised approximately 10-15% of wild type wax mixtures. The abundances of both branched wax constituents and accompanying unbranched compounds were reduced on the cer6, cer3 and <em>cer1</em> mutants but not cer4, indicating that branched compounds are in part synthesized by the same machinery as unbranched compounds. In contrast, the abundances of unbranched, but not branched, wax constituents were reduced on the cer2 and cer26 mutants, suggesting that the pathways to both types of compounds deviate in later steps of chain elongation. Finally, the abundances of branched, but not unbranched, wax compounds were reduced on the <em>cer1</em>6 mutant, and the (uncharacterized) CER16 protein may therefore be controlling the relative abundances of iso-alkanes and iso-alcohols on Arabidopsis surfaces.