The successful <em>differentiation</em> of human <em>embryonic</em> stem cells (hESCs) to fibrochondrocyte-like cells and characterization of these differentiated cells is a critical step toward tissue engineering of musculoskeletal fibrocartilages (e.g., knee meniscus, temporomandibular joint disc, and intervertebral disc). In this study, <em>growth</em> <em>factors</em> and primary cell cocultures were applied to hESC embryoid bodies (EBs) for 3 weeks and evaluated for their effect on the synthesis of critical fibrocartilage matrix components: glycosaminoglycans (GAG) and collagens (types I, II, and VI). Changes in surface markers (CD<em>1</em>05, CD44, SSEA, PDGFR alpha) after the <em>differentiation</em> treatments were also analyzed. The study was conducted in three phases: (<em>1</em>) examination of <em>growth</em> <em>factors</em> (TGF-beta 3, BMP-2, BMP-4, BMP-6, PDGF-BB, sonic hedgehog protein); (2) comparison of two cocultures (primary chondrocytes or fibrochondrocytes); and (3) the combination of the most effective <em>growth</em> <em>factor</em> and coculture regimen. TGF-beta 3 with BMP-4 yielded EBs positive for collagens I, II, and VI, with up to 6.7- and 4.8-fold increases in GAG and collagen, respectively. Analysis of cell surface markers showed a significant increase in CD44 with the TGF-beta 3 + BMP-4 treatment compared to the controls. Coculture with fibrochondrocytes resulted in up to a 9.8-fold increase in collagen II production. The combination of the <em>growth</em> <em>factors</em> BMP-4 + TGF-beta 3 with the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. This study determined two powerful treatments for inducing fibrocartilaginous <em>differentiation</em> of hESCs and provides a foundation for using flow cytometry to purify these differentiated cells.

We have developed a differential display screening approach to identify mesoderm-specific genes, relying upon the <em>differentiation</em> of <em>embryonic</em> stem (ES) cells in vitro. Using this strategy, we have isolated a novel murine gene that encodes a secreted molecule containing a variant epidermal <em>growth</em> <em>factor</em>-like (EGF) motif. We named this gene Cryptic, based on its predicted protein sequence similarity with Cripto, which encodes an EGF-related <em>growth</em> <em>factor</em>. Based on their strong sequence similarities, we propose that Cryptic, Cripto, and the Xenopus FRL-<em>1</em> gene define a new family of <em>growth</em> <em>factor</em>-like molecules, which we name the 'CFC' (Cripto, Frl-<em>1</em>, and Cryptic) family. Analysis of Cryptic expression by in situ hybridization shows that it is expressed during gastrulation in two spatial domains that correspond to the axial and lateral mesoderm. In the first domain of expression, Cryptic expression is progressively localized to the anterior primitive streak, the head process, and the node and notochordal plate. In the second domain, Cryptic expression is initially concentrated in the lateral region of the egg cylinder, and is later found circumferentially in the intermediate and lateral plate mesoderm. Furthermore, Cryptic expression can also be detected at the early head-fold stage in the midline neuroectoderm, and consequently is an early marker for the prospective floor plate of the neural tube. Expression of Cryptic ceases at the end of gastrulation, and has not been observed in later <em>embryonic</em> stages or in adult tissues. Thus, Cryptic encodes a putative signaling molecule whose expression suggests potential roles in mesoderm and/or neural patterning during gastrulation.

Using both a radioimmunoassay and a radioreceptor assay, we have estimated the content of mouse epidermal <em>growth</em> <em>factor</em>-urogastrone (EGF-URO) in fetal mice at <em>1</em><em>1</em>(<em>1</em>/2) to <em>1</em>7(<em>1</em>/2) days of gestation. Concurrently, the amount of specific EGF-URO receptor binding was determined in crude membrane fractions from the embryos. EGF-URO receptor binding is readily detected in membranes from the youngest embryos (day <em>1</em><em>1</em>(<em>1</em>/2)) and rises steadily up to parturition (<em>1</em>8 days); the rise is more marked in embryo membranes derived from a potential target tissue, such as the maxilla and secondary palate. In the <em>embryonic</em> extracts, EGF-URO proved to be labile, requiring the presence of soybean trypsin inhibitor and sodium azide to stabilize the recovery of added EGF-URO in test samples. Even with added stabilizing agents, immunoreactive EGF-URO was barely detectable before day <em>1</em>4(<em>1</em>/2) (less than 20 fmol per embryo), whereas a substantial increase was observed from day <em>1</em>5(<em>1</em>/2) to <em>1</em>7(<em>1</em>/2) (from 70 to 200 fmol per embryo). In contrast, the radioreceptor assay detected appreciable amounts of an EGF-URO-like substance at <em>1</em><em>1</em>(<em>1</em>/2) days (50 fmol per embryo); the values estimated by radioreceptor assay (about <em>1</em>0-fold higher than by radioimmunoassay) also increase markedly between days <em>1</em>5(<em>1</em>/2) and <em>1</em>7(<em>1</em>/2) (on average from 500 to 3000 fmol per embryo). We conclude that during fetal mouse development there is an increase both in the receptors for EGF-URO and in a substance (presumably a fetal <em>growth</em> <em>factor</em>) that can occupy the receptor. The <em>differences</em> between the radioreceptor and radioimmunoassay estimates for the EGF-URO content suggest that the fetal form of mouse EGF-URO differs from the adult molecule.

MicroRNAs (miRs) play an important role in cell <em>differentiation</em> and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage <em>differentiation</em>. Human <em>embryonic</em> stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34(+) EB cells. miRs-<em>1</em>26/<em>1</em>26* were the most enriched of the 7 miRs that were up-regulated in CD34(+) cells, and their expression paralleled the kinetics of hematopoietic transcription <em>factors</em> RUNX<em>1</em>, SCL, and PU.<em>1</em>. To define the role of miRs-<em>1</em>26/<em>1</em>26* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-<em>1</em>26/<em>1</em>26* and analyzed their hematopoietic <em>differentiation</em>. Induction of miRs-<em>1</em>26/<em>1</em>26* during both EB <em>differentiation</em> and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-<em>1</em>26/<em>1</em>26* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for <em>growth</em> and expansion of erythroid cells, is one target of miR-<em>1</em>26. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-<em>1</em>26/<em>1</em>26*. Our results define an important function of miRs-<em>1</em>26/<em>1</em>26* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic <em>differentiation</em> of hESCs.

Syp is a protein tyrosine phosphatase implicated in insulin and <em>growth</em> <em>factor</em> signaling. To evaluate the role of syp in insulin's regulation of plasma glucose, we generated knockout mice. Homozygous knockout mice die prior to day <em>1</em>0.5 of <em>embryonic</em> development. Hemizygous mice express half the levels of syp protein compared with their wild type littermates but do not display any gross morphological changes. Total body weight (age 2-<em>1</em>0 weeks) and plasma insulin and glucose levels both in fasting and glucose-challenged states were comparable in the wild type and the hemizygous mice. No <em>differences</em> were observed in insulin-induced glucose uptake in soleus muscle and epididymal fat; insulin inhibition of lipolysis was also similar. We injected insulin into the portal vein of the mice to examine upstream events of the insulin signaling cascade. Tyrosine phosphorylation of insulin receptor and insulin receptor substrate-<em>1</em> (IRS-<em>1</em>) from hemizygous tissue was similar to that of wild type tissue. Association of the p85 subunit of phosphatidylinositol 3-kinase to IRS-<em>1</em> increased an average of 2-fold in both groups. We did not observe an increase of IRS-<em>1</em>/syp association after insulin administration, but we did note a significant basal association in both wild type and hemizygous tissue. Our results do not support a major role for syp in the acute in vivo metabolic actions of insulin.

Neural stem cells (NSCs) have been proposed as a promising cellular source for the treatment of diseases in nervous systems. NSCs can self-renew and generate major cell types of the mammalian central nervous system throughout adulthood. NSCs exist not only in the embryo, but also in the adult brain neurogenic region: the subventricular zone (SVZ) of the lateral ventricle. <em>Embryonic</em> stem (ES) cells acquire NSC identity with a default mechanism. Under the regulations of leukemia inhibitory <em>factor</em> (LIF) and fibroblast <em>growth</em> <em>factors</em>, the NSCs then become neural progenitors. Neurotrophic and <em>differentiation</em> <em>factors</em> that regulate gene expression for controlling neural cell fate and function determine the <em>differentiation</em> of neural progenitors in the developing mammalian brain. For clinical application of NSCs in neurodegenerative disorders and damaged neurons, there are several critical problems that remain to be resolved: <em>1</em>) how to obtain enough NSCs from reliable sources for autologous transplantation; 2) how to regulate neural plasticity of different adult stem cells; 3) how to control <em>differentiation</em> of NSCs in the adult nervous system. In order to understand the mechanisms that control NSC <em>differentiation</em> and behavior, we review the ontogeny of NSCs and other stem cell plasticity of neuronal <em>differentiation</em>. The role of NSCs and their regulation by neurotrophic <em>factors</em> in CNS development are also reviewed.

Remodeling of the chromatin template by inhibition of histone deacetylase (HDAC) activities represents a major goal for transcriptional therapy in neoplastic diseases. Recently, a number of specific and potent HDAC-inhibitors that modulate in vitro cell <em>growth</em> and <em>differentiation</em> have been developed. In this study we analyzed the effect of trichostatin A (TSA), a specific and potent HDAC-inhibitor, on mouse embryos developing in vivo. When administered i.p. to pregnant mice (at a concentration of 0.5-<em>1</em> mg/kg) at postimplantation stages (<em>embryonic</em> day 8 to <em>embryonic</em> day <em>1</em>0), TSA was not toxic for the mother and did not cause any obvious malformation during somitogenesis or at later stages of development. Treated embryos were born at similar frequency and were indistinguishable from control animals, developed normally, and were fertile. Interestingly, embryos from TSA-treated mice killed during somitogenesis were modestly but consistently larger than control embryos and presented an increased (+2 to +6) number of somites. This correlated with an increased acetylation of histone H4, the number of somites expressing the myogenic <em>factor</em> Myf-5, and the expression of Notch, RARalpha2, and RARbeta2 mRNAs. These data indicate that the effects of TSA on transcription: (a) are not toxic for the mother; (b) transiently accelerated <em>growth</em> in mouse embryos without perturbing embryogenesis; and (c) do not result in teratogenesis, at least in rodents. Thus, TSA might represent a nontoxic and effective agent for the transcriptional therapy of neoplasia.

Pluripotent <em>embryonic</em> stem (ES) cells are a potential source of all types of cells for regenerative medicine. ES cells maintain pluripotency through a complex interplay of different signaling pathways and transcription <em>factors</em>, including leukemia inhibitory <em>factor</em> (LIF), Nanog, Sox2, and Oct3/4. Nanog, however, plays a key role in maintaining the pluripotency of mouse and human ES cells. Phosphoinositde 3-kinase (PI3K) signaling pathway which is activated in response to <em>growth</em> <em>factors</em> and cytokines also plays a critical role in promoting the survival and proliferation of ES cells. Our earlier studies revealed that retinol, the alcohol form of vitamin A, enhances the expression of Nanog and prevents <em>differentiation</em> of ES cells in long-term cultures. Normally vitamin A/retinol is associated with cell <em>differentiation</em> via its potent metabolite, retinoic acid. Thus far, no direct function has been ascribed to retinol itself. In this study, we demonstrate for the first time that retinol directly activates phosphoinositide three (PI3) kinase signaling pathway through IGF-<em>1</em> receptor/insulin receptor substrate one (IRS-<em>1</em>) by engaging Akt/PKB-mTORC<em>1</em> mammalian target of rapamycin-2 (mammalian target of rapamycin complex 2), indicating a <em>growth</em> <em>factor</em>-like function of vitamin A. Furthermore, ES cells do not express enzymes to metabolize retinol into retinoic acid and lack receptors for retinol transport into the cytoplasm, indicating that retinol signaling is independent of retinoic acid. This study presents a novel system to investigate how extracellular signals control the self renewal of ES cells which will be important for high-quality ES cells for regenerative medicine.

Gene-targeting studies have shown that Delta-like 4 (Dll4) is required for normal <em>embryonic</em> vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are not well defined. We generated primary human umbilical vascular endothelial cells that express Dll4 protein to study Dll4 function and previously showed that Dll4 down-regulates vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor 2 and NRP<em>1</em> expression and inhibits VEGF function. We now report that expression of Dll4 in endothelial cells inhibited attachment and migration to stromal-derived <em>growth</em> <em>factor</em> <em>1</em> (SDF<em>1</em>) chemokine. Cell surface, total protein, and mRNA levels of CXCR4, principal signaling receptor for SDF<em>1</em>, were significantly decreased in Dll4-transduced endothelial cells, attributable to a significant reduction of CXCR4 promoter activity. An immobilized recombinant extracellular portion of Dll4 (rhDLL4) was sufficient to down-regulate CXCR4 mRNA and protein, whereas protein levels of SDF<em>1</em>, VEGF, and RDC<em>1</em> were unchanged. The gamma-secretase inhibitor L-685,458 significantly reconstituted CXCR4 mRNA in rhDLL4-stimulated endothelial cells. CXCR4 mRNA levels were significantly reduced in mouse xenografts of Dll4-transduced human gliomas compared with control gliomas, and vascular CXCR4 was not detected by immunohistochemistry in the enlarged vessels within the Dll4 gliomas. Thus, Dll4 may contribute to vascular <em>differentiation</em> and inhibition of the angiogenic response by regulating multiple receptor pathways.

There is increasing appreciation for the neurodevelopmental underpinnings of many psychiatric disorders. Disorders that begin in childhood such as autism, language disorders or mental retardation as well as adult-onset mental disorders may have origins early in neurodevelopment. Neural stem cells (NSCs) can be defined as self-renewing, multipotent cells that are present in both the <em>embryonic</em> and adult brain. Several recent research findings demonstrate that psychiatric illness may begin with abnormal specification, <em>growth</em>, expansion and <em>differentiation</em> of <em>embryonic</em> NSCs. For example, candidate susceptibility genes for schizophrenia, autism and major depression include the signaling molecule Disrupted In Schizophrenia-<em>1</em> (DISC-<em>1</em>), the homeodomain gene engrailed-2 (EN-2), and several receptor tyrosine kinases, including brain-derived <em>growth</em> <em>factor</em> and fibroblast <em>growth</em> <em>factors</em>, all of which have been shown to play important roles in NSCs or neuronal precursors. We will discuss here stem cell biology, signaling <em>factors</em> that affect these cells, and the potential contribution of these processes to the etiology of neuropsychiatric disorders. Hypotheses about how some of these <em>factors</em> relate to psychiatric disorders will be reviewed.

Heparan sulfate proteoglycans (HSPGs) have essential functions during <em>embryonic</em> development and throughout postnatal life. To exert these functions, they undergo a series of processing reactions by heparan-sulfate-modifying enzymes (HSMEs), which endows them with highly modified heparan sulfate (HS) domains that provide specific docking sites for a large number of bioactive molecules. The development and antigen-dependent <em>differentiation</em> of normal B lymphocytes, as well as the <em>growth</em> and progression of B-lineage malignancies, are orchestrated by an array of <em>growth</em> <em>factors</em>, cytokines and chemokines many of which display HS binding. As discussed in this review, tightly regulated HSPG expression is a requirement for normal B cell maturation, <em>differentiation</em> and function. In addition, the HSPG syndecan-<em>1</em> functions as a versatile co-receptor for signals from the bone marrow microenvironment, essential for the survival of long-lived plasma cells and multiple myeloma (MM) plasma cells. Targeting of HSMEs or HS chains on MM cells increases their sensitivity to drugs currently used in MM treatment, including bortezomib, lenalidomide or dexamethasone. Taken together, these findings render the HS biosynthetic machinery a promising target for MM treatment.

Fine tuning of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) signaling is critical in endothelial cell (EC) <em>differentiation</em> and vascular development. Nevertheless, the system for regulating the sensitivity of VEGF signaling has remained unclear. Previously, we established an <em>embryonic</em> stem cell culture reproducing early vascular development using Flk<em>1</em> (VEGF receptor-2)+ cells as common progenitors, and demonstrated that cyclic adenosine monophosphate (cAMP) enhanced VEGF-induced EC <em>differentiation</em>. Here we show that protein kinase A (PKA) regulates sensitivity of Flk<em>1</em>+ vascular progenitors to VEGF signaling for efficient EC <em>differentiation</em>. Blockade of PKA perturbed EC <em>differentiation</em> and vascular formation in vitro and ex vivo. Overexpression of constitutive active form of PKA (CA-PKA) potently induced EC <em>differentiation</em> and vascular formation. Expression of Flk<em>1</em> and Neuropilin-<em>1</em> (NRP<em>1</em>), which form a selective and sensitive receptor for VEGF(<em>1</em>65), was increased only in CA-PKA-expressing progenitors, enhancing the sensitivity of the progenitors to VEGF(<em>1</em>65) by more than <em>1</em>0 times. PKA activation induced the formation of a VEGF(<em>1</em>65), Flk<em>1</em>, and NRP<em>1</em> protein complex in vascular progenitors. These data indicate that PKA regulates <em>differentiation</em> potential of vascular progenitors to be endothelial competent via the dual induction of Flk<em>1</em> and NRP<em>1</em>. This new-mode mechanism regulating "progenitor sensitivity" would provide a novel understanding in vascular development and regeneration.

