Osteoporosis, a disorder of skeletal fragility, is common in the elderly, and its prevalence is increasing as more individuals with low bone mineral density (BMD), the strongest predictor of fracture risk, are detected. Previous basic and clinical studies imply there is a significant role for insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) in determining BMD. Recently, polymorphisms upstream of the P<em>1</em> promoter region of the human IGF-I gene have been found to be associated with serum levels of IGF-I, BMD and fracture risk in various ethnic groups. Multiple quantitative trait loci (QTLs) have been identified that underlie serum IGF-I in a mouse intercross between two inbred strains. The most promising QTL on mouse chromosome 6 has provided clues for unraveling the molecular mechanisms that regulate osteoblast <em>differentiation</em>. Genomic engineering resulting in IGF-I deficient mice, and mice with targeted over-expression of IGF-I reinforce the essential role of IGF-I in bone development at both the <em>embryonic</em> and postnatal stages. Thus, it is apparent that significant new insights into the role of the IGF-I gene in bone remodeling occur through several distinct mechanisms: (<em>1</em>) the skeletal IGF regulatory system; (2) the systemic <em>growth</em> hormone/IGF-I axis; (3) parathyroid hormone signaling; (4) sex steroids; and (5) the OPG/RANKL/RANK cytokine system. Molecular dissection of the IGF regulatory system and its signaling pathway in bone may reveal novel therapeutic targets for the treatment of osteoporosis.

Aberrant cell proliferation and <em>differentiation</em> after toxic injury to airway epithelium can lead to the development of various lung diseases including cancer. The activator protein-<em>1</em> (AP-<em>1</em>) transcription <em>factor</em>, composed of mainly Jun-Jun and Jun-Fos protein dimers, acts as an environmental biosensor to various external toxic stimuli and regulates gene expression involved in various biological processes. Gene disruption studies indicate that the AP-<em>1</em> family members c-jun, junB, and fra<em>1</em> are essential for <em>embryonic</em> development, whereas junD, c-fos, and fosB are required for normal postnatal <em>growth</em>. However, broad or target-specific transgenic overexpression of the some of these proteins gives very distinct phenotype(s), including tumor formation. This implies that, although they are required for normal cellular processes, their abnormal activation after toxic injury can lead to the pathogenesis of the lung disease. Consistent with this view, various environmental toxicants and carcinogens differentially regulate Jun and Fos expression in cells of the lung both in vivo and in vitro. Moreover, Jun and Fos proteins distinctly bind to the promoter regions of a wide variety of genes to differentially regulate their expression in epithelial injury, repair, and <em>differentiation</em>. Importantly, lung tumors induced by various carcinogens display a sustained expression of certain AP-<em>1</em> family members. Therefore a better understanding of the mechanisms of regulation and functional role(s), as well as identification of target genes of members of the AP-<em>1</em> family in airway epithelial cells, will provide additional insight into toxicant-induced lung diseases. These studies might offer a unique opportunity to use AP-<em>1</em> family members and transactivation as potential diagnostic markers or drug targets for early detection and/or prevention of various lung diseases.

Cellular resistance to multiple proapoptotic stimuli and invasion of surrounding brain tissue by migrating tumor cells are main obstacles to an effective therapy for human malignant glioma. Here, we report that the Wnt family of <em>embryonic</em> <em>differentiation</em> genes modulate <em>growth</em> of malignant glioma cells in vitro and in vivo and inhibit cellular migration in vitro. sFRPs (soluble Frizzled-related proteins) are soluble proteins that bind to Wnt and interfere with Wnt signaling. We find that sFRP-<em>1</em> and sFRP-2 are produced by the majority of longterm and ex vivo malignant glioma cell lines. Glioma cells that ectopically express sFRPs exhibit increased clonogenicity and enhanced resistance to serum starvation. In contrast, sFRPs do not modulate glioma cell susceptibility to apoptosis induced by the cytotoxic cytokines, CD95 (Fas/APO-<em>1</em>) ligand (CD95L) or Apo2 ligand/tumor necrosis <em>factor</em>-related apoptosis-inducing ligand (Apo2L/TRAIL), or various cytotoxic drugs. sFRP-2 strongly promotes the <em>growth</em> of intracranial glioma xenografts in nude mice. In contrast, enhanced expression of sFRPs inhibits the motility of glioma cells in vitro. sFRP-mediated effects on glioma cells are accompanied by decreased expression and activity of matrix metalloproteinase-2 (MMP-2) and decreased tyrosine phosphorylation of beta-catenin. Thus, sFRPs promote survival under non-supportive conditions and inhibit the migration of glioma cells. We suggest that the regulation of these cellular processes involves expression of MMP-2 and tyrosine phosphorylation of beta-catenin. These data support a function for Wnt signaling and its modulation by sFRPs in the biology of human gliomas. Oncogene (2000) <em>1</em>9, 42<em>1</em>0 - 4220

In multiple sclerosis lesions resident oligodendrocyte progenitor cells (OPCs) are present, but fail to remyelinate. In the current study we examined whether neural precursor cell (NPC) transplantation can facilitate host brain-derived remyelination. We used the chronic cuprizone-induced demyelination model in aged mice, in which slow remyelination follows cuprizone removal. NPCs were transplanted to the lateral ventricles (intracerebroventricular) of cuprizone-induced demyelinated brains. In this experimental setup, transplanted cells remained mostly in the periventricular area in an undifferentiated state. The extent of demyelination, remyelination, and proliferation of host brain regenerative cell population were examined at <em>1</em> week posttransplantation in the splenium of the corpus callosum, which was devoid of any transplanted cells. Transplantation of NPCs, but not of control, human <em>embryonic</em> kidney cells, significantly enhanced remyelination compared with sham-operated mice. Remyelination was performed exclusively by host brain OPCs. The proregenerative effect of transplanted NPCs was related to an increase in the proliferation of host brain OPCs. To examine the mechanism that underlies the proregenerative effect of NPCs in vitro, we used an NPC-OPC coculture system. These experiments indicated that NPCs induced the proliferation of OPCs and facilitated their <em>differentiation</em> into mature oligodendrocytes. The mitogenic effect of NPCs was mediated by platelet-derived <em>growth</em> <em>factor</em>-AA and fibroblast <em>growth</em> <em>factor</em>-2. In conclusion, NPC transplantation enhances host-derived myelin regeneration following chronic demyelination. This trophic effect may stimulate resident OPCs to overcome the remyelination failure in multiple sclerosis.

Insulin-like <em>growth</em> <em>factor</em>-<em>1</em> (IGF-<em>1</em>) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse <em>embryonic</em> stem cell (ESC) suspension with IGF-<em>1</em> might enhance cellular engraftment and host organ-specific <em>differentiation</em> after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF-<em>1</em> to the injected cell population. Green fluorescent protein (GFP)-labeled sv<em>1</em>29 ESCs (2.5 x <em>1</em>0(5)) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF-<em>1</em> (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF-<em>1</em> (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF-<em>1</em> group (p = .025). There was a trend toward higher fractional shortening in the IGF-treated group. Histological analysis demonstrated that IGF-<em>1</em> promoted expression of alpha-sarcomeric actin (p = .0<em>1</em>5) and major histocompatibility complex class I (p = .0<em>1</em>). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF-<em>1</em> promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

The transcriptional intermediary <em>factor</em> <em>1</em> (TIF<em>1</em>) family protein TIF<em>1</em>beta is a corepressor for Krüppel-associated box (KRAB)-domain-containing zinc finger proteins and plays a critical role in early embryogenesis. Here, we examined TIF<em>1</em>beta distribution in the nucleus of mouse <em>embryonic</em> carcinoma F9 cells during retinoic-acid-induced primitive endodermal <em>differentiation</em>. Using confocal immunofluorescence microscopy, we show that, although TIF<em>1</em>beta is diffusely distributed throughout the nucleoplasm of undifferentiated cells, it relocates and concentrates into distinct foci of centromeric heterochromatin in differentiated cells characterized by a low proliferation rate and a well developed cytokeratin network. This relocation was not observed in isoleucine-deprived cells, which are <em>growth</em> arrested, or in compound RXR alpha(-/-)/RAR gamma(-/-) null mutant cells, which are resistant to RA-induced <em>differentiation</em>. Amino-acid substitutions in the PxVxL motif of TIF<em>1</em>beta, which abolish interaction with members of the heterochromatin protein <em>1</em> (HP<em>1</em>) family, prevent its centromeric localization in differentiated cells. Collectively, these data provide compelling evidence for a dynamic nuclear compartmentalization of TIF<em>1</em>beta that is regulated during cell <em>differentiation</em> through a mechanism that requires HP<em>1</em> interaction.

<em>Embryonic</em> stem (ES) cells have the capacity to differentiate into all cells of the developing embryo and may provide a renewable resource for future cell replacement therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ES cell culture has previously been shown to induce transcription <em>factors</em>, signaling molecules, and cell adhesion proteins expressed during mesoderm specification of the embryo. Here, we show the dynamics of primitive streak mesoderm <em>differentiation</em> in ES cells is comparable between serum and serum-free embryoid body (EB) cultures, supplemented with BMP4. Furthermore, we show a delayed wave of expression of a cohort of genes (Pax2, WT<em>1</em>, podocalyxin, pod-<em>1</em>, and nephrin), which play important roles during <em>embryonic</em> kidney development. The paired box transcription <em>factor</em>, Pax2, is one of the earliest genes expressed during kidney organogenesis and is required for normal urogenital development. ES cell lines containing either a modified Pax2 promoter-lacZ or bacterial artificial chromosome-green fluorescent protein (GFP) transgene were generated, which enabled the quantitative analysis of kidney rather than neuronal Pax2 expression within EBs. Both beta-galactosidase activity and GFP expression were detected by immunohistochemical and flow cytometric analysis following <em>1</em>6 days of EB culture, which correlated with an increase in Pax2 transcript levels. Together, these results suggest a spontaneous kidney gene expression program develops in mature EBs grown in both serum and serum-free conditions, when supplemented with BMP4. Further, the recombinant <em>growth</em> <em>factors</em> BMP2, BMP4, and BMP7 strongly influence gene expression within mesoderm induced EBs. BMP4 promotes ventral (blood) and intermediate (kidney) mesoderm gene expression, whereas BMP2 and BMP7 promote kidney outcomes at the expense of hematopoietic commitment. This induction assay and these unique ES cell lines will be useful for the generation of mesoderm-derived cell populations with implications for future cell therapeutic/integration assays.

Fibroblast <em>growth</em> <em>factors</em> (FGFs) are structurally related mitogens that can regulate the <em>differentiation</em> of a wide variety of cells. As a step towards elucidating the developmental roles played by one of these <em>factors</em>, we have used in situ hybridization methods to examine expression of the murine F gf-5 gene during embryogenesis. F gf-5 RNA was detected at seven distinct sites in the developing mouse embryo: (<em>1</em>) postimplantation epiblast (<em>embryonic</em> day 5 <em>1</em>/4-7 <em>1</em>/2), (2) lateral splanchnic mesoderm (E9 <em>1</em>/2-<em>1</em>0 <em>1</em>/2), (3) lateral somatic mesoderm (E<em>1</em>0 <em>1</em>/2-<em>1</em>2 <em>1</em>/2), (4) myotomes (E<em>1</em>0 <em>1</em>/2-<em>1</em>2 <em>1</em>/2), (5) mastication muscle (E<em>1</em><em>1</em> <em>1</em>/2-<em>1</em>4 <em>1</em>/2), (6) limb mesenchyme (E<em>1</em>2 <em>1</em>/2-<em>1</em>4 <em>1</em>/2), and (7) acoustic ganglion (E<em>1</em>2 <em>1</em>/2-<em>1</em>4 <em>1</em>/2). At several of these sites, expression is spatially restricted within the tissues. We offer several hypotheses regarding the roles of FGF-5 in murine development.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was expressed in developing respiratory epithelial cells under control of the promoter from the human surfactant protein C (SP-C) gene. SP-C-VEGF transgenic mice did not survive after birth. When obtained by hysterectomy on <em>embryonic</em> day <em>1</em>5 (E<em>1</em>5) or <em>1</em>7 (E<em>1</em>7), abnormalities in the transgenic mice were confined to the lung and were correlated with the expression of transgene mRNA as revealed by in situ hybridization. On E<em>1</em>5 and E<em>1</em>7, marked abnormalities in lung morphogenesis were observed in transgenic mice. Lungs consisted of large dilated tubules with increased peritubular vascularity. The mRNA levels of the VEGF receptor, Flk-<em>1</em>, and the endothelial cell specific receptor tyrosine kinase, Tie-<em>1</em>, were increased in lung mesenchyme of the transgenic mice. The numbers of acinar tubules and the abundance of mesenchyme were decreased. Endogenous VEGF mRNA was expressed in the respiratory epithelial cells of the developing lungs, and the levels of VEGF mRNA were increased in the SP-C-VEGF transgenic mice. Although the normal pattern of immunostaining for SP-C and Clara cell secretory protein (CCSP) indicated that epithelial cell <em>differentiation</em> was relatively unaltered by the transgene, electron microscopic analysis revealed a lack of alveolar Type I cell <em>differentiation</em> at E<em>1</em>8. Expression of VEGF in the developing respiratory epithelium of transgenic mice increased <em>growth</em> of the pulmonary blood vessels, disrupted branching morphogenesis of the lung and inhibited Type I cell <em>differentiation</em>.

<em>Growth</em> <em>factor</em> independence-<em>1</em>B (Gfi-<em>1</em>B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI<em>1</em>B in mice leads to <em>embryonic</em> lethality resulting from failure to produce definitive erythrocytes, hindering the study of Gfi-<em>1</em>B function in adult hematopoiesis. We here show that, in humans, Gfi-<em>1</em>B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and <em>differentiation</em> of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming <em>growth</em> <em>factor</em>-beta receptor gene, TGFBR3, as a direct target of Gfi-<em>1</em>B. Knockdown of Gfi-<em>1</em>B results in altered transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary <em>factor</em> <em>1</em>-gamma (TIF<em>1</em>-gamma). Because the Smad2/TIF<em>1</em>-gamma complex is known to specifically regulate erythroid <em>differentiation</em>, we propose that, by repressing TGF-beta type III receptor (TbetaRIotaII) expression, Gfi-<em>1</em>B favors the Smad2/TIF<em>1</em>-gamma interaction downstream of TGF-beta signaling, allowing immature progenitors to differentiate toward the erythroid lineage.

Neuregulin-<em>1</em> (NRG-<em>1</em>) <em>growth</em> and <em>differentiation</em> <em>factors</em> and their erbB receptors are hypothesized to promote <em>embryonic</em> hippocampal neuron <em>differentiation</em> via as yet unknown mechanisms. We have found that NRG-<em>1</em>beta increases the out<em>growth</em> of primary neurites, neuronal area, total neurite length, and neuritic branching in E<em>1</em>8 hippocampal neurons. NRG-<em>1</em>beta effects on neurite extension and arborization are similar to, but not additive with, those of brain-derived neurotrophic <em>factor</em> and reflect direct NRG-<em>1</em> action on hippocampal neurons as these cells express the NRG-<em>1</em> receptors erbB2 and erbB4, the erbB-specific inhibitor PD<em>1</em>58780 decreases NRG-<em>1</em>beta induced neurite out<em>growth</em>, and NRG-<em>1</em>beta stimulation induces p42/44 ERK phosphorylation. Pharmacological inhibition of p42/44 ERK and protein kinase C (PKC), but not PI3K or p38 MAP kinase, inhibits NRG-<em>1</em>beta-induced neurite extension and elaboration. We conclude that NRG-<em>1</em>beta stimulates hippocampal neurite extension and arborization via a signaling pathway that involves erbB membrane tyrosine kinases (erbB2 and/or erbB4), p42/44 ERK, and PKC.

The 90-kDa ribosomal S6 kinases (RSK<em>1</em>-3) are important mediators of <em>growth</em> <em>factor</em> stimulation of cellular proliferation, survival, and <em>differentiation</em> and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-<em>1</em> (PDK<em>1</em>). Here we performed the functional characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for <em>growth</em> <em>factor</em> stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372, and Ser389, and the low basal ERK activity in serum-starved cells appeared to be sufficient for induction of approximately 50% of the constitutive RSK4 activity. Finally experiments in mouse <em>embryonic</em> stem cells with targeted deletion of the PDK<em>1</em> gene suggested that PDK<em>1</em> was not required for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK<em>1</em>. The unusual regulation and <em>growth</em> <em>factor</em>-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent findings suggesting that RSK4 can participate in non-<em>growth</em> <em>factor</em> signaling as for instance p53-induced <em>growth</em> arrest.

Human ES (hES) cell lines have only recently been generated, and <em>differences</em> between human and mouse ES cells have been identified. In this manuscript we describe the properties of two human ES cell lines, BG0<em>1</em> and BG02. By immunocytochemistry and reverse transcription polymerase chain reaction, undifferentiated cells expressed markers that are characteristic of ES cells, including SSEA-3, SSEA-4, TRA-<em>1</em>-60, TRA-<em>1</em>-8<em>1</em>, and OCT-3/4. Both cell lines were readily maintained in an undifferentiated state and could differentiate into cells of all three germ layers, as determined by expression of beta-tubulin III neuron-specific molecule (ectoderm), cardiac troponin I (cardiomyocytes, mesoderm), and alpha-fetoprotein (endoderm). A large-scale microarray (<em>1</em>6,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG0<em>1</em> and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE0<em>1</em>, GE09, and pooled samples derived from GE0<em>1</em>, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming <em>growth</em> <em>factor</em>-beta superfamily were employed to examine differentially expressed genes. Several <em>growth</em> <em>factors</em>, receptors, and components of signaling pathways that regulate <em>embryonic</em> development, in particular the nodal signaling pathway, were detected in both BG0<em>1</em> and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG0<em>1</em> and BG02 human ES cell lines.

OBJECTIVE

To examine the potential of NIH-maintained human <em>embryonic</em> stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-<em>1</em>/IGF-<em>1</em>/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression.

METHODS

hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs). Wnt (Integration <em>1</em> and wingless) blocking morphogens Dickkopf-<em>1</em> (Dkk-<em>1</em>) and Insulin-like growth factor <em>1</em> (IGF-<em>1</em>) were used to direct NPs to a rostral neural fate, and fibroblast growth factor 9 (FGF9)/fibroblast growth factor-basic (bFGF) were added to bias the differentiation of developing anterior neuroectoderm cells to neural retina (NR) rather than retinal pigment epithelium (RPE). Cells were dissociated and grafted into the subretinal and epiretinal space of young adult (4-6-week-old) mice (C57BL/6J x<em>1</em>29/Sv mixed background). Remaining cells were replated for (i) immunocytochemical analysis and (ii) used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Mice were sacrificed 3 weeks or 3 months after grafting, and the grafts were examined by histology and immunohistochemistry for survival of hESC-RPCs, presence of mature neuronal and retinal markers, and the dynamics of in vivo maturation and integration into the host retina.

RESULTS

At the time of grafting, hESC-RPCs exhibited immature neural/neuronal immunophenotypes represented by nestin and neuronal class III β-tubulin, with about half of the cells positive for cell proliferation marker Kiel University -raised antibody number 67 (Ki67), and no recoverin-positive (recoverin [+]) cells. The grafted cells expressed eye field markers paired box 6 (PAX6), retina and anterior neural fold homeobox (RAX), sine oculis homeobox homolog 6 (SIX6), LIM homeobox 2 (LHX2), early NR markers (Ceh-<em>1</em>0 homeodomain containing homolog [CHX<em>1</em>0], achaete-scute complex homolog <em>1</em> [MASH<em>1</em>], mouse atonal homolog 5 [MATH5], neurogenic differentiation <em>1</em> [NEUROD<em>1</em>]), and some retinal cell fate markers (brain-specific homeobox/POU domain transcription factor 3B [BRN3B], prospero homeobox <em>1</em> [PROX<em>1</em>], and recoverin). The cells in the subretinal grafts matured to predominantly recoverin [+] phenotype by 3 months and survived in a xenogenic environment without immunosuppression as long as the blood-retinal barrier was not breached by the transplantation procedure. The epiretinal grafts survived but did not express markers of mature retinal cells. Retinal integration into the retinal ganglion cell (RGC) layer and the inner nuclear layer (INL) was efficient from the epiretinal but not subretinal grafts. The subretinal grafts showed limited ability to structurally integrate into the host retina and only in cases when NR was damaged during grafting. Only limited synaptogenesis and no tumorigenicity was observed in grafts.

CONCLUSIONS

Our studies show that (i) immunosuppression is not mandatory to xenogenic graft survival in the retina, (ii) the subretinal but not the epiretinal niche can promote maturation of hESC-RPCs to photoreceptors, and (iii) the hESC-RPCs from epiretinal but not subretinal grafts can efficiently integrate into the RGC layer and INL. The latter could be of value for long-lasting neuroprotection of retina in some degenerative conditions and glaucoma. Overall, our results provide new insights into the technical aspects associated with cell-based therapy in the retina.

The pituitary gland represents the endocrine core, governing the body's hormonal landscape by adapting its cellular composition to changing demands. It is assumed that stem/progenitor cells are involved in this remodeling. Recently, we uncovered a candidate stem/progenitor cell population in the anterior pituitary. Here, we scrutinized this "side population" (SP) and show that, unexpectedly, not the subset expressing high levels of "stem cell antigen-<em>1</em>" (Sca<em>1</em>(high)) but the remainder non-Sca<em>1</em>(high) fraction clusters the pituitary progenitor cells. Transcriptomal interrogation revealed in the non-Sca<em>1</em>(high) SP upregulated expression of the pituitary stem/progenitor cell markers Sox2 and Sox9, and of multiple <em>factors</em> critically involved in pituitary embryogenesis. The non-Sca<em>1</em>(high) SP encloses the cells that generate spheres and display multipotent hormone <em>differentiation</em> capacity. In culture conditions selecting for the non-Sca<em>1</em>(high) subset within the SP, stem cell <em>growth</em> <em>factors</em> that induce SP expansion, affect transcription of <em>embryonic</em> <em>factors</em>, suggesting impact on a developmental program that unfolds within this SP compartment. Non-Sca<em>1</em>(high) SP cells, revealed by Sox2 expression, are observed in the postulated periluminal stem/progenitor cell niche, but also in small groups scattered over the gland, thereby advocating the existence of multiple niches. In early postnatal mice undergoing a pituitary <em>growth</em> wave, Sox2(+) cells are more abundant than in adults, concordant with a larger SP and higher non-Sca<em>1</em>(high) proportion. Together, we tracked down pituitary progenitor cells by SP phenotype, and thus provide a straightforward method to isolate and scrutinize these cells from the plastic pituitary ex vivo, as well as a culture system for in-depth exploration of their regulatory network.

Insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) is essential for cell <em>growth</em>, <em>differentiation</em> and postnatal development. A null mutation in igf-<em>1</em> causes intrauterine <em>growth</em> retardation and perinatal lethality. The present study was designed to test the lower limit of igf-<em>1</em> gene dosage that ensures survival and postnatal <em>growth</em> by using the Cre/loxP system. Mice with variable reductions in IGF-I levels were generated by crossing EIIa-cre transgenic mice and mice with loxP-flanked igf-<em>1</em> locus (igf-<em>1</em>/flox). EIIa-cre mice express bacteriophage P<em>1</em> Cre (causes recombination) recombinase under the adenovirus promoter EIIa, during early <em>embryonic</em> development before implantation, and cause genomic recombination of the igf-<em>1</em>/flox locus. Mice with the most extensive recombination die immediately after birth, while the survivors have significant <em>growth</em> retardation in proportion to the reduction in their igf-<em>1</em> gene. Interestingly, this gene dosage effect on body weight was not very significant before weaning. However, when the young animals were weaned at 3 weeks, the igf-<em>1</em> gene dosage was the only independent predictor of the weight gain between 3 and 6 weeks among the parameters tested. Although <em>growth</em> retarded, mice with Cre-induced partial igf-<em>1</em> deficiency were fertile and gave birth to null mice. Thus Cre-induced genomic recombination using the EIIa promoter occurs during development and creates distinct phenotypes compared with the conventional null mutation. This variability allows for postnatal survival and will enable one to begin to explore the role of the endocrine vs. paracrine effects of IGF-I.

The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin <em>1</em> (ngn<em>1</em>). An immediate downstream gene is the bHLH gene neuronal <em>differentiation</em> (NeuroD). Targeted null mutations of ngn<em>1</em> results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn<em>1</em> null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites <em>growth</em> to sensory epithelia is initially independent of trophic <em>factors</em> released from developing sensory epithelia, but becomes rapidly dependent on those <em>factors</em>. Null mutations of specific neurotrophic <em>factors</em> lose distinct primary neuron populations which undergo rapid <em>embryonic</em> cell death.

Hepatocyte nuclear <em>factors</em> 3 (HNF-3) belong to an evolutionarily conserved family of transcription <em>factors</em> that are critical for diverse biological processes such as development, <em>differentiation</em>, and metabolism. To study the physiological role of HNF-3alpha, we generated mice that lack HNF-3alpha by homologous recombination in <em>embryonic</em> stem cells. Mice homozygous for a null mutation in the HNF-3alpha gene develop a complex phenotype that is characterized by abnormal feeding behavior, progressive starvation, persistent hypoglycemia, hypotriglyceridemia, wasting, and neonatal mortality between days 2 and <em>1</em>4. Hypoglycemia in HNF-3alpha-null mice leads to physiological counter-regulatory responses in glucocorticoid and <em>growth</em> hormone production and an inhibition of insulin secretion but fails to stimulate glucagon secretion. Glucagon-producing pancreatic alpha cells develop normally in HNF-3alpha-/- mice, but proglucagon mRNA levels are reduced 50%. Furthermore, the transcriptional levels of neuropeptide Y are also significantly reduced shortly after birth, implying a direct role of HNF-3alpha in the expression of these genes. In contrast, mRNA levels were increased in HNF-3 target genes phosphofructo-2-kinase/fructose-2,6-bisphophatase, insulin <em>growth</em> <em>factor</em> binding protein-<em>1</em>, and hexokinase I of HNF-3alpha-null mice. Mice lacking one or both HNF-3alpha alleles also show impaired insulin secretion and glucose intolerance after an intraperitoneal glucose challenge, indicating that pancreatic beta-cell function is also compromised. Our results indicate that HNF-3alpha plays a critical role in the regulation of glucose homeostasis and in pancreatic islet function.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to <em>differentiation</em>. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and <em>differentiation</em> potential similar to <em>embryonic</em> stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f<em>1</em> (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription <em>factor</em> Pou5f<em>1</em> and c-Kit receptor. Only the POU5F<em>1</em>+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of <em>growth</em> <em>factors</em>, generates cell lines that express pluripotent ES markers, i.e., Pou5f<em>1</em>, Nanog, Sox2, Rex<em>1</em>, Dppa5, SSEA-<em>1</em>, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

BACKGROUND

The Foxa family (a1, a2, and a3) of proteins are transcription factors that are central to endodermal development. Recently, Foxa1 has been shown to regulate the transcription of several murine and human prostate specific genes involved in differentiated function by interacting with DNA promoter sequences and androgen receptors. Currently, the developmental expression pattern of Foxa proteins in the murine prostate is unknown.

METHODS

Male CD-1 mice (embryonic, prepubertal, pubertal, and adult) were used for immunohistochemical analysis of Foxa1, a2, and a3. Immunofluorescence was also performed for androgen receptor and cytokeratin 14 expression. Prostate tissue from pre-pubertal, pubertal, and adult mice were analyzed by Western blot and RT-PCR analysis for Foxa1, a2, and a3 expression.

RESULTS

Strong Foxa1 immunoreactivity was observed in epithelial cells throughout prostate development, growth, and adult differentiation. Prominent Foxa2 protein expression was only observed in the early stages of prostate development and was exclusively localized to epithelial cells of the forming buds. RT-PCR analysis identified low Foxa2 mRNA expression levels in the ventral and dorsolateral lobes of the adult prostate, with Foxa2 epithelial cell expression being localized to periurethral regions of the murine adult prostatic complex. Foxa3 expression was not observed in the murine prostate.

CONCLUSIONS

Foxa proteins represent epithelial cell markers in the murine prostate gland. The early expression of Foxa1 and a2 proteins in prostate formation suggests that these proteins play an important role in normal prostate development, in addition to differentiated secretory function.

Bone morphogenetic protein-2 (BMP-2), a member of the transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) superfamily, is characterized by its ability to induce cartilage and bone formation. We have recently demonstrated that the multipotential, murine <em>embryonic</em> mesenchymal cell line, C3H<em>1</em>0T<em>1</em>/2, when cultured at high density, is induced by BMP-2 or TGF-beta <em>1</em> to undergo chondrogenic <em>differentiation</em>. The high-cell-density requirement suggests that specific cell-cell interactions, such as those mediated by cell adhesion molecules, are important in the chondrogenic response. In view of our recent finding that N-cadherin, a Ca(2+)-dependent cell adhesion molecule, is functionally required in normal <em>embryonic</em> limb mesenchyme cellular condensation and chondrogenesis, we examine here whether N-cadherin is also involved in BMP-2 induction of chondrogenesis in C3H<em>1</em>0T<em>1</em>/2 cells. BMP-2 stimulation of chondrogenesis in high-density micromass cultures of C3H<em>1</em>0T<em>1</em>/2 cells was evidenced by Alcian blue staining, elevated [35S]sulfate incorporation, and expression of the cartilage matrix markers, collagen type II and cartilage proteoglycan link protein. With BMP-2 treatment, N-cadherin mRNA expression was stimulated 4-fold within 24 h, and by day 5, protein levels were stimulated 8-fold. An N-cadherin peptidomimic containing the His-Ala-Val sequence to abrogate homotypic N-cadherin interactions inhibited chondrogenesis in a concentration-dependent manner. To analyze the functional role of N-cadherin further, C3H<em>1</em>0T<em>1</em>/2 cells were stably transfected with expression constructs of either full-length N-cadherin or a dominant negative, N-terminal deletion mutant of N-cadherin. Moderate (2-fold) overexpression of full-length N-cadherin augmented, whereas higher (4-fold) overexpression inhibited the BMP-2-chondrogenic effect. On the other hand, expression of the dominant negative N-cadherin mutant dramatically inhibited BMP-2 stimulated chondrogenesis. These data strongly suggest that upregulation of N-cadherin expression, at defined critical levels, is a candidate mechanistic component of BMP-2 stimulation of mesenchymal chondrogenesis.

Transforming <em>growth</em> <em>factor</em>-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, <em>differentiation</em>, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, <em>growth</em>/<em>differentiation</em> <em>factor</em>-<em>1</em>5 (GDF-<em>1</em>5). GDF-<em>1</em>5 is identical to macrophage inhibitory cytokine-<em>1</em> (MIC-<em>1</em>). GDF-<em>1</em>5/MIC-<em>1</em> mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-<em>1</em>5/MIC-<em>1</em> is a potent survival promoting and protective <em>factor</em> for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the <em>embryonic</em> rat midbrain floor. The trophic effect of GDF-<em>1</em>5/MIC-<em>1</em> was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-<em>1</em>5/MIC-<em>1</em> probably acts directly on neurons. GDF-<em>1</em>5/MIC-<em>1</em> also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-<em>1</em>5/MIC-<em>1</em> into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least <em>1</em> month. Administration of 5 microgram of GDF-<em>1</em>5/MIC-<em>1</em> in the same paradigm also provided significant neuroprotection. GDF-<em>1</em>5/MIC-<em>1</em> also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-<em>1</em>5/MIC-<em>1</em> is a novel neurotrophic <em>factor</em> with prominent effects on DAergic and serotonergic neurons. GDF-<em>1</em>5/MIC-<em>1</em> may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve <em>growth</em> <em>factor</em> (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-<em>1</em> specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-<em>1</em>-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-<em>1</em>, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-<em>1</em>-stimulated cardiomyocytes caused NGF-mediated PC<em>1</em>2 cell <em>differentiation</em>. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-<em>1</em>-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-<em>1</em>-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late <em>embryonic</em> stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-<em>1</em>-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-<em>1</em> regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.

<em>Growth</em>/<em>differentiation</em> <em>factor</em> <em>1</em> (GDF-<em>1</em>) is a recently described member of the transforming <em>growth</em> <em>factor</em> beta superfamily isolated from a day-8.5 mouse embryo cDNA library. Northern (RNA) analysis of <em>embryonic</em> mRNA detected two GDF-<em>1</em> transcripts [<em>1</em>.4 kilobases (kb) and 3.0 kb in length] displaying distinct temporal patterns of expression. Only the 3.0-kb transcript was detected in adult tissues, where its expression was restricted almost exclusively to the central nervous system. Comparison of murine and human brain cDNA sequences corresponding to the 3.0-kb transcript revealed high conservation of two nonoverlapping open reading frames with poor conservation of the intervening spacer region and the putative 5' and 3' untranslated sequences. By immunohistochemical analysis, the protein encoded by the downstream open reading frame (GDF-<em>1</em>) was detected exclusively in the brain, spinal cord, and peripheral nerves in day-<em>1</em>4.5 mouse embryos. The upstream open reading frame encodes a protein of unknown function containing multiple putative membrane-spanning domains. These findings raise the possibility that this mRNA may give rise to two different proteins.