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BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo

At E4.0 the inner cell mass of the mouse blastocyst consists of a core of embryonic ectoderm cells surrounded by an outer layer of primitive (extraembryonic) endoderm, which subsequently gives rise to both visceral endoderm and parietal endoderm. Shortly after blastocyst implantation, the solid mass of ectoderm cells is converted by a process known as cavitation into a pseudostratified columnar epithelium surrounding a central cavity. The trophectoderm-derived extraembryonic ectoderm undergoes a similar cavitation process at a slightly later stage. Cavitation, this type of morphogenetic conversion, also occurs during the formation of other hollow (tubular) structures that arise from solid primordia, such as the ducts of various exocrine glands. In early postimplantation mouse development, cavitation prepares the embryo for gastrulation, the process by which the three germ layers are formed. Two cell lines, which form embryoid bodies that do (PSA1) or do not (S2) cavitate, have been used as an in vitro model system for studying the mechanism of cavitation in the early embryo. Evidence is provided that cavitation is the result of both programmed cell death and selective cell survival, and that the process depends on signals from visceral endoderm. Bmp2 and Bmp4 are expressed in PSA1 embryoid bodies and embryos at the stages when visceral endoderm differentiation and cavitation are occurring, and blocking BMP signaling via expression of a transgene encoding a dominant negative mutant form of BMP receptor IB inhibits expression of the visceral endoderm marker, Hnf4, and prevents cavitation in PSA1 embryoid bodies. Furthermore, addition of BMP protein to cultures of S2 embryoid bodies induces expression of Hnf4 and other visceral endoderm markers and also cavitation. Taken together, these data indicate that BMP signaling is both capable of promoting, and required for differentiation of, visceral endoderm and cavitation of embryoid bodies. Based on these and other data, a model is proposed for the role of BMP signaling during peri-implantation stages of mouse embryo development. It is suggested that BMP4 produced in the ectoderm acts on the primitive endoderm to promote visceral endoderm differentiation. BMP2 produced in the endoderm may also play a role in the process. BMP4 produced in the ectoderm and/or BMP2 produced in the visceral endoderm act(s) on the ectoderm [perhaps in conjunction with other, visceral endoderm-derived signals(s)] to promote programmed cell death of the inner cells and the differentiation of the outer layer of columnar cells (Coucouvanis, 1999).

DPP homologs are involved in the generation of primordial germ cells

Before gastrulation, the mouse embryo consists of three distinct cell lineages that were established in the blastocyst during the peri-implantation period, that is, epiblast, extraembryonic endoderm, and trophectoderm. The epiblast, from which the entire fetus will form, as well as the extraembryonic mesoderm and amnion ectoderm, is a cup-shaped epithelium apposed on its open end to the extraembryonic ectoderm, a trophectoderm derivative. Both epiblast and extraembryonic ectoderm are covered by visceral endoderm, which is part of the extraembryonic endoderm lineage. The primordial germ cells (PGCs) of the mouse embryo are derived from part of the population of epiblast cells that mainly will give rise to the extraembryonic mesoderm. Precursors of the PGCs are located before gastrulation in the extreme proximal region of the epiblast adjacent to the extraembryonic ectoderm, and have descendants not only in the germ line, but also in extraembryonic structures, that is, the allantois, blood islands, and yolk sac mesoderm, as well as both layers of the amnion. At embryonic day (E) 6.0, these precursors lie scattered in a ring that extends up to three cell diameters from the junction with the extraembryonic ectoderm. Early in gastrulation, they converge toward the primitive streak in the posterior of the embryo and translocate through it. Allocation to the germ cell lineage is thought to occur in ~45 cells around E7.2, after the precursors have passed through the streak and have come to reside in the extraembryonic mesoderm. This is about the time when the putative PGCs can first be identified morphologically in a cluster posterior to the primitive streak in a position that will later become the base of the allantois. PGCs stain strongly in a characteristic pattern for alkaline phosphatase (AP) activity, which by this stage is due to tissue nonspecific AP. The PGCs continue to express AP during their proliferation in the developing hindgut and migration into the genital ridges. Transplantation studies have shown that genetically marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to PGCs and extraembryonic mesoderm when grafted to the proximal epiblast. These results raise the possibility that PGC precursors are induced by extracellular factors and/or cell interactions present locally at the junction between the extraembryonic ectoderm and epiblast (Lawson, 1999 and references).

Thus, inductive cell interactions are required around gastrulation to establish the germ line. Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of beta-galactosidase activity in Bmp4lacZneo (a reporter inserted into the first intron of Bmp4) embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage (Lawson, 1999).

The primordial germ cells (PGCs) of the mouse are derived from proximal epiblast cells that are adjacent to the extraembryonic ectoderm during gastrulation. Previous studies have demonstