The endothelin system is a family of three similar small peptides, two G-protein-coupled receptors and two proteinases. Endothelins have several physiological roles, notably in <em>embryonic</em> <em>differentiation</em> and vascular homeostasis. Numerous types of tumour express endothelins and their regulation is often aberrant when compared with the normal tissue from which the tumour arose. However, endothelin expression is tumour-type specific, and in some instances, expression of individual members of the endothelin system will be upregulated, while in other tumour types, they may be downregulated. Endothelins have numerous potential roles in tumours including modulating angiogenesis, inducing mitogenesis and invasion of tumour cells, and protecting cells from apoptosis. Expression of endothelins is controlled by the tumour microenvironment, whilst the endothelins themselves modify that environment; a case in point is that hypoxia stimulates endothelin expression via hypoxia-inducible <em>factor</em> (HIF)-<em>1</em>, while endothelins stabilise HIF-<em>1</em> leading to expression of, for instance, vascular endothelial <em>growth</em> <em>factor</em>. This review highlights the potential roles of endothelins in various cancers and describes the pre-clinical and clinical progress that has been made in several tumour types - notably prostate, ovary, melanoma and breast cancer. The interactions between the endothelin network and HIF-<em>1</em> are highlighted.

BACKGROUND

Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood.

METHODS

This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells).

RESULTS

EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14.

CONCLUSIONS

EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.

Endothelial cells are required to initiate pancreas development from the endoderm. They also control the function of endocrine islets after birth. Here we investigate in developing pancreas how the endothelial cells become organized during branching morphogenesis and how their development affects pancreatic cell <em>differentiation</em>. We show that endothelial cells closely surround the epithelial bud at the onset of pancreas morphogenesis. During branching morphogenesis, the endothelial cells become preferentially located near the central (trunk) epithelial cells and remain at a distance from the branch tips where acinar cells differentiate. This correlates with predominant expression of the angiogenic <em>factor</em> vascular endothelial <em>growth</em> <em>factor</em>-A (VEGF-A) in trunk cells. In vivo ablation of VEGF-A expression by pancreas-specific inactivation of floxed Vegfa alleles results in reduced endothelial development and in excessive acinar <em>differentiation</em>. On the contrary, acinar <em>differentiation</em> is repressed when endothelial cells are recruited around tip cells that overexpress VEGF-A. Treatment of <em>embryonic</em> day <em>1</em>2.5 explants with VEGF-A or with VEGF receptor antagonists confirms that acinar development is tightly controlled by endothelial cells. We also provide evidence that endothelial cells repress the expression of Ptf<em>1</em>a, a transcription <em>factor</em> essential for acinar <em>differentiation</em>, and stimulate the expression of Hey-<em>1</em> and Hey-2, two repressors of Ptf<em>1</em>a activity. In explants, we provide evidence that VEGF-A signaling is required, but not sufficient, to induce endocrine <em>differentiation</em>. In conclusion, our data suggest that, in developing pancreas, epithelial production of VEGF-A determines the spatial organization of endothelial cells which, in turn, limit acinar <em>differentiation</em> of the epithelium.

Cripto-<em>1</em> (CR-<em>1</em>)/Teratocarcinoma-derived <em>growth</em> <em>factor</em><em>1</em> (TDGF-<em>1</em>) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal <em>growth</em> <em>factor</em> (EGF)/Cripto-<em>1</em>-FRL-<em>1</em>-Cryptic (CFC) family, CR-<em>1</em> functions as an obligatory co-receptor for the transforming <em>growth</em> <em>factor</em>-β (TGF-β) family members, Nodal and <em>growth</em> and <em>differentiation</em> <em>factors</em> <em>1</em> and 3 (GDF<em>1</em>/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-<em>1</em> can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78kDa glucose regulated protein (GRP78). Finally, CR-<em>1</em> can facilitate signaling through the canonical Wnt/β-catenin and Notch/Cbf-<em>1</em> pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-<em>1</em> is essential for early <em>embryonic</em> development and maintains <em>embryonic</em> stem cell pluripotentiality. CR-<em>1</em> performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-<em>1</em> can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-<em>1</em> may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer.

We have cloned the bovine homologue of Mater (maternal antigen that embryos require) cDNA, potentially the first germ cell-specific maternal-effect gene in this species. The 3297 base-pair longest open reading frame encodes a putative protein of <em>1</em>098 amino acids with a domain organization similar to its human counterpart. By reverse transcription coupled to polymerase chain reaction, we have analyzed the spatiotemporal expression of MATER, along with other potential markers of germ cells or oocytes: ZAR<em>1</em> (zygotic arrest <em>1</em>), GDF9 (<em>growth</em> and <em>differentiation</em> <em>factor</em> 9), BMP<em>1</em>5 (bone morphogenetic protein <em>1</em>5), and VASA. In agreement with a preferential oocyte origin, MATER, ZAR<em>1</em>, GDF9, and BMP<em>1</em>5 transcripts were detected in the oocyte itself at a much higher level than in the gonads, while no significant expression was detected in our panel of somatic tissues (uterus, heart, spleen, intestine, liver, lung, mammary gland, muscle). In situ hybridization confirmed oocyte-restricted expression of MATER and ZAR<em>1</em> within the ovary, as early as preantral follicle stages. VASA was highly represented in the testis and the ovary, and still present in the oocyte from antral follicles. Maternal MATER, ZAR<em>1</em>, GDF9, and BMP<em>1</em>5 transcripts persisted during oocyte in vitro maturation and fertilization and in preimplantation embryo until the five- to eight-cell or morula stage, but transcription was not reactivated at the time of <em>embryonic</em> genome activation.

TNF-like weak inducer of apoptosis (TWEAK) and fibroblast <em>growth</em> <em>factor</em> (FGF)-inducible <em>1</em>4 (Fn<em>1</em>4) are a TNF superfamily ligand-receptor pair involved in many cellular processes including proliferation, migration, <em>differentiation</em>, inflammation, and angiogenesis. The Fn<em>1</em>4 receptor is expressed at relatively low levels in normal tissues, but it is known to be dramatically elevated in a number of tumor types, including brain and breast tumors. Thus, it seems to be an excellent candidate for therapeutic intervention. We first analyzed Fn<em>1</em>4 expression in human tumor cell lines. Fn<em>1</em>4 was expressed in a variety of lines including breast, brain, bladder, skin, lung, ovarian, pancreatic, colon, prostate, and cervical cancer cell lines. We then developed an immunoconjugate containing a high-affinity anti-Fn<em>1</em>4 monoclonal antibody (ITEM-4) conjugated to recombinant gelonin (rGel), a highly cytotoxic ribosome-inactivating N-glycosidase. Both ITEM-4 and the conjugate were found to bind to cells to an equivalent extent. Confocal microscopic analysis showed that ITEM4-rGel specifically and rapidly (within 2 hours) internalized into Fn<em>1</em>4-positive T-24 bladder cancer cells but not into Fn<em>1</em>4-deficient mouse <em>embryonic</em> fibroblasts. Cytotoxicity studies against 22 different tumor cell lines showed that ITEM4-rGel was highly cytotoxic to Fn<em>1</em>4-expressing cells and was 8- to 8 × <em>1</em>0(4)-fold more potent than free rGel. ITEM4-rGel was found to kill cells by inducing apoptosis with high-mobility group box <em>1</em> protein release. Finally, ITEM4-rGel immunoconjugate administration promoted long-term tumor <em>growth</em> suppression in nude mice bearing T-24 human bladder cancer cell xenografts. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the <em>growth</em> of Fn<em>1</em>4-expressing tumors.

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse <em>embryonic</em> stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-<em>1</em>, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast <em>growth</em> <em>factor</em>-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and <em>differentiation</em> and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.

GH and PRL have been shown to stimulate proliferation and insulin production in islets of Langerhans. To identify genes regulated by GH/PRL in islets, we performed differential screening of a complementary DNA library from neonatal rat islets cultured for 24 h with human GH (hGH). One hGH-induced clone had 96% identity with mouse preadipocyte <em>factor</em>-<em>1</em> (Pref-<em>1</em>, or delta-like protein (Dlk)]. The size of Pref-<em>1</em> messenger RNA (mRNA) in islets was <em>1</em>.6 kilobases, with two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-<em>1</em> mRNA content of islets from adult rats was only <em>1</em>% of that in neonatal islets. Pref-<em>1</em> mRNA was markedly up-regulated in islets from pregnant rats from day <em>1</em>2 to term compared with those from age-matched female rats. Two peaks in mRNA expression were observed during gestation, one on day <em>1</em>4 and the other at term, whereafter it decreased to nonpregnant levels. Pref-<em>1</em> mRNA was up-regulated 3- to 4-fold in neonatal rat islets of Langerhans after 48-h culture with hGH, as found also with bovine GH or ovine PRL. During the development of pancreas from <em>embryonic</em> day <em>1</em>2 (E<em>1</em>2) to postnatal day 4, we observed a 2-fold increase in Pref-<em>1</em> mRNA on E<em>1</em>7 and a 5-fold increase at birth, followed by a rapid decline on postnatal day 4. Pref-<em>1</em> immunoreactivity was found in a subpopulation of insulin cells of neonatal islets of Langerhans. At an early embryonal stage (E<em>1</em>3), most cells of the pancreatic anlage were Pref-<em>1</em> positive, becoming predominantly restricted to the insulin-producing cells during development. In conclusion, these findings suggest that Pref-<em>1</em> is involved in both <em>differentiation</em> and <em>growth</em> of beta-cells.

<em>Embryonic</em> stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of <em>differentiation</em> in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-<em>1</em>-positive (Sca-<em>1</em>(+)) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC <em>differentiation</em>, Sca-<em>1</em>(+) progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in <em>differentiation</em> medium with or without platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB for 6-90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-<em>1</em>(+) progenitors with antibodies against integrin alpha(<em>1</em>), alpha(v), and beta(<em>1</em>), but not beta(3), inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC <em>differentiation</em>. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor-beta significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC <em>differentiation</em> and that integrin (alpha(<em>1</em>), beta(<em>1</em>), and alpha(v))-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor-beta signaling pathways are involved in SMC <em>differentiation</em>.

<em>Embryonic</em> stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-<em>1</em> and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific <em>embryonic</em> antigen-<em>1</em>, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than <em>1</em>7 passages, some ES-like cells differentiated to neural precursor cells in the presence of basic fibroblast <em>growth</em> <em>factor</em> (bFGF), epidermal <em>growth</em> <em>factor</em> and platelet-derived <em>growth</em> <em>factor</em>. We also developed a protocol that resulted in the <em>differentiation</em> of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell <em>factor</em> and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.

Beta transforming <em>growth</em> <em>factor</em> (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-<em>1</em> for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-<em>1</em> and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-<em>1</em> and TGF beta-2 inhibited cell proliferation in MvILu with no significant <em>differences</em> in the ED50 (3<em>1</em> +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-<em>1</em> in inhibiting DNA synthesis in AEC (63<em>1</em>0 +/- 985 pg/ml vs <em>1</em>0<em>1</em> +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-<em>1</em> and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs <em>1</em>0 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal <em>growth</em> <em>factor</em> (EGF), platelet derived <em>growth</em> <em>factor</em> (PDGF), acidic and basic fibroblast <em>growth</em> <em>factors</em> (FGF), skeletal <em>growth</em> <em>factor</em> (SGF)/insulin-like <em>growth</em> <em>factor</em>-II (IGF-II), and insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the <em>growth</em> <em>factors</em> were added to maximal inhibiting concentrations of TGF beta-<em>1</em>, both acidic and basic FGF significantly reduced TGF beta-<em>1</em> inhibition in AEC. We conclude that (<em>1</em>) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-<em>1</em>, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-<em>1</em> from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-<em>1</em> and TGF beta-2, and (4) both TGF beta-<em>1</em> and TGF beta-2 are potent mitogens for chicken <em>embryonic</em> calvarial bone cells.

Ewing's sarcoma is a highly malignant bone tumor found in children and adolescents, and the origin of this malignancy is not well understood. Here, we introduced a Ewing's sarcoma-associated genetic fusion of the genes encoding the RNA-binding protein EWS and the transcription <em>factor</em> ETS (EWS-ETS) into a fraction of cells enriched for osteochondrogenic progenitors derived from the <em>embryonic</em> superficial zone (eSZ) of long bones collected from late gestational murine embryos. EWS-ETS fusions efficiently induced Ewing's sarcoma-like small round cell sarcoma formation by these cells. Analysis of the eSZ revealed a fraction of a precursor cells that express <em>growth</em>/<em>differentiation</em> <em>factor</em> 5 (Gdf5), the transcription <em>factor</em> Erg, and parathyroid hormone-like hormone (Pthlh), and selection of the Pthlh-positive fraction alone further enhanced EWS-ETS-dependent tumor induction. Genes downstream of the EWS-ETS fusion protein were quite transcriptionally active in eSZ cells, especially in regions in which the chromatin structure of the ETS-responsive locus was open. Inhibition of β-catenin, poly (ADP-ribose) polymerase <em>1</em> (PARP<em>1</em>), or enhancer of zeste homolog 2 (EZH2) suppressed cell <em>growth</em> in a murine model of Ewing's sarcoma, suggesting the utility of the current system as a preclinical model. These results indicate that eSZ cells are highly enriched in precursors to Ewing's sarcoma and provide clues to the histogenesis of Ewing's sarcoma in bone.

Induced pluripotent stem cells (iPSCs) have generated hope and excitement because of the potential they possess for generating patient-specific <em>embryonic</em>-like stem cells (ESCs). Although many hurdles remain to be solved before the cells can be applied clinically; studies directed toward understanding <em>factors</em> that control <em>differentiation</em> of the cells toward various cell lineages are prerequisites for their future application. In the present study, we generated murine iPSC and assessed their <em>differentiation</em> toward osteogenic lineage. Murine tail tip fibroblasts were reprogrammed into <em>embryonic</em>-like state by transduction with defined <em>factors</em> (Oct3/4, Sox2, c-Myc, and klf4) carried in a retroviral vector. The reprogrammed cells expressed ESC markers, gave rise to three germ layers as demonstrated by teratoma formation and immunofluorescence staining. These data confirmed that the reprogrammed cells exhibited ESC-like state. Treatment of iPSCs-derived embryoid bodies (EBs) with transforming <em>growth</em> <em>factor</em> beta <em>1</em> (TGF-beta<em>1</em>) in the presence of retinoic acid enhanced generation of MSC-like cells. The MSCs-like cells expressed putative makers associated with MSCs; the cells deposited calcium in vitro when cultured in osteogenic medium. Interestingly MSCs-like cells generated from iPSC directed EBs by treatment with retinoic acid and TGF-beta<em>1</em> deposited more calcium in vitro than cells derived without TGF-beta<em>1</em> treatment. Taken together, the data demonstrate that iPSC give rise to MSCs-like state and that the cells have potential to differentiate toward osteoblasts. In addition, brief treatment of iPSC-derived EBs with TGF-beta<em>1</em> may be an approach for directing iPSC toward MSC-like state.

BACKGROUND

Pluripotent stem cells represent a powerful model system to study the early steps of cardiac specification for which the molecular control is largely unknown. The EGF-CFC (epidermal <em>growth</em> <em>factor</em>-Cripto/FRL-<em>1</em>/Cryptic) Cripto protein is essential for cardiac myogenesis in <em>embryonic</em> stem cells (ESCs).

OBJECTIVE

Here, we study the role of apelin and its G protein-coupled receptor, APJ, as downstream targets of Cripto both in vivo and in ESC differentiation.

RESULTS

Gain-of-function experiments show that APJ suppresses neuronal differentiation and restores the cardiac program in Cripto(-/-) ESCs. Loss-of-function experiments point for a central role for APJ/apelin in the gene regulatory cascade promoting cardiac specification and differentiation in ESCs. Remarkably, we show for the first time that apelin promotes mammalian cardiomyogenesis via activation of mitogen-activated protein kinase/p70S6 through coupling to a Go/Gi protein.

CONCLUSIONS

Together our data provide evidence for a previously unrecognized function of APJ/apelin in the Cripto signaling pathway governing mesoderm patterning and cardiac specification in mammals.

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell <em>differentiation</em> and islet development while islet cell-derived angiogenic <em>factors</em> promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic <em>factors</em> in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic <em>factor</em> transgene) to increase the β cell production of vascular endothelial <em>growth</em> <em>factor</em>-A (VEGF-A), angiopoietin-<em>1</em> (Ang<em>1</em>), or angiopoietin-2 (Ang2) during islet cell <em>differentiation</em> and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as <em>embryonic</em> day <em>1</em>2.5 (E<em>1</em>2.5) and increased dramatically by E<em>1</em>6.5. While α and β cells formed islet cell clusters in control embryos at E<em>1</em>6.5, the increased EC population perturbed endocrine cell <em>differentiation</em> and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day <em>1</em>. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang<em>1</em> or Ang2 in β cells in developing or adult islets did not alter islet <em>differentiation</em>, development, or morphology, but altered islet EC ultrastructure. These data indicate that (<em>1</em>) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.

The importance of canonical transforming <em>growth</em> <em>factor</em> beta (TGF-beta) and bone morphogenetic protein (BMP) signaling during cartilage and joint development is well established, but the necessity for noncanonical (SMAD-independent) signaling during these processes is largely unknown. TGF-beta activated kinase <em>1</em> (TAK<em>1</em>) is a MAP3K activated by TGF-beta, BMP, and other mitogen-activated protein kinase (MAPK) signaling components. We set out to define the potential role for noncanonical, TAK<em>1</em>-mediated signaling in cartilage and joint development via deletion of Tak<em>1</em> in chondrocytes (Col2Cre;Tak<em>1</em>(f/f)) and the developing limb mesenchyme (Prx<em>1</em>Cre;Tak<em>1</em>(f/f)). Deletion of Tak<em>1</em> in chondrocytes resulted in novel <em>embryonic</em> developmental cartilage defects including decreased chondrocyte proliferation, reduced proliferating chondrocyte survival, delayed onset of hypertrophy, reduced Mmp<em>1</em>3 expression, and a failure to maintain interzone cells of the elbow joint, which were not observed previously in another Col2Cre;Tak<em>1</em>(f/f) model. Deletion of Tak<em>1</em> in limb mesenchyme resulted in widespread joint fusions likely owing to the <em>differentiation</em> of interzone cells to the chondrocyte lineage. The Prx<em>1</em>Cre;Tak<em>1</em>(f/f) model also allowed us to identify novel columnar chondrocyte organization and terminal maturation defects owing to the interplay between chondrocytes and the surrounding mesenchyme. Furthermore, both our in vivo models and in vitro cell culture studies demonstrate that loss of Tak<em>1</em> results in impaired activation of the downstream MAPK target p38, as well as diminished activation of the BMP/SMAD signaling pathway. Taken together, these data demonstrate that TAK<em>1</em> is a critical regulator of both MAPK and BMP signaling and is necessary for proper cartilage and joint development.

Recent studies have suggested that docosahexaenoic acid (DHA) enhances neuronal <em>differentiation</em> of neural stem cells (NSCs) isolated from rat <em>embryonic</em> day <em>1</em>4.5. However the underlying mechanism remains largely unknown. One hypothesis supported by DHA controls the expression level of basic helix-loop-helix (bHLH) transcription <em>factors</em>, such as hairy and enhancer of split <em>1</em> (Hes<em>1</em>), Mash<em>1</em>, neurogenin<em>1</em>, and NeuroD; another is that previous studies in retinal progenitor cells DHA affects the cell cycle. In this study, we show that treatment with DHA under <em>differentiation</em> conditions without basic fibroblast <em>growth</em> <em>factor</em>, (<em>1</em>) increases Tuj-<em>1</em> and MAP2 positive cells in NSCs, (2) that the expression level of Hes<em>1</em> mRNA and protein decreased significantly from day <em>1</em> to day 4, on the other hand, the NeuroD mRNA expression level increased from day <em>1</em> to day 4 after treatment with DHA and (3) decreased the percentage of S-phase cells, which correlated with prolonged expression of cyclin-dependent kinase inhibitor p27(kip<em>1</em>), suggesting that DHA enhances neuronal <em>differentiation</em> of NSCs, in part, by controlling the bHLH transcription <em>factors</em> and promoting cell cycle exit. We therefore speculate that DHA is one of the essential key molecules for neuronal <em>differentiation</em> of NSCs.

This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small <em>embryonic</em>-like stem cells (VSELs) that were SCA-<em>1</em>+/Lin-/CD45-, positive for nuclear octamer-binding transforming <em>factor</em> 4 (OCT-4), Nanog, and cell surface stage-specific <em>embryonic</em> antigen <em>1</em>, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/<em>growth</em> <em>differentiation</em> <em>factor</em> 9-positive oocyte-like structure were observed by day 6, probably arising as a result of <em>differentiation</em> of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and <em>differentiation</em> into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific <em>differentiation</em>. These results show relevance to women who undergo premature ovarian failure because of oncotherapy.

We show that alpha 6 integrin function was required for normal lens cell <em>differentiation</em> by using an antisense construct to suppress alpha 6 integrin expression. To elucidate the mechanism by which this integrin functions in the regulation of the lens cell <em>differentiation</em> process, we determined the molecular composition of alpha 6 integrin signaling complexes at distinct stages of <em>differentiation</em> in vivo. Because both alpha 6 integrin and insulin-like <em>growth</em> <em>factor</em>-<em>1</em> (IGF-<em>1</em>) have been implicated in signaling lens cell <em>differentiation</em>, we examined the possibility that they formed a signaling complex in the <em>embryonic</em> lens. Coprecipitation analysis revealed that alpha 6 integrin/IGF-<em>1</em> receptor complexes were present and that their association was greatest in the equatorial zone, the region of the <em>embryonic</em> lens in which lens cells proliferate and then initiate their <em>differentiation</em>. These results provide in vivo support for the formation of integrin/<em>growth</em> <em>factor</em> receptor signaling complexes. We also found that extracellular signal-regulated kinase (ERK), a downstream effector of both integrin and <em>growth</em> <em>factor</em> receptor signaling pathways, was associated with the alpha 6 integrin signaling complexes in the <em>embryonic</em> lens. This result was supported by our findings that activated ERK, in addition to its nuclear location, localized to lens cell membranes in specific regions of cell-matrix and cell-cell contact. A connection between integrin ligand engagement and ERK activation was shown in vitro after lens cell attachment to laminin. These results demonstrate that alpha 6 integrin function is required for the early stages of lens cell <em>differentiation</em> most likely through its association with the IGF-<em>1</em> receptor and the activation of ERK.

Morphogenesis is regulated by intrinsic <em>factors</em> within cells and by inductive signals transmitted through direct contact, diffusible molecules, and gap junctions. In addition, connected tissues <em>growing</em> at different rates necessarily generate complicated distributions of physical deformations (strains) and pressures. In this Perspective we present the hypothesis that <em>growth</em>-generated strains and pressures in developing tissues regulate morphogenesis throughout development. We propose that these local mechanical cues influence morphogenesis by: (<em>1</em>) modulating <em>growth</em> rates; (2) modulating tissue <em>differentiation</em>; (3) influencing the direction of <em>growth</em>; and (4) deforming tissues. It is in this context that we review concepts and experiments of cell signaling and gene expression in various mechanical environments. Tissue and organ culture experiments are interpreted in light of the developmental events associated with the <em>growth</em> of the limb buds and provide initial support for the presence and morphological importance of <em>growth</em>-generated strains and pressures. The concepts presented are used to suggest future lines of research that may give rise to a more integrated mechanobiological view of early <em>embryonic</em> musculoskeletal morphogenesis.

We previously characterized a genetically engineered mouse astrocytoma model with <em>embryonic</em> astrocyte-specific, activated (<em>1</em>2)V-Ha-RAS (GFAP-RAS) transgenesis. The GFAP-RAS line Ras-B8 appears normal at birth, but 50% of mice die by 4 months from low- and high-grade astrocytomas. We examined the development and progression of astrocytomas in the Ras-B8 genetically engineered mouse. At <em>embryonic</em> day <em>1</em>6.5 (E<em>1</em>6.5), there were no pathological <em>differences</em> compared to control littermates, aside from transgene expression. Diffuse astroglial hyperplasia was the first distinguishing feature in the <em>1</em>-week-old Ras-B8 mice; however, these astrocytes were not transformed in vitro or in vivo. From 3 to 8 weeks the incidence of low-grade astrocytomas progressively increased with 85% of <em>1</em>2-week-old mice harboring low- or high-grade astrocytomas, the latter characterized by increased proliferation, nuclear atypia, and angiogenesis. Tp 53 mutations were detected in both astrocytoma grades, with high-grade astrocytomas expressing elevated levels of epidermal <em>growth</em> <em>factor</em> receptor and vascular endothelial <em>growth</em> <em>factor</em>, plus decreased levels of PTEN and p<em>1</em>6, similar to human astrocytomas. We postulate that expression of (<em>1</em>2)V-Ha-RAS in astroglial precursors induces astroglial hyperplasia, but transformation and subsequent progression requires additional molecular alterations resulting from aberrant activated p2<em>1</em>-RAS. Of interest, many of these acquired alterations occur in human astrocytomas, further validating GFAP-RAS as a useful model for studying astrocytoma development and progression.

Chicken NF-M transcription <em>factor</em>, in cooperation with either c-Myb or v-Myb, is active in the combinatorial activation of myeloid-cell-specific genes in heterologous cell types, such as <em>embryonic</em> fibroblasts. In humans, similar effects were observed with homologous members of the CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators, especially the human homolog of chicken NF-M, C/EBP-beta (NF-IL6). However, the NF-IL6 gene is expressed in a variety of nonmyeloid cell types and is strongly inducible in response to inflammatory stimuli, making it an unlikely candidate to have an exclusive role as a combinatorial <em>differentiation</em> switch during myelopoiesis in human cells. By using a reverse transcription-PCR-based approach and a set of primers specific for the DNA-binding domains of highly homologous members of the C/EBP family of transcriptional regulators, we have cloned a novel human gene encoding a member of the C/EBP gene family, identified as the human homolog of CRP<em>1</em>, C/EBP-epsilon. A <em>1</em>.2-kb cDNA encoding full-length human C/EBP-epsilon was cloned from a promyelocyte-late myeloblast-derived lambda gt<em>1</em><em>1</em> library. Molecular analysis of the cDNA and genomic clones indicated the presence of two exons encoding a protein with an apparent molecular mass of 32 kDa and a pI of 9.5. Primer extension analysis of C/EBP-epsilon mRNA detected a single major transcription start site approximately 200 bp upstream of the start codon. The putative promoter area is similar to those of several other myeloid-cell-specific genes in that it contains no TATAAA box but has a number of purine-rich stretches with multiple sites for the <em>factors</em> of the Ets family of transcriptional regulators. Northern blot analyses indicated a highly restricted mRNA expression pattern, with the strongest expression occurring in promyelocyte and late-myeloblast-like cell lines. Western blot and immunoprecipitation studies using rabbit anti-C/EBP-epsilon antibodies raised against the N-terminal portion of C/EBP-epsilon (amino acids <em>1</em> to <em>1</em><em>1</em>5) showed that C/EBP-epsilon is a 32-kDa nuclear phosphoprotein. The human C/EBP-epsilon protein exhibited strong and specific binding to double-stranded DNA containing consensus C/EBP sites. Cotransfection of the C/EBP-epsilon sense and antisense expression constructs together with chloramphenicol acetyltransferase reporter vectors containing myeloid-cell-specific c-mim and human myeloperoxidase promoters suggested a role for C/EBP-epsilon transcription <em>factor</em> in the regulation of a subset of myeloid-cell-specific genes. Transient tranfection of a promyelocyte cell line (NB4) with a C/EBP-epsilon expression plasmid increased cell <em>growth</em> by sevenfold, while antisense C/EBP-epsilon caused a fivefold decrease in clonal <em>growth</em> of these cells.