Hedgehog ligands bind to protein patched homologue <em>1</em> (PTC), a conserved receptor that activates the GLI family of transcription <em>factors</em>, which are involved in development, disease and skeletal repair processes. During <em>embryonic</em> development, hedgehog signalling helps to pattern the limbs and plays a critical part in regulating chondrocyte <em>differentiation</em> and osteogenesis during the longitudinal <em>growth</em> of long bones. This signalling pathway also regulates mesenchymal cell <em>differentiation</em> during skeletal repair and regeneration. In pathologic and degenerative processes, such as osteoarthritis or cartilaginous tumour formation, hedgehog signalling is dysregulated. Several pharmacologic strategies can modulate hedgehog signalling, and targeting this pathway could lead to the development of novel therapeutic approaches. For example, by precisely regulating the level of activity of the hedgehog signalling pathway, the pace of degeneration in osteoarthritis could be slowed, bone repair could be enhanced, and cartilaginous tumour cell viability could be inhibited. As such, regulation of hedgehog signalling could be manipulated to safeguard skeletal health.

In skin, fibroblasts of the connective tissue play a decisive role in epidermal homeostasis and repair by contributing to the regulation of keratinocyte proliferation and <em>differentiation</em>. The AP-<em>1</em> transcription <em>factor</em> subunit JUN plays a crucial role in this mesenchymal-epithelial interplay by regulating the expression of two critical paracrine-acting cytokines, keratinocyte <em>growth</em> <em>factor</em> (KGF) and granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF). We have performed gene expression profiling of wild-type and Jun(-/-) mouse <em>embryonic</em> fibroblasts to identify additional players involved in this complex network, and have found pleiotrophin (PTN) and the stromal cell-derived <em>factor</em> <em>1</em> (SDF-<em>1</em>) as novel JUN-regulated <em>factors</em>. Both cytokines are expressed by dermal fibroblasts in vivo, as shown by semi-quantitative RT-PCR and in situ hybridization on murine skin sections. Using a heterologous feeder layer co-culture system, we demonstrated that PTN and SDF-<em>1</em> exert a mitogenic effect on primary human keratinocytes. Moreover, SDF-<em>1</em>-induced keratinocyte proliferation could be specifically inhibited by neutralizing antibodies against SDF-<em>1</em> or its receptor, CXCR4. Consistent with its role in promoting keratinocyte <em>growth</em>, PTN was upregulated during cutaneous wound healing in vivo. Interestingly, co-cultivation with keratinocytes stimulated PTN expression but repressed SDF-<em>1</em> production in fibroblasts, demonstrating the complexity of the paracrine regulatory cytokine networks that control skin homeostasis and regeneration.

BACKGROUND

New blood vessel formation, or angiogenic switch, is an essential event in the development of solid tumors and their metastatic growth. Tumor blood vessel formation and remodeling is a complex and multi-step processes. The differentiation and recruitment of mural cells including vascular smooth muscle cells and pericytes are essential steps in tumor angiogenesis. However, the role of tumor cells in differentiation and recruitment of mural cells has not yet been fully elucidated. This study focuses on the role of human tumor cells in governing the differentiation of mouse mesenchymal stem cells (MSCs) to pericytes and their recruitment in the tumor angiogenesis process.

RESULTS

We show that C3H/10T1/2 mouse embryonic mesenchymal stem cells, under the influence of different tumor cell-derived conditioned media, differentiate into mature pericytes. These differentiated pericytes, in turn, are recruited to bind with capillary-like networks formed by endothelial cells on the matrigel under in vitro conditions and recruited to bind with blood vessels on gel-foam under in vivo conditions. The degree of recruitment of pericytes into in vitro neo-angiogenesis is tumor cell phenotype specific. Interestingly, invasive cells recruit less pericytes as compared to non-invasive cells. We identified tumor cell-secreted platelet-derived growth factor-B (PDGF-B) as a crucial factor controlling the differentiation and recruitment processes through an interaction with neuropilin-1 (NRP-1) in mesenchymal stem cells.

CONCLUSIONS

These new insights into the roles of tumor cell-secreted PDGF-B-NRP-1 signaling in MSCs-fate determination may help to develop new antiangiogenic strategies to prevent the tumor growth and metastasis and result in more effective cancer therapies.

OBJECTIVE

To document the transcriptome of the pancreatic islet during the early and late development of the mouse pancreas and highlight the qualitative and quantitative features of gene expression that contribute to the specification, growth, and differentiation of the major endocrine cell types. A further objective was to identify endocrine cell biomarkers, targets of diabetic autoimmunity, and regulatory pathways underlying islet responses to physiological and pathological stimuli.

METHODS

mRNA expression profiling was performed by microarray analysis of e12.5-18.5 embryonic pancreas from neurogenin 3 (Ngn3)-null mice, a background that abrogates endocrine pancreatic differentiation. The intersection of this data with mRNA expression in isolated adult pancreatic islets and pancreatic endocrine tumor cell lines was determined to compile lists of genes that are specifically expressed in endocrine cells.

RESULTS

The data provided insight into the transcriptional and morphogenetic factors that may play major roles in patterning and differentiation of the endocrine lineage before and during the secondary transition of endocrine development, as well as genes that control the glucose responsiveness of the beta-cells and candidate diabetes autoantigens, such as insulin, IA-2 and Slc30a8 (ZnT8). The results are presented as downloadable gene lists, available at https://www.cbil.upenn.edu/RADQuerier/php/displayStudy.php?study_id=1330, stratified by predictive scores of relative cell-type specificity.

CONCLUSIONS

The deposited data provide a rich resource that can be used to address diverse questions related to islet developmental and cell biology and the pathogenesis of type 1 and 2 diabetes.

Myelopoiesis and lymphopoiesis are controlled by haematopoietic <em>growth</em> <em>factors</em>, including cytokines, and chemokines that bind to G-protein-coupled receptors (GPCRs). Regulators of G-protein signalling (RGSs) are a protein family that can act as GTPase-activating proteins for G(alphai)- and G(alphaq)-class proteins. We have identified a new member of the R4 subfamily of RGS proteins, RGS<em>1</em>8. RGS<em>1</em>8 contains clusters of hydrophobic and basic residues, which are characteristic of an amphipathic helix within its first 33 amino acids. RGS<em>1</em>8 mRNA was most highly abundant in megakaryocytes, and was also detected specifically in haematopoietic progenitor and myeloerythroid lineage cells. RGS<em>1</em>8 mRNA was not detected in cells of the lymphoid lineage. RGS<em>1</em>8 was also highly expressed in mouse <em>embryonic</em> <em>1</em>5-day livers, livers being the principal organ for haematopoiesis at this stage of fetal development. RGS<em>1</em>, RGS2 and RGS<em>1</em>6, other members of the R4 subfamily, were expressed in distinct progenitor and mature myeloerythroid and lymphoid lineage blood cells. RGS<em>1</em>8 was shown to interact specifically with the G(alphai-3) subunit in membranes from K562 cells. Furthermore, overexpression of RGS<em>1</em>8 inhibited mitogen-activated-protein kinase activation in HEK-293/chemokine receptor 2 cells treated with monocyte chemotactic protein-<em>1</em>. In yeast cells, RGS<em>1</em>8 overexpression complemented a pheromone-sensitive phenotype caused by mutations in the endogeneous yeast RGS gene, SST2. These data demonstrated that RGS<em>1</em>8 was expressed most highly in megakaryocytes, and can modulate GPCR pathways in both mammalian and yeast cells in vitro. Hence RGS<em>1</em>8 might have an important role in the regulation of megakaryocyte <em>differentiation</em> and chemotaxis.

CONCLUSIONS

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from <em>1</em><em>1</em>- to <em>1</em>2-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial <em>differentiation</em> (CD3<em>1</em>, CD34, von Willebrand <em>factor</em> [vWF], and fms-like tyrosine kinase-<em>1</em> [Flk-<em>1</em>]/vascular endothelial <em>growth</em> <em>factor</em> receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial <em>differentiation</em>. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD3<em>1</em>-), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD3<em>1</em> and vWF. Furthermore, the CD34+/CD3<em>1</em>+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human <em>embryonic</em> aorta is a rich source of CD34+/CD3<em>1</em>- endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.

Septation of the single tubular <em>embryonic</em> outflow tract into two outlet segments in the heart requires the precise integration of proliferation, <em>differentiation</em> and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor alpha knockout (Rxra(-/-)) mouse. Rxra(-/-) embryos exhibit lethality between <em>embryonic</em> day (E) <em>1</em>3.5 and <em>1</em>5.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E<em>1</em>2.5, before the <em>embryonic</em> lethality, we found no qualitative <em>difference</em> between wild type and Rxra(-/-) proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E<em>1</em>2.5 embryos demonstrated elevated levels of transforming <em>growth</em> <em>factor</em> beta2 (TGFbeta2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E<em>1</em><em>1</em>.5 embryos treated with TGFbeta2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra(-/-). TGFbeta2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra(-/-) embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E<em>1</em>2.5 and E<em>1</em>3.5. The data suggests that elevated levels of TGFbeta2 can (<em>1</em>) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.

Formation of new beta cells can take place by two pathways: replication of already differentiated beta cells or neogenesis from putative islet stem cells. Under physiological conditions both processes are most pronounced during the fetal and neonatal development of the pancreas. In adulthood little increase in the beta cell number seems to occur. In pregnancy, however, a marked hyperplasia of the beta cells is observed both in rodents and man. Increased mitotic activity has been seen both in vivo and in vitro in islets exposed to placental lactogen (PL), prolactin (PRL) and <em>growth</em> hormone (GH). Receptors for both GH and PRL are expressed in islet cells and are upregulated during pregnancy. By mutational analysis we have identified different functional domains of the cytoplasmic part of the GH receptor. Thus the mitotic signaling only requires the membrane proximal part of the receptor and activation of the tyrosine kinase JAK2 and the transcription <em>factors</em> STAT<em>1</em> and 3. The activation of the insulin gene however also requires the distal part of the receptor and activation of calcium uptake and STAT5. In order to identify putative autocrine <em>growth</em> <em>factors</em> or targets for <em>growth</em> <em>factors</em> we have cloned a novel GH/PRL stimulated rat islet gene product, Pref-<em>1</em> (preadipocyte <em>factor</em>-<em>1</em>). This protein contains six EGF-like motifs and may play a role both in <em>embryonic</em> pancreas <em>differentiation</em> and in beta cell <em>growth</em> and function. In summary, the increasing knowledge about the mechanisms involved in beta cell <em>differentiation</em> and proliferation may lead to new ways of forming beta cells for treatment of diabetes in man.

The effects of vasoactive intestinal peptide (VIP) on the proliferation of central nervous system (CNS) and cancer cells were investigated. VIP has important actions during CNS development. During neurogenesis, VIP stimulates the proliferation and <em>differentiation</em> of brain neurons. Addition of VIP to <em>embryonic</em> mouse spinal cord cultures increases neuronal survival and activity dependent neurotrophic <em>factor</em> (ADNF) secretion from astroglial cells. VIP is an integrative regulator of brain <em>growth</em> and development during neurogenesis and embryogenesis. Also, VIP causes increased proliferation of human breast and lung cancer cells in vitro. VIP binds with high affinity to cancer cells, elevates the cAMP and increases gene expression of c-fos, c-jun, c-myc and vascular endothelial cell <em>growth</em> <em>factor</em>. The effects of VIP on cancer cells are reversed by VIPhybrid, a synthetic VPAC(<em>1</em>) receptor antagonist. VIPhyb inhibits the basal <em>growth</em> of lung cancer cells in vitro and tumors in vivo and potentiates the ability of chemotherapeutic drugs to kill cancer cells. Due to the high density of VPAC(<em>1</em>) receptors in cancer cells, VIP has been radiolabeled with <em>1</em>23I, <em>1</em>8F and 99mTc to image tumors. It remains to be determined if radiolabeled VIP analogs will be useful agents for early detection of cancer in patients.

Transforming <em>growth</em> <em>factor</em> beta 3 (TGF-beta 3) has been cloned from humans, chickens, pigs, and mice. Although the specific in vivo roles of this form of TGF-beta are unknown, the pattern of <em>embryonic</em> and tissue-specific expression of TGF-beta 3 suggests that it is involved in embryogenesis and cell <em>differentiation</em>. We have cloned and sequenced the TGF-beta 3 5'-flanking region to study the transcriptional regulation of this gene. Characterization of the 5'-flanking region showed a <em>1</em><em>1</em>04-base pair 5'-untranslated region, a TATA box 2<em>1</em> bp upstream from the transcription start site, and cAMP-responsive element (CRE) and AP-2 binding site consensus sequences starting at <em>1</em>2 and 24 bp, respectively, upstream from the TATA box. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter plasmid to study functional activity. Basal transcriptional activity of the promoter was regulated by multiple upstream elements including the CRE and the AP-2 site. The CRE was important for both basal and forskolin induction of promoter activity. The TGF-beta 3 promoter was found to be strikingly dissimilar to the TGF-beta <em>1</em> promoter. Since the TGF-beta s have activity in promoting or inhibiting proliferation and <em>differentiation</em> of multiple cell types, it seems likely that the differential and tissue-specific transcriptional regulation of these genes is of fundamental importance in the induction and maintenance of differentiated cell types in various tissues.

The Pituitary adenylate cyclase-activating peptide (PACAP) ligand/type <em>1</em> receptor (PAC<em>1</em>) system regulates neurogenesis and gliogenesis. It has been well established that the PACAP/PAC<em>1</em> system induces <em>differentiation</em> of neural progenitor cells (NPCs) through the Gs-mediated cAMP-dependent signaling pathway. However, it is unknown whether this ligand/receptor system has a function in proliferation of NPCs. In this study, we identified that PACAP and PAC<em>1</em> were highly expressed and co-localized in NPCs of mouse cortex at <em>embryonic</em> day <em>1</em>4.5 (E<em>1</em>4.5) and found that the PACAP/PAC<em>1</em> system potentiated <em>growth</em> <em>factor</em>-induced proliferation of mouse cortical NPCs at E<em>1</em>4.5 via Gq-, but not Gs-, mediated PLC/IP3-dependent signaling pathway in an autocrine manner. Moreover, PAC<em>1</em> activation induced elongation of cellular processes and a stellate morphology in astrocytes that had the bromodeoxyuridine (BrdU)-incorporating ability of NPCs. Consistent with this notion, we determined that the most BrdU positive NPCs differentiated to astrocytes through PAC<em>1</em> signaling. These results suggest that the PACAP/PAC<em>1</em> system may play a dual role in neural/glial progenitor cells not only <em>differentiation</em> but also proliferation in the cortical astrocyte lineage via Ca2+-dependent signaling pathways through PAC<em>1</em>.

When cultured in type I collagen gels, two kidney-derived cell lines, Madin-Darby canine kidney (MDCK) cells and murine inner medullary collecting duct (mIMCD3) cells, from branching tubular structures in the presence of Swiss 3T3 conditioned medium, in which hepatocyte <em>growth</em> <em>factor</em> (HGF) is the major branching tubule inducing <em>factor</em>. However, upon incubation with transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) in the presence of 3T3 conditioned medium, MDCK tubulogenesis and branching was markedly inhibited. In contrast, mIMCD3 cells, which are much less susceptible to <em>growth</em> and tubulogenesis inhibition by TGF-beta, formed long straight tubulelike structures in presence of TGF-beta, suggesting a dissociation between tubulogenesis and branching morphogenesis. Interestingly, those long tubules that did branch often superficially resembled the early branching ureteric bud in <em>embryonic</em> kidneys. Quantitation of branching events revealed a selective branch-inhibiting effect of TGF-beta on mIMCD3 cells at concentrations between 0.02 and 2 ng/ml. There was no qualitative or quantitative <em>difference</em> among TGF-beta <em>1</em>, -beta 2, and -beta 3 on inhibition of branching events, suggesting existence of potentially redundant mechanisms for modulating branching morphogenesis. Concentrations of TGF-beta that resulted in long nonbranching tubules also altered the profile of extracellular matrix-degrading proteases and their inhibitors expressed by developing tubules. Ratios of urokinase type plasminogen activator (u-PA) to plasminogen activator inhibitor (PAI-l) and matrix metalloprotease (MMP)-<em>1</em> to tissue inhibitor of metalloprotease (TIMP)-<em>1</em> were both markedly decreased. In addition, apart from a direct effect on epithelial cell branching morphogenesis, TGF-beta downregulated the expression of HGF mRNA in Swiss 3T3 cells. Thus TGF-beta exerts at least three distinct effects relevant to tubulogenesis and branching morphogenesis inhibition of branching morphogenesis alone (mIMCD3 cells), inhibition of both tubulogenesis and branching morphogenesis (MDCK cells), and inhibition of the expression of <em>growth</em> <em>factor</em> which induce tubulogenesis and branching morphogenesis (3T3 cells). In the context of epithelial tissue development, which requires tightly regulated branching tubulogenesis of epithelial cells, the data suggest a model where branching patterns are regulated by a precise temporal and spatial balance between branching morphogens such as HGF and inhibitory morphogens such as members of the TGF-beta superfamily [e.g., TGF-beta isoforms, certain bone morphogenetic proteins].