BACKGROUND

A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type.

METHODS

The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets.

RESULTS

Highly significant <em>differences</em> in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast <em>growth</em> <em>factor</em> receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-<em>1</em> (CTAG<em>1</em>A/B), undifferentiated <em>embryonic</em> cell transcription <em>factor</em> <em>1</em> (UTF<em>1</em>) and synaptosomal-associated protein, 9<em>1</em> kDa homolog (SNAP9<em>1</em>) were shown to represent specific biomarkers of human spermatogonia.

CONCLUSIONS

These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.

As the brain develops, a homogeneous population of mitotically active progenitors generates the molecularly heterogeneous post-mitotic cells of the mature brain. The balance between cell division, <em>growth</em> arrest and <em>differentiation</em> of these progenitors undoubtedly requires the activation of a vast array of genes. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. Within the nervous system, PACAP has been shown to stimulate neurite out<em>growth</em>, regulate neurotransmitter production and neuronal survival. These diverse biological actions are mediated through interaction with two types of receptors, a PACAP-selective receptor (PAC(<em>1</em>)-R) and receptors which interact almost equally with both VIP and PACAP. Since several lines of evidence suggest that PACAP acts as a neurotrophic <em>factor</em>, we sought to characterize PACAP and PAC(<em>1</em>)-R expression in the developing rat nervous system. The PAC(<em>1</em>)-R is expressed at very high levels in ventricular zones throughout the neuraxis. In addition to the <em>embryonic</em> enrichment in proliferative zones, PAC(<em>1</em>)-R expression is maintained in areas of neurogenesis in the adult central nervous system (CNS), namely, the subventricular zone of the ol<em>factor</em>y bulb and hippocampal dentate gyrus. In contrast, PACAP is expressed primarily in the post-mitotic parenchyma. This temporal regulation and cellular distribution suggests that PACAP, through its interaction with the PAC(<em>1</em>)-R, may play a role in mammalian neurogenesis.

Fibroblast <em>growth</em> <em>factor</em> (FGF)/FGF receptor (FGFR) signaling plays a crucial role in mesoderm formation and patterning. Heartless mutant studies in Drosophila suggest that FGFR<em>1</em>, among the different FGFRs, may play a role in cardiogenesis. However, fgfr<em>1</em>-/- mice die during gastrulation before heart formation. To establish the contribution of FGFR<em>1</em> in cardiac development, we investigated the capacity of murine fgfr<em>1</em>+/- and fgfr<em>1</em>-/- <em>embryonic</em> stem (ES) cells to differentiate to cardiomyocytes in vitro. Clusters of pulsating cardiomyocytes were observed in >90% of 3-dimensional embryoid bodies (EBs) originated from fgfr<em>1</em>+/- ES cells at day 9 to <em>1</em>0 of <em>differentiation</em>. In contrast, <em>1</em>0% or less of fgfr<em>1</em>-/- EBs showed beating foci at day <em>1</em>6. Accordingly, fgfr<em>1</em>-/- EBs were characterized by impaired expression of early cardiac transcription <em>factors</em> Nkx2.5 and d-Hand and of late structural cardiac genes myosin heavy chain (MHC)-alpha, MHC-beta, and ventricular myosin light chain. Homozygous fgfr<em>1</em> mutation resulted also in alterations of the expression of mesoderm-related early genes, including nodal, BMP2, BMP4, T(bra), and sonic hedgehog. Nevertheless, fgfr<em>1</em>+/- and fgfr<em>1</em>-/- EBs similarly express cardiogenic precursor, endothelial, hematopoietic, and skeletal muscle markers, indicating that fgfr<em>1</em>-null mutation exerts a selective effect on cardiomyocyte development in differentiating ES cells. Accordingly, inhibitors of FGFR signaling, including the FGFR<em>1</em> tyrosine kinase inhibitor SU 5402, the MEK<em>1</em>/2 inhibitor U0<em>1</em>26, and the protein kinase C inhibitor GF<em>1</em>09 all prevented cardiomyocyte <em>differentiation</em> in fgfr<em>1</em>+/- EBs without affecting the expression of the hematopoietic/endothelial marker flk-<em>1</em>. In conclusion, the data point to a nonredundant role for FGFR<em>1</em>-mediated signaling in cardiomyocyte development.

Fibroblast <em>growth</em> <em>factor</em> receptor <em>1</em> (FGFR<em>1</em>) has critical roles in cellular proliferation and <em>differentiation</em> during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4-<em>1</em>), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR<em>1</em>. Nedd4-<em>1</em> binds directly to and ubiquitylates activated FGFR<em>1</em>, by interacting primarily via its WW3 domain with a novel non-canonical sequence (non-PY motif) on FGFR<em>1</em>. Deletion of this recognition motif (FGFR<em>1</em>-Δ6) abolishes Nedd4-<em>1</em> binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4-<em>1</em> knockdown. Accordingly, FGFR<em>1</em>-Δ6, or Nedd4-<em>1</em> knockdown, exhibits sustained FGF-dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk<em>1</em>/2, and PLCγ). Expression of FGFR<em>1</em>-Δ6 in human <em>embryonic</em> neural stem cells strongly promotes FGF2-dependent neuronal <em>differentiation</em>. Furthermore, expression of this FGFR<em>1</em>-Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR<em>1</em> signalling. These results identify Nedd4-<em>1</em> as a key regulator of FGFR<em>1</em> endocytosis and signalling during neuronal <em>differentiation</em> and <em>embryonic</em> development.

As for any other cell population, the development, cell fate, and properties of mesencephalic dopaminergic (mesDA) neurons are ultimately controlled at the transcriptional level. The genes for two transcription <em>factors</em> Engrailed-<em>1</em> ( En<em>1</em>) and Engrailed-2 ( En2) play an essential role in the development and maintenance of these cells. They belong to a family of genes that have been investigated in Drosophila for more than half a century. The products of these genes are all characterized by homeotic tissue transformation and a highly conserved protein sequence, the homeobox. En<em>1</em> and En2 act upon at least two steps of the <em>differentiation</em> of mesDA neurons. They take part in the regionalization event, which gives rise to the neuroepithelium that provides the precursor cells in the ventral midbrain with the fibroblast <em>growth</em> <em>factor</em> 8 signal necessary for their induction. Additionally, these genes are required in postmitotic mesDA neurons in which they are expressed from <em>embryonic</em> day <em>1</em>2 continuously into adulthood. In mutant mice homozygous null for En<em>1</em> and En2, the neurons are generated in the ventral midbrain, become postmitotic, and begin to express their neurotransmitter phenotype. However, thereafter, they rapidly die by apoptosis. Cell mixing experiments in vitro and in vivo have demonstrated that the engrailed requirement for the survival of mesDA neurons is cell-autonomous. The inactivation of engrailed by RNA interference induces apoptosis in less than 24 h. These data suggest that the engrailed genes control an essential mechanism for the survival of mesDA neurons.

This paper identifies a new, developmental role for transcription <em>factor</em> AP-2 in the activation of amphibian <em>embryonic</em> epidermal keratin gene expression. Keratin transcription <em>factor</em> KTF-<em>1</em> is shown by several criteria to be identical or closely related to AP-2. KTF-<em>1</em>/AP-2 is shown to be tissue-specific from its first transcription in Xenopus embryos, and restricted to a small number of adult tissues, including skin. Epidermis-specific keratin transcription closely follows specification of the <em>embryonic</em> ectoderm in Xenopus, and is subject to regulation by <em>growth</em> <em>factors</em> and <em>embryonic</em> induction. We further show that in mouse basal keratinocytes, a KTF-<em>1</em>/AP-2-like <em>factor</em> is present and binds to a DNA sequence previously shown to be important in the regulation of the keratin K<em>1</em>4 gene, which is actively expressed in these cells. Thus, the study of AP-2 and its role in the regulation of keratin gene transcription should enhance our understanding of both amphibian <em>embryonic</em> development and mammalian skin <em>differentiation</em>.

Human <em>embryonic</em> stem (hES) cells as a renewable cell source have great prospective applications in both developmental biology research and regenerative medicine. To realize these potentials, the development of effective and safe genetic manipulation methods in hES cells is an obvious demand. We report here that baculoviral vectors were able to transduce hES cells efficiently. In transient transduction experiments, a recombinant baculoviral vector equipped with a human elongation <em>factor</em> <em>1</em>-alpha promoter and a woodchuck hepatitis post-transcriptional regulatory element transduced up to 80% of cells in hES cell clumps and embryoid bodies. For prolonged transgene expression, hybrid baculoviral vectors that have incorporated a rep gene and inverted terminal repeat sequences from adeno-associated virus were produced. These hybrid vectors yielded stable transgene expression during the prolonged undifferentiated proliferation of hES cells and after <em>differentiation</em>. Baculoviral transduction did not affect the normal <em>growth</em>, phenotype, and pluripotency of hES cells. Thus, baculoviral vectors suitable for both transient overexpression and long-term stable expression are an attractive option for genetic manipulation of hES cells.

We have analysed the function of transforming <em>growth</em> <em>factor</em> beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extra<em>embryonic</em> mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from <em>embryonic</em> stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (<em>1</em>) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for <em>differentiation</em> of extra<em>embryonic</em> mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the <em>differentiation</em> of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extra<em>embryonic</em> mesoderm alone is sufficient to account for the extra<em>embryonic</em> phenotype reported previously in TGF-beta<em>1</em>(-/-) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (<em>1</em>995) Development <em>1</em>2<em>1</em>, <em>1</em>845-<em>1</em>854).

Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (<em>differentiation</em>); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast <em>growth</em> <em>factor</em> (FGF) regulates multiple <em>embryonic</em> events, including germ layer <em>differentiation</em> and morphogenesis; the cellular components that direct FGF signaling to evoke these different responses remain largely unknown. We show here that the copper transporter <em>1</em> (Ctr<em>1</em>) protein is a critical router of FGF signals during early embryogenesis. Ctr<em>1</em> both promotes the <em>differentiation</em> and inhibits the morphogenesis of mesoderm and neurectoderm in embryos of the frog Xenopus laevis, thereby coordinating normal development. Signal sorting by Ctr<em>1</em> involves the activation of the Ras-MAP kinase cascade and appears to be independent of its role in copper transport. Mouse <em>embryonic</em> stem (ES) cells deficient for Ctr<em>1</em> (Ctr<em>1</em>(-/-)) retain characteristics of pluripotency under conditions that favor <em>differentiation</em> in wild-type ES cells, indicating a conserved role for Ctr<em>1</em> during amphibian and mammalian cell fate determination. Our studies support a model in which vertebrate Ctr<em>1</em> functions as a key regulator of the <em>differentiation</em> capacity of both stem and progenitor cell populations.

Bone morphogenetic proteins (BMPs) are morphogens implicated in <em>embryonic</em> and regenerative odontogenic <em>differentiation</em>. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have optimized the gene transfer of <em>Growth</em>/<em>differentiation</em> <em>factor</em> <em>1</em><em>1</em> (Gdf<em>1</em><em>1</em>)/Bmp<em>1</em><em>1</em> plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5-<em>1</em>0% Optison (Molecular Biosystems Inc., San Diego, CA) and stimulated by ultrasound (<em>1</em> MHz, 0.5 W/cm(2), 30 sec) showed significant efficiency of gene transfer and high level of protein production selectively in the local region, within 500 microm of the amputated site of the pulp tissue. The Gdf<em>1</em><em>1</em> cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of dentin sialoprotein (Dsp), a <em>differentiation</em> marker for odontoblasts. The transfection of Gdf<em>1</em><em>1</em> by sonoporation stimulated a large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs using ultrasound-mediated gene therapy for endodontic dental treatment.

Hepatocyte <em>growth</em> <em>factor</em> activator inhibitor type <em>1</em> (HAI-<em>1</em>)/serine protease inhibitor, Kunitz type <em>1</em> (SPINT<em>1</em>) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte <em>growth</em> <em>factor</em> activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal <em>differentiation</em> and function. In this study, skin and hair phenotypes of mice lacking the Hai-<em>1</em>/Spint<em>1</em> gene were characterized. Previously, we reported that the homozygous deletion of Hai-<em>1</em>/Spint<em>1</em> in mice resulted in <em>embryonic</em> lethality attributable to impaired placental development. To test the role of Hai-<em>1</em>/Spint<em>1</em> in mice, the placental function of Hai-<em>1</em>/Spint<em>1</em>-mutant mice was rescued. Injection of Hai-<em>1</em>/Spint<em>1</em>(+/+) blastocysts with Hai-<em>1</em>/Spint<em>1</em>(-/-) <em>embryonic</em> stem cells successfully generated high-chimeric Hai-<em>1</em>/Spint<em>1</em>(-/-) embryos (B6Hai-<em>1</em>(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that <em>embryonic</em> lethality of Hai-<em>1</em>/Spint<em>1</em>(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-<em>1</em>(-/-High) mice showed <em>growth</em> retardation and died by <em>1</em>6 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-<em>1</em>/Spint<em>1</em>-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-<em>1</em>/Spint<em>1</em> has critical roles in the regulated keratinization of the epidermis and hair development.

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage <em>growth</em> properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a <em>differentiation</em> response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory <em>factor</em> (LIF) to maintain <em>embryonic</em> stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell <em>differentiation</em> by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific <em>embryonic</em> antigen (SSEA)-<em>1</em> expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-<em>growth</em> rate but significantly alters the probability of a cell undergoing a self-renewal vs. a <em>differentiation</em> division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous <em>factors</em>, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell <em>differentiation</em> control.

BACKGROUND

The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.

RESULTS

In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.

CONCLUSIONS

Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.

GATA-<em>1</em> and GATA-2 transcription <em>factors</em> are required for effective hematopoiesis. These regulatory proteins present overlapping yet distinct patterns of expression in hematopoietic cells. Absence of GATA-2 leads to defective hematopoiesis and an <em>embryonic</em> lethal phenotype. Disruption of GATA-<em>1</em> results in a compensatory increase in GATA-2 in early erythroid cells and incomplete erythropoiesis with embryos dying at <em>1</em><em>1</em>.5 days. We examine the specific role of GATA-2 later in hematopoiesis, during erythroid <em>differentiation</em>. Stable K562 cell lines expressing various levels of GATA-2 were generated using a GATA-2 expression plasmid. Overexpression of GATA-2 transcripts was determined by quantitative polymerase chain reaction (PCR). Cytospin smears, <em>growth</em> curve analysis, PCR, and flow cytometry were used to examine the effects of increased levels of GATA-2 in altering cell phenotype and activation of megakaryocytic markers. Human progenitor erythroid cells also were transfected with a GATA-2 expression vector. <em>Growth</em> curve analysis, benzidine staining, and high-performance liquid chromatographic analysis were used to study the effects of GATA-2 on erythroid maturation and proliferation.K562/GATA-2 cell lines expressing high levels of GATA-2 mRNA showed a marked decrease in proliferation and a shift in phenotype toward the megakaryocyte lineage. Ploidy analyses showed that these cell lines developed a multinuclear phenotype, including tetraploids and octaploids. PCR analysis showed activation of megakaryocyte-specific genes including thrombopoietin receptor (c-mpl). Surface expression of platelet glycoprotein receptors Ib/IX (CD42b/CD42a) and IIb/IIIa (CD4<em>1</em>/CD6<em>1</em>) also was demonstrated by flow cytometry. In primary human adult erythroid cultures transfected with a GATA-2 expression vector, production of total hemoglobin and cell proliferation decreased in a dose-dependent manner.GATA-2 plays an important role in deciding cell lineage throughout hematopoiesis, and increased expression of GATA-2 determines megakaryocytic <em>differentiation</em>. Downregulation of GATA-2 is required for erythroid <em>differentiation</em>.

Epigenetic control of genes that are silent in <em>embryonic</em> stem cells, but destined for expression during <em>differentiation</em>, includes distinctive hallmarks, such as simultaneous activating/repressing (bivalent) modifications of chromatin and DNA hypomethylation at enhancers of gene expression. Although alpha-fetoprotein (Afp) falls into this class of genes, as it is silent in pluripotent stem cells and activated during <em>differentiation</em> of endoderm, we find that Afp chromatin lacks bivalent histone modifications. However, critical regulatory sites for Afp activation, overlapping Foxa<em>1</em>/p53/Smad-binding elements, are located within a 300-bp region lacking DNA methylation, due to transposed elements underrepresented in CpG sequences: a short interspersed transposable element and a medium reiterated sequence <em>1</em> element. Forkhead family member Foxa<em>1</em> is activated by retinoic acid treatment of <em>embryonic</em> stem cells, binds its DNA consensus site within the short interspersed transposable/medium reiterated sequence <em>1</em> elements, and displaces linker histone H<em>1</em> from silent Afp chromatin. Small interfering RNA depletion of Foxa<em>1</em> showed that Foxa<em>1</em> is essential in providing chromatin access to transforming <em>growth</em> <em>factor</em> beta-activated Smad2 and Smad4 and their subsequent DNA binding. Together these transcription <em>factors</em> establish highly acetylated chromatin and promote expression of Afp. Foxa<em>1</em> acts as a pioneer transcription <em>factor</em> in de novo activation of Afp, by exploiting a lack of methylation at juxtaposed transposed elements, to bind and poise chromatin for intersection with transforming <em>growth</em> <em>factor</em> beta signaling during <em>differentiation</em> of <em>embryonic</em> stem cells.

The three zones of adrenal cortex are thought to arise from a single multipotential stem cell, but the mechanisms underlying the zonal <em>differentiation</em> during <em>embryonic</em> development of adrenal cortex are poorly understood. Employing subtraction cloning strategy, we isolated three distinct clones that were specifically expressed in the rat glomerulosa zone. One clone, named zona glomerulosa specific clone, encoded a membrane-spanning protein with a signal peptide at the N-terminus, six epidermal <em>growth</em> <em>factor</em>-like repeat motifs, and a transmembrane domain near the C-terminus. It was identified as a rat homolog of preadipocyte <em>factor</em>-<em>1</em> (Pref-<em>1</em>), a <em>factor</em> involved in maintaining the undifferentiated status of preadipocyte. Immunohistochemical studies confirmed the presence of Pref-<em>1</em> protein in the glomerulosa zone. Detailed examination revealed that the zone is divided into two layers; the first is a few-cells-thick layer present underneath the capsule (expressing both Pref-<em>1</em> protein and aldosterone synthase cytochrome P450), and the second layer is beneath the first (containing Pref-<em>1</em> protein but not aldosterone synthase). Moreover, another cell layer was found beneath the second layer and above the fasciculata zone, whose cells contained no Pref-<em>1</em> protein, aldosterone synthase, or <em>1</em><em>1</em>beta-hydroxylase. These findings suggest that a recently reported aldosterone synthase- and <em>1</em><em>1</em>beta-hydroxylase-less cell layer between the two zones is composed of two kinds of cell: Pref-<em>1</em> protein-positive and -negative cells. The level of Pref-<em>1</em> message in the adrenal glands of animals having various pituitary-adrenal axis activities, as well as various plasma salt concentrations, correlated with the total number of glomerulosa cells. However, the specific content of Pref-<em>1</em> message in a cell was fairly constant. When the adrenal gland was surgically enucleated and the remaining capsule regenerated, the level of Pref-<em>1</em> transcript was significantly suppressed at the early phase. At this phase, only a minor population of the cortical cells expressed Pref-<em>1</em> protein, most of these cells already expressing a fasciculata/reticularis-specific marker, inner zone antigen. These findings suggest that the capsular cells, mostly composed of the glomerulosa cells, may have potential for differentiating into other zones' cells, and the down-regulation of Pref-<em>1</em> expression may be an important step in the adrenal zonal <em>differentiation</em>.

Neurotrophic support is generally believed to result from a direct action of <em>growth</em> <em>factors</em> on developing neurons. However, there is increasing evidence that <em>growth</em> <em>factors</em> can indirectly affect neuronal development by glial-mediated processes. To investigate a possible role of glia in mediating neurotrophic effects on dopaminergic neurons, four purified <em>growth</em> <em>factors</em> were screened for dual effects on the survival and <em>differentiation</em> of dopaminergic neurons and on the proliferation of mesencephalic glial cells in vitro. Dissociated <em>embryonic</em> day <em>1</em>4.5 rat mesencephalon was grown at low cell density without serum, conditions under which both glial <em>growth</em> and neuronal survival are not optimal. Treatment of these cultures with acidic fibroblast <em>growth</em> <em>factor</em> (aFGF) or basic fibroblast <em>growth</em> <em>factor</em> (bFGF) increased the number of surviving tyrosine hydroxylase-immunoreactive (TH-IR) neurons by 90-<em>1</em><em>1</em>0% [corrected] at 8 d in vitro in a dose-dependent manner. The effects of these <em>factors</em> were not additive. High-affinity dopamine uptake was increased by bFGF, but not by aFGF. Length of TH-IR neurites was not affected by either aFGF or bFGF. Both <em>growth</em> <em>factors</em> induced proliferation of mesencephalic astrocytes as demonstrated by autoradiographic labeling with 3H-thymidine combined with immunocytochemistry for glial fibrillary acidic protein (GFAP). In contrast, platelet-derived <em>growth</em> <em>factor</em> (PDGF) and interleukin-<em>1</em> had no effect on the survival or <em>differentiation</em> of dopaminergic neurons or the proliferation of mesencephalic astrocytes. Inhibition of glial proliferation abolished the neurotrophic effects exerted by aFGF or bFGF on dopaminergic neurons. Moreover, conditioned medium derived from mesencephalic glial cultures replated in the virtual absence of neurons also contained neurotrophic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasculogenesis, the <em>differentiation</em> of mesodermal cells to angioblasts and the subsequent formation of blood islands and blood vessels by angioblasts in the conceptus, is a dynamic process modulated, in part, by cell-extracellular matrix and cell-cell interactions in the presence of a variety of <em>growth</em> <em>factors</em> and morphogens. In this report we demonstrate differential tyrosine phosphorylation of platelet-endothelial cell adhesion molecule-<em>1</em> (PECAM-<em>1</em>) during the formation of blood islands and vessels from clusters of extra<em>embryonic</em> and <em>embryonic</em> angioblasts in the murine conceptus. In addition, we identify the phosphorylation of a particular tyrosine residue in the PECAM-<em>1</em> cytoplasmic domain, Tyr686, which has the potential of mediating binding to Src homology 2 domain-containing proteins, affecting PECAM-<em>1</em> cellular localization and endothelial cell migration.

Mouse <em>embryonic</em> stem-derived cells were recently shown to differentiate into endothelial and smooth muscle cells. In the present study, we investigated whether human umbilical vein endothelium-derived cells retain the potential to differentiate into smooth muscle cells. Examination of biochemical markers, including basic calponin, SM22alpha, prostaglandin E synthase, von Willebrand <em>factor</em>, and PECAM-<em>1</em>, as well as cell contractility, showed that whereas endothelium-derived cells cultured with fibroblast <em>growth</em> <em>factor</em> can be characterized as endothelial cells, when deprived of fibroblast <em>growth</em> <em>factor</em>, a significant fraction differentiates into smooth muscle-like cells. Reapplication of fibroblast <em>growth</em> <em>factor</em> reversed this <em>differentiation</em>. Activin A was up-regulated in fibroblast <em>growth</em> <em>factor</em>-deprived, endothelium-derived cells; moreover, the inhibitory effects of exogenous follistatin and overexpressed Smad7 on smooth muscle-like <em>differentiation</em> confirmed that the <em>differentiation</em> was driven by activin A signaling. These findings indicate that when deprived of fibroblast <em>growth</em> <em>factor</em>, human umbilical vein endothelium-derived cells are capable of differentiating into smooth muscle-like cells through activin A-induced, Smad-dependent signaling, and that maintenance of the endothelial cell phenotype and <em>differentiation</em> into smooth muscle-like cells are reciprocally controlled by fibroblast <em>growth</em> <em>factor</em>-<em>1</em> and activin A.

Previous experiments showed that transgenic mice expressing a secreted self-activating transforming <em>growth</em> <em>factor</em> (TGF) -beta<em>1</em> did not show a phenotype in the lens and cornea until postnatal day 2<em>1</em>, when anterior subcapsular cataracts, sporadic thickening of the corneal stroma, and thinning of the corneal epithelium were noted (Srinivasan et al., <em>1</em>998). To examine the effects of higher concentrations of TGF-beta<em>1</em> on the lens and cornea, we constructed transgenic mice harboring the strong, lens-specific chicken betaB<em>1</em>-crystallin promoter driving an activated porcine TGF-beta<em>1</em> gene. In contrast to the earlier study, the transgenic mice had microphthalmic eyes with closed eyelids. Already at <em>embryonic</em> day (E) <em>1</em>3.5, the future cornea of the transgenic mice was threefold thicker than that of wild-type littermates due to increased proliferation of corneal stromal mesenchyme cells. Staining of fibronectin and thrombospondin-<em>1</em> was increased in periocular mesenchyme. At E<em>1</em>7.5, the thickened transgenic corneal stroma was vascularized and densely populated by abundant star-shaped, neural cell adhesion molecule-positive cells of mesenchymal appearance surrounded by irregular swirls of collagen and extracellular matrix. The corneal endothelium, anterior chamber, and stroma of iris/ciliary body did not develop, and the transgenic cornea was opaque. Fibronectin, perlecan, and thrombospondin-<em>1</em> were elevated, whereas type VI collagen decreased in the transgenic corneal stroma. Stromal mesenchyme cells expressed alpha-smooth muscle actin as did lens epithelial cells and cells of the retinal pigmented epithelium. By E<em>1</em>7.5, lens fiber cells underwent apoptotic cell death that was followed by apoptosis of the entire anterior lens epithelium between E<em>1</em>8.5 and birth. Posteriorly, the vitreous humor was essentially absent; however, the retina appeared relatively normal. Thus, excess TGF-beta<em>1</em>, a mitogen for <em>embryonic</em> corneal mesenchyme, severely disrupts corneal and lens <em>differentiation</em>. Our findings profoundly contrast with the mild eye phenotype observed with presumably lower levels of ectopic TGF-beta and illustrate the complexity of TGF-beta utilization and the importance of dose when assessing the effects of this <em>growth</em> <em>factor</em>.

We and others have previously demonstrated that retinal cells can be derived from human <em>embryonic</em> stem cells (hESCs) and induced pluripotent stem cells under defined culture conditions. While both cell types can give rise to retinal derivatives in the absence of inductive cues, this requires extended culture periods and gives lower overall yield. Further understanding of this innate <em>differentiation</em> ability, the identification of key <em>factors</em> that drive the <em>differentiation</em> process, and the development of clinically compatible culture conditions to reproducibly generate functional neural retina is an important goal for clinical cell based therapies. We now report that insulin-like <em>growth</em> <em>factor</em> <em>1</em> (IGF-<em>1</em>) can orchestrate the formation of three-dimensional ocular-like structures from hESCs which, in addition to retinal pigmented epithelium and neural retina, also contain primitive lens and corneal-like structures. Inhibition of IGF-<em>1</em> receptor signaling significantly reduces the formation of optic vesicle and optic cups, while exogenous IGF-<em>1</em> treatment enhances the formation of correctly laminated retinal tissue composed of multiple retinal phenotypes that is reminiscent of the developing vertebrate retina. Most importantly, hESC-derived photoreceptors exhibit advanced maturation features such as the presence of primitive rod- and cone-like photoreceptor inner and outer segments and phototransduction-related functional responses as early as 6.5 weeks of <em>differentiation</em>, making these derivatives promising candidates for cell replacement studies and in vitro disease modeling.

Although the role of <em>1</em>alpha,25-dihydroxyvitamin D3 in calcium homeostasis of bone tissue is clear, evidence of the involvement of vitamin D3 in the central nervous system functions is increasing. In fact, vitamin D3 regulates vitamin D receptor and nerve <em>growth</em> <em>factor</em> expression, modulates brain development, and reverses experimental autoimmune encephalomyelitis. Only few studies, however, address vitamin D3 effect on <em>embryonic</em> hippocampal cell <em>differentiation</em>. In this investigation, the HN9.<em>1</em>0e cell line was used as experimental model; these cells, that are a somatic fusion product of hippocampal cells from <em>embryonic</em> day-<em>1</em>8 C57BL/6 mice and N<em>1</em>8TG2 neuroblastoma cells, show morphological and cytoskeletal features similar to their neuronal precursors. By this model, we have studied the time course of vitamin D3 localization in the nucleus and its effect on proteins involved in proliferation and/or <em>differentiation</em>. We found that the translocation of vitamin D3 from cytoplasm to the nucleus is transient, as the maximal nuclear concentration is reached after <em>1</em>0 h of incubation with (3)H-vitamin D3 and decreases to control values by <em>1</em>2 h. The appearance of <em>differentiation</em> markers such as Bcl2, NGF, STAT3, and the decrease of proliferation markers such as cyclin-<em>1</em> and PCNA are late events. Moreover, physiological concentrations of vitamin D3 delay cell proliferation and induce cell <em>differentiation</em> of <em>embryonic</em> cells characterized by modification of soma lengthening and formation of axons and dendrites.

OBJECTIVE

The early events of reproduction involve a carefully modulated complex system of oocyte maturation, fertilization, and proliferation. The aim of the study was to measure the presence of cytokines, namely interleukin <em>1</em> (IL-<em>1</em>), interleukin 6 (IL-6), colony-stimulating <em>factor</em> <em>1</em> (CSF-<em>1</em>), and tumor necrosis <em>factor</em> (TNF) in the conditioned medium (CM) of the oocytes, granulosa cells, cumulus cells, one to eight-cell embryos and sperm.

METHODS

The material was obtained from men and women undergoing in vitro fertilization therapy.

METHODS

We hypothesized that cytokines might affect embryonic growth and differentiation as they show a pleotropic effect on immune cells.

RESULTS

All these cytokines are present in significant quantities in the CM and were shown to be expressed in a sequential manner; thus, some are present in the oocyte and its vestment, the corona-cumulus complex (IL-<em>1</em>, IL-6, and CSF-<em>1</em>), whereas TNF appears only at the stage of six to eight-cell embryos. Inflammatory cytokines could not be detected in sperm samples.

CONCLUSIONS

It is possible that these cytokines have a role in the regulation of embryonic development, maternal immunological recognition of pregnancy, and maintenance of proper hormonal environment.

BACKGROUND

Mammalian fetal skin injury heals scarlessly. The intrinsic differences between embryonic and adult fibroblasts that underlie this observation are poorly understood. Several studies have linked Wnt proteins with skin morphogenesis. The authors' study aimed to establish a correlation between beta-catenin-dependent (canonical) Wnt protein, transforming growth factor (TGF)-beta1, and the expression of hyaluronan synthesis enzymes during scarless versus scarring wound healing.

METHODS

Wnt signaling was quantified after 1.5-mm skin wounds were created in BAT-gal fetal (e16.5) and postnatal (p1) mice. Canonical Wnt signals were localized by X-gal staining and quantified with quantitative real-time polymerase chain reaction. Primary embryonic and postnatal mouse dermal fibroblasts were treated with recombinant Wnt3a or TGF-beta1. Proliferation was assayed by bromodeoxyuridine incorporation. Gene expression of enzymes that regulate hyaluronan production and turnover was examined by quantitative real-time polymerase chain reaction (hyaluronan synthases or HAS1-3, hyaluronadase-2), as well as other target genes for Wnt and TGF-beta (Axin2, TGF-beta1, TGF-beta3, type 1 collagen, proliferating cell nuclear antigen).

RESULTS

Canonical Wnt signaling increased following wounding in postnatal, but not fetal, mice. In vitro, rmWnt3a increased postnatal fibroblast proliferation but not in embryonic cells. Both Wnt3a and TGF-beta1 induced HAS2 and HAS3 gene expression in embryonic fibroblasts, while HAS1 and Hyal2 were induced in postnatal fibroblasts. Finally, rmWnt3a significantly increased type I collagen expression, particularly in postnatal fibroblasts, and influenced expression of TGF-beta isoforms.

CONCLUSIONS

Increased canonical Wnt signaling occurs during postnatal but not fetal cutaneous wound repair. Fetal and postnatal fibroblasts have a disparate response to rmWnt3a in vitro. rmWnt3a affects postnatal fibroblasts in a similar fashion to rhTGF-beta1, a known profibrotic cytokine.