Stromal cell-derived <em>factor</em> (SDF)-<em>1</em>/CXCL<em>1</em>2, released by murine <em>embryonic</em> stem (ES) cells, enhances survival, chemotaxis, and hematopoietic <em>differentiation</em> of murine ES cells. Conditioned medium (CM) from murine ES cells <em>growing</em> in the presence of leukemia inhibitory <em>factor</em> (LIF) was generated while the ES cells were in an undifferentiated Oct-4 expressing state. ES cell-CM enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed <em>growth</em> <em>factor</em> addition in vitro and decreased apoptosis of murine bone marrow c-kit(+)lin- cells. ES CM contained interleukin (IL)-<em>1</em>alpha, IL-<em>1</em>0, IL-<em>1</em><em>1</em>, macrophage-colony stimulating <em>factor</em> (CSF), oncostatin M, stem cell <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em>, as well as a number of chemokines and other proteins, some of which are known to enhance survival/anti-apoptosis of progenitors. Irradiation of ES cells enhanced release of some proteins and decreased release of others. IL-6, FGF-9, and TNF-alpha, not detected prior to irradiation was found after ES cells were irradiated. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells <em>growing</em> in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other <em>growth</em> modulatory proteins, results which may be of physiological and/or practical significance.

The highly conserved Wnt genes belong to a widely distributed family of presumptive signaling molecules that have been implicated not only in the regulation of normal pattern formation during embryogenesis and <em>differentiation</em> of cell lineages, but also in oncogenic events. All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted <em>growth</em> <em>factors</em>: a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In this study, we report the cloning and sequencing of several overlapping cDNAs encoding approximately 4.<em>1</em> kb of the human homologue of Wnt-5A. The mature protein contained 343 residues (M(r) approximately 38,000 excluding any post-translational modifications) with a>> 93% homology to the reported sequences of other Wnt-5A proteins >> 99% homologous to mouse Wnt-5A). This protein maintained certain features--a hydrophobic signal sequence, the Wnt-<em>1</em> family "signature sequence" (CKCHGvSGSC), and a number of other conserved amino acid residues: 24 cysteine residues, 4 asparagine-linked oligosaccharide consensus sequences, and a tyrosine sulfation site--that have been found in all other Wnt-5A proteins. Reverse transcriptase PCR analysis of RNA from a variety of human <em>embryonic</em>, neonatal, and adult cells and/or tissues showed that human Wnt-5A expression was detected only in neonatal heart and lung. It may be relevant, however, that the 3'-untranslated region contained numerous AT-rich motifs that could be involved in the rapid degradation of mRNA. Finally, using a combination of Southern blotting, PCR amplification, and in situ hybridization, the human Wnt-5A (WNT5A) gene was mapped to chromosome 3p<em>1</em>4-p2<em>1</em>.