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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.04.028 Byline: Yasuhiro Takashima (1)(3), Takumi Era (1), Kazuki Nakao (2), Saki Kondo (4), Masato Kasuga (3), Austin G. Smith (5), Shin-Ichi Nishikawa (1) Keywords: STEMCELL Abstract: Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1.sup.+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1.sup.+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1.sup.+ cells give rise to oligodendrocytes by expression of PDGFR[beta] and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins. Author Affiliation: (1) Laboratory for Stem Cell Biology, Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan (2) Laboratory for Animal Resources and Genetic Engineering, Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan (3) Department of Clinical Molecular Medicine, Division of Diabetes and Digestive and Kidney Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunokimachi, Chuo-ku, Kobe, 650-0017, Japan (4) Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan (5) Institute for Stem Cell Biology, University of Cambridge, Cambridge, CB2, 1QT, UK Article History: Received 14 September 2006; Revised 16 February 2007; Accepted 9 April 2007 Article Note: (miscellaneous) Published: June 28, 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.016 Byline: Atsushi Takebe (a)(c), Takumi Era (a), Mitsuhiro Okada (a)(b), Lars Martin Jakt (a)(b), Yoshikazu Kuroda (c), Shin-Ichi Nishikawa (a) Keywords: ES cell; In vitro differentiation; DNA microarray; PDGFR[alpha]; ARID3b Abstract: An inherent difficulty in using DNA microarray technology on the early mouse embryo is its relatively small size. In this study, we investigated whether use of ES cell differentiation culture, which has no theoretical limit in the number of cells that can be generated, can improve this situation. Seven distinct ES-cell-derived populations were analyzed by DNA microarray and examined for genes whose distribution patterns are similar to those of PDGFR[alpha], a gene implicated in differentiation of mesoderm/mesenchymal lineages. Using software developed in our laboratory, we formed a group of 30 genes which showed the highest similarity to PDGFR[alpha], 18 of these genes were shown to be involved in development of either mesodermal, mesenchymal or neural crest cells. This list also contains several genes whose role in embryogenesis has not yet been fully identified. One such molecule is mARID3b. The mARID3b expression is found in the paraxial mesoderm and cranial mesenchyme. mARID3b-null mouse showed early embryonic lethality, and most phenotypes of this mutant appear to develop from a failure to generate a sufficient number of cranial mesenchymal cells. These results demonstrate the potential use of ES cell differentiation culture in identifying novel genes playing an indispensable role in embryogenesis. Author Affiliation: (a) Laboratory for Stem Cell Biology, RIKEN Center for Development Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (b) The Foundation for Biomedical Research and Innovation, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (c) Division of Gastroenterological Surgery, Graduate School of Medicine, Kobe University, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan Article History: Received 27 June 2005; Revised 18 November 2005; Accepted 6 December 2005

Identification of novel genes that are similar to platelet-derived growth factor receptor, using DNA microarray studies on mouse embryonic cells, to find their role in embryogenesis is presented.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.025 Byline: Siu-Shan Mak (a), Mariko Moriyama (a), Eri Nishioka (b), Masatake Osawa (a), Shin-Ichi Nishikawa (a) Keywords: bcl2; bcl2 null; Melanoblast; c-Kit; Hair follicle development; Melanocyte stem cell; Melanocyte survival; Stem cell development; Stem cell factor Abstract: Bcl2 null mice display a characteristic loss of pigmentation demonstrating the importance of Bcl2 in the melanocyte (Mc) lineage. It was recently reported that this abnormal phenotype is due to the failure of melanocyte stem cell (MSC) maintenance and that Bcl2 is selectively important for the survival of MSCs. However, in our analysis of the same mouse, we observe a reduction in melanoblast (Mb) number in both epidermal and follicular populations. More importantly, there is a complete absence of MSCs. SCF downregulation in the epidermis is concomitant with the dramatic reduction in Mb numbers observed in the Bcl2 null, suggesting that Bcl2 is indispensable for the survival of Mbs in the absence of c-Kit signaling. Consistently, abrogation of c-Kit signaling in Bcl2 null mice depletes all Mbs and Mcs, whereas continuous expression of SCF in epidermal keratinocytes rescues the MSCs. Our results demonstrate that Bcl2 has a general role in Mb and Mc survival and is essential for the emergence of MSCs. Moreover, the results indicate that the first wave of Mcs that provide hair pigmentation is derived directly from epidermal Mbs bypassing MSCs. Furthermore, a Bcl2-independent mechanism of action of SCF in the Mc lineage is revealed as SCF c-Kit signaling is functional in the absence of Bcl2. Author Affiliation: (a) RIKEN, Center for Developmental Biology (CDB), Laboratory for Stem Cell Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (b) Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Article History: Received 29 July 2005; Revised 6 December 2005; Accepted 9 December 2005

The Foxl1 gene, which encodes a winged helix transcriptional regulator, is expressed in the mesenchymal layer of developing and mature gastrointestinal tract. Foxl1-deficient mice exhibit various defects not only in the epithelial layer of the gastrointestinal tract but also in gut-associated lymphoid tissues. In the small intestine of Foxl1-deficient mice, the formation of Peyer's patches is affected, particularly in the caudal region. This alteration is shown to be due to the delayed formation of Peyer's patches organizing centers as revealed by the expressions of VCAM1 and IL-7 receptor [alpha]-chain at 17.5 days postcoitus. Peyer's patch defects are concordant with the significantly decreased expression of Lymphotoxin [beta]-receptor in the caudal region of fetal intestine. Foxl1 is suggested to regulate the responsiveness of fetal intestinal mesenchymal cells to inductive signals mediated by Lymphotoxins during Peyer's patch organogenesis. In addition, constitutive outgrowth of colonic patches due to defects in radioresistant stromal components of colonic patches are seen in Foxl1-deficient mice. Because of the functional similarities of hypertrophic colonic patches to those seen in hapten-induced experimental colitis, this hypertrophy is suggested to involve Lymphotoxin [beta]-receptor signaling. Together, the data suggest that Foxl1 might be involved in cellular responses of gut-associated lymphoid tissues dependent upon the Lymphotoxins/Lymphotoxin [beta]-receptor axis. Keywords: Foxl1; Peyer's patch; Colonic patch; Gut mesenchyme; Lymphotoxins; Mouse

Research has been conducted on the matrix metalloproteinase important for the extracellular matrix remodeling. Results indicate that membrane-anchored glycoprotein RECK regulates metalloproteinases involved in cancer progression and the results are reported.

Melanocytes (Mc) and their progenitors melanoblasts (Mb) are derived from the neural crest and migrate along the dorsolateral pathway to colonize the dermis, the epidermis, and finally the hair matrix. To examine the involvement of cadherins in the migration of Mc lineage cells, we combined flow cytometric analysis of dissociated live cells with immunohistochemical staining of tissue sections to quantify the level of cadherin expression on the surface of Mb/Mc. At 11.5 days postcoitum, Mb are in the dermis and are E-cadherin(super -)P-cadherin(super -) (E-cad(super -)P-cad(super -)). During the next 48 h, a 200-fold increase of E-cadherin expression is induced on the surface of Mb prior to their entry into the epidermis, thereby forming a homogeneous E-cad(super high)p-cad(super -/low) population. The cadherin expression pattern then diversifies, giving rise to three populations, an E-cad(super -)P-cad(super -) dermal population, E-cad(super high)P-cad(super low) epidermal population, and E-cad(super -)P-cad(super med.high) follicular population. In all three populations, the patterns of expression are region-specific, being identical with those of surrounding cells such as keratinocytes and fibroblasts, and are preserved before and after pigmentation. While most of the epidermal Mb/Mc disappear after the neonatal stage in normal mice, forced expression of steel factor in the epidermis of transgenic mice promotes survival of epidermal Mb/Mc, maintaining epidermal-type cadherin expression pattern (E-cad(super high)P-cad(super low)) throughout the postnatal life. These findings indicate the involvement of extrinsic cues in coordinating the cadherin expression pattern of Mb/Mc and suggest a role for E- and P-cadherins in guiding Mc progenitors to their final destinations. Key Words: melanocyte; melanoblast; cadherins; E-cadherin; P-cadherin; expression; migration; diversification; colonization; quantitative; qualitative; microenvironment; steel factor (SLF); flow cytometry; epidermis; dermis; hair follicles; hair matrix.

Ovarian follicle development is controlled by the cycling variation of gonadotrophins derived from the central nervous system. Intragonadal signals are also required, especially in the autonomous development of small follicles. Receptor tyrosine kinase c-kit and its ligand SLF (Steel factor) are expressed on the surface of specific populations of follicle-forming cells in a contiguous manner and are thought to have important roles in follicular development. We blocked the interaction of c-kit and its ligand by administering the function-blocking antibody ACK2 to developing mice at various times after birth and monitored ovarian follicle development. A blockade of c-kit function disturbed the onset of primordial follicle development, primary follicle growth, follicular fluid formation of preantral follicles, and Penultimate-stage ovarian follicle maturation before ovulation. Ovarian follicle growth was dependent on c-kit during the first 5 days after birth when the functional FSH receptor is not yet expressed in mouse ovary. In contrast, primordial follicle formation and survival, small preantral or antral follicle development, ovulation, and luteinization of the ovulated follicle were not affected by this antibody. These findings indicate the stepwise requirement of c-kit and its ligand interaction system in the developing ovarian follicle and that c-kit with its ligand supports the autonomous development of ovarian follicle independent of gonadotrophins.

In mice, coat pigmentation requires a stem cell (SC) system in which the survival, proliferation, and differentiation of melanocytes (MCs) are regulated by microenvironments in hair follicles (HFs). In vitro systems are required to analyze the behavior of single melanocyte stem cells (MCSCs) and their potential to form SC systems in vivo. We describe here an experimental system for the isolation, self-renewal, and differentiation of MCSCs, as well as an in vivo reconstitution assay for assessing their potential. Using Dcttm1(Cre)Bee/CAG-CAT-GFP mice, we show that, in the presence of stem cell factor and basic fibroblast growth factor and the XB2 feeder cell line, purified MCSCs can undergo clonogenic proliferation, resulting in c-Kitlow side scatterlow cells. In culture, these cells maintain their capacity to differentiate and reconstitute an MCSC system in HFs. As these cells are present in the upper part of the HF near the bulge region, express only low levels of housekeeping genes, and are resistant to neonatal treatment with ACK2, it is likely that only MCSCs that are quiescent in vivo have clonogenic activity in vitro. We also found that MCSCs can be purified from wild-type mice by fluorescent cell sorting and can be characterized in vitro. [ABSTRACT FROM AUTHOR]

Previous studies indicate that c-Kit is required for postnatal melanocyte development. To understand the precise mechanisms of c-Kit dependence, we studied melanocyte development in newborn C57BL/6 mice by means of peritoneal injection of a monoclonal anti-c-Kit antibody (ACK2), which blocks c-Kit functions. The mice were injected once or more with ACK2 at various intervals after birth. In experiment 1, skin samples were examined on day 10 post-partum and in experiment 2 they were examined daily until day 10 post-partum. We studied melanocytes in the hair follicles, epidermis, and dermis by light and electron microscopy with dopa reactions and immunohistochemistry. Epidermal melanocytes in untreated mice were dopa negative and c-Kit positive on day 0 post-partum but became dopa positive soon thereafter. In ACK2-treated mice, the earlier the mice received ACK2 injections after birth, the fewer melanocytes they had, not only in the epidermis, but also in follicles. In these mice, melanocytes that had undergone apoptosis in the dermis and the follicles were detected ultrastructurally. Some appeared to have produced tyrosinase, because they had dopa-positive melanosomes. These results suggest that melanocytes in newborn mice are c-Kit dependent and undergo apoptosis when c-Kit receptors are blocked by ACK2 in the early days after birth. During this c-Kit-dependent period, melanocytes differentiate from dopa negative to positive and migrate from the epidermis to hair follicles. [ABSTRACT FROM AUTHOR]

Summary: Current antiangiogenic therapy is limited by its cytostatic nature and systemic side effects. To address these limitations, we have unveiled the role of RhoJ, an endothelial-enriched Rho GTPase, during tumor progression. RhoJ blockade provides a double assault on tumor vessels by both inhibiting tumor angiogenesis and disrupting the preformed tumor vessels through the activation of the RhoA-ROCK (Rho kinase) signaling pathway in tumor endothelial cells, consequently resulting in a functional failure of tumor vasculatures. Moreover, enhanced anticancer effects were observed when RhoJ blockade was employed in concert with a cytotoxic chemotherapeutic agent, angiogenesis-inhibiting agent, or vascular-disrupting agent. These results identify RhoJ blockade as a selective and effective therapeutic strategy for targeting tumor vasculature with minimal side effects. [Copyright &y& Elsevier]

The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα+ mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis. [ABSTRACT FROM AUTHOR]

The formation of functional skin entails multiple key signals that are implicated repeatedly in distinct processes during embryogenesis. Although Eph receptors and their membrane-bound ephrin ligands play a role in a wide variety of embryonic processes, their function in skin development has not been addressed. Here, we show that ephrin B2 is transiently expressed in hair buds during embryogenesis and in dermal mesenchymal cells during the perinatal period. Keratinocyte-specific ephrin B2-targeted mutant mice exhibit no skin phenotype, whereas postnatal systemic ephrin B2 ablation results in the enhancement of keratinocyte proliferation. Although the same treatment results in a defect of vascular remodeling, our analyses showed that the keratinocyte phenotype is not caused by hypoxia due to vascular defects. Interestingly, we found an enhanced expression of IL-1 family molecules, which have been implicated in the regulation of keratinocyte proliferation. On the basis of these observations, we propose that the transient expression of ephrin B2 in perinatal dermal mesenchymal cells plays a role in adjusting the activity of the mesenchymal microenvironment that regulates proliferation of keratinocytes. [ABSTRACT FROM AUTHOR]

Summary: Recent studies have shown that Notch signaling plays an important role in epidermal development, but the underlying molecular mechanisms remain unclear. Here, by integrating loss- and gain-of-function studies of Notch receptors and Hes1, we describe molecular information about the role of Notch signaling in epidermal development. We show that Notch signaling determines spinous cell fate and induces terminal differentiation by a mechanism independent of Hes1, but Hes1 is required for maintenance of the immature state of spinous cells. Notch signaling induces Ascl2 expression to promote terminal differentiation, while simultaneously repressing Ascl2 through Hes1 to inhibit premature terminal differentiation. Despite the critical role of Hes1 in epidermal development, Hes1 null epidermis transplanted to adult mice showed no obvious defects, suggesting that this role of Hes1 may be restricted to developmental stages. Overall, we conclude that Notch signaling orchestrates the balance between differentiation and immature programs in suprabasal cells during epidermal development. [Copyright &y& Elsevier]

Elucidation of the molecular mechanisms underlying stem cell regulation is of great importance both for basic biology and for clinical applications. Melanocyte stem cells (MSCs) are an excellent model in which to study the molecular basis of stem cell regulation, as the genetic alterations involved in the maintenance of the stem cells are readily identifiable by a premature hair graying phenotype. Research on MSCs has been hampered by the lack of a reliable system to assay their function. Here, by co-culturing highly purified melanoblasts (MBs) with XB2 keratinocytes, we describe an efficient culture method that allows the expansion of immature MBs in vitro. These MBs are also capable of undergoing terminal differentiation into mature melanocytes (MCs) when differentiation is induced. Furthermore, by performing a hair-follicle reconstitution assay in which expanded MBs in a mixture of epidermal and dermal cells were grafted to reconstitute a hair follicle, we demonstrate that the expanded MBs retain their capacity to become incorporated into newly developed hair follicles and repopulate the MC stem cell population there. Thus, by integrating genetic manipulations in cultured MBs in vitro, this method provides a powerful tool with which to study the molecular basis of stem cell regulation.Journal of Investigative Dermatology (2008) 128, 408–420; doi:10.1038/sj.jid.5700997; published online 26 July 2007 [ABSTRACT FROM AUTHOR]

Abstract: Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1+ mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development. [Copyright &y& Elsevier]

: ObjectiveThe aim of this study was to assess the potential of FLK1+ and vascular endothelial (VE)-cadherin+ populations from different stages of embryonic development to generate hematopoietic cells ex vivo and to contribute to the hematopoietic systems of recipient mice.: Materials and MethodsFLK1+ of VE-cadherin+ cells were isolated from 7.5- to 9.5-dpc concepti and cultured ex vivo on OP9 stromal cells and hematopoietic development examined. VE-cadherin+CD45− cells from 8.5- and 9.5-dpc concepti were injected intrahepatically into newborn busulfan-treated SCID recipients and engraftment monitored.: ResultsVE-cadherin+ cells from 7.5- and 8.5-dpc concepti can readily generate hematopoi-etic cells ex vivo compared to FLK1+ VE-cadherin− cells. Similar hematopoietic potential can be found in the VE-cadherin+ cells from the 8.5-dpc yolk sac. When VE-cadherin+CD45− cells were injected into SCID recipients, long-term engraftment, particularly within the lymphoid system, was observed. This potential was observed in VE-cadherin+CD45− cells from 9.5-dpc embryo or yolk sac but from tissues from younger concepti.: ConclusionsFLK1+VE-cadherin− cells, possibly representing the lateral plate mesoderm, are not as effective at generating hematopoietic cells compared to similarly staged VE-cadherin+ cells. VE-cadherin+CD45− cells can also contribute to the hematolymphoid system of intrahepatically injected newborn SCID recipients, suggesting that cells bearing an endothelial phenotype are capable of generating long-term hematopoietic precursors. [Copyright &y& Elsevier]

The first wave of coat hair development is initiated around embryonic day 14 in the mouse. Whereas ectodysplasin and ectodermal dysplasia receptor, tumor necrosis factor and tumor necrosis factor receptor family molecules, respectively, were identified to be signals triggering this process, not much was known regarding their downstream molecular targets. In this report, we show that mucosal addressin cell adhesion molecule 1 and intercellular adhesion molecule 1 are induced in the keratinocytes of the hair placode as a direct consequence of ectodermal dysplasia receptor signal, and tumor-necrosis-factor-receptor-associated factor 6 is involved in this mucosal addressin cell adhesion molecule 1 expression. Experiments using an in vitro culture of skin fragments demonstrated that ectodermal-dysplasia-receptor-induced mucosal addressin cell adhesion molecule 1 expression occurs at the initial phase of follicle development before involvement of Sonic hedgehog signal. Follicle development in this culture was also suppressed to some extent, though not completely, by addition of soluble mucosal addressin cell adhesion molecule 1/IgG-Fc chimeric protein, whereas monoclonal antibody that can inhibit mucosal addressin cell adhesion molecule 1 interaction with integrin α4β7 had no effect on this process. These results demonstrated for the first time that the structural proteins, mucosal addressin cell adhesion molecule 1 and intercellular adhesion molecule 1, are induced by ectodermal dysplasia receptor signal and suggested the potential involvement of mucosal addressin cell adhesion molecule 1 in the morphogenesis of follicular keratinocytes. [ABSTRACT FROM AUTHOR]

: ObjectiveOf various types of xenograft assays, the use of NOD/SCID mice has been the most popular method for quantitating candidate human stem cells. Limitations of the assay include low levels of engraftment, except when large numbers of cells are injected, and the development of thymic lymphoma, which precluded observation of long-term engraftment. In order to establish an assay that allows long-term in vivo engraftment and higher engraftment levels by a reasonable number of human cells, we tested a model based on “conditioned newborn” NOD/SCID or NOD/SCID/β2-microglobulinnull (BMGnull) mice.: Materials and MethodsUsing human cord blood mononuclear cells, we tested various nonradiation conditioning regimens and cell injection routes. Conditioning with a combination of 5-fluorouracil (5FU) and anti-mouse c-kit blocking antibody (Ack-2) or a combination of busulfan (BU) and cyclophosphamide (CY) and the use of facial vein for the cell injection route yielded the highest levels of multilineage engraftment.: ResultsAt 4–5 months posttransplantation, the median of engraftment level in bone marrow with 5FU/Ack-2 and BU/CY regimens were 0.9% (range: 0.2–40.5%) and 2.1% (range: 0.3–2.4%) in NOD/SCID mice, and 11.3% (range: 0.7–38%) and 14.1% (range: 0.8–52%) in NOD/SCID/BMGnull mice, respectively. Multilineage engraftment was demonstrated by flow cytometry and by the growth of multilineage colonies in methylcellulose culture. Secondary transplantation of the isolated human CD45+ cells, also performed at 4–5 months posttransplantation, revealed engraftment at the levels of 1.5 ± 0.42% at 2 months after secondary transplantation.: ConclusionOur assay may provide a quantitative method for analysis of human hematopoietic stem cells. [Copyright &y& Elsevier]

Hair follicles develop and are maintained by multiple rounds of inductive events involving interactions among various cell types within the follicles and the adjacent mesenchyme. Although evidence suggests that several growth factors, cell adhesion molecules, and transcriptional regulators are involved in those cell-cell interactions, the molecular mechanisms regulating each pivotal step of hair follicle development, such as formation of the hair germ, root sheath, sebaceous gland, and hair canal, remain largely unknown. In this study, we established the antagonistic monoclonal antibody APA5 against platelet-derived growth factor (PDGF) receptor-α (PDGFR-α) and used it to investigate the role of PDGFR-α in neonatal skin development. In addition to the dermal mesenchyme, a known site of PDGFR-α expression, immunohistologic staining of neonatal skin detected transient expression of PDGFR-α in the perinatal epidermis for several days. On the other hand, ligands for PDGFR-α were detected in epithelial cells and sebaceous glands of hair follicles. To determine whether this contiguous expression of PDGF and PDGFR-α in neonatal skin plays a functional role, we injected APA5 into neonates to block the function of PDGFR-α. Consistent with the PDGF/PDGFR-α expression in the neonatal skin, two defects were induced by this procedure. First, hair canal formation in the epidermis was severely suppressed. Second, the growth of dermal connective tissues and of hair follicles of pelage hairs was suppressed. These results indicate that PDGF signals are involved in both the epidermis-follicle interaction and the dermal mesenchyme-follicle interaction required for hair canal formation and the growth of the dermal mesenchyme, respectively. [ABSTRACT FROM AUTHOR]

Hematopoietic cells (HPCs) develop from hemogenic endothelial cells (ECs), a specialized type of ECs undergoing hematopoietic transition. However, the mesoderm origin for hemogenic ECs or HPCs has not been clarified. To examine the origin for hemogenic mesoderm, we inactivated Etv2, a master regulator for EC/HPC commitment, in specific regions. Region-specific Etv2 ablation in early mesoderm caused local EC differentiation block, resulting in the loss of specific vascular beds without compensatory migration of residual ECs into avascular area. This feature of local EC/HPC differentiation block was correlated to the hemogenic potential of each mesoderm subset. We found that caudal-lateral mesoderm of E7.5-8.5 embryos represent the pre-committed population critical for generating hemogenic ECs. Etv2 ablation in caudal-lateral mesoderm by Hoxb6 Cre or Hoxb6CreER transgene affected vitelline plexus formation and intra-aortic hematopoietic clusters. In differentiated embryonic stem cells, this mesoderm subset marked by Hoxb6-lateral mesoderm promoter showed enriched T lymphopoietic potential among Flk-1+ cells, which could be regarded as a characteristic for definitive HPCs. These findings indicate that critical mesoderm precursors possibly for definitive type hemogenic ECs are regionally specified in primitive mesoderm, suggesting that Hoxb6+ caudal-lateral mesoderm represents the critical source of HPCs, which are potentially useful to enrich definitive HPCs from embryonic stem cells. [ABSTRACT FROM AUTHOR]

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin+/CD45+ cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis. [Copyright &y& Elsevier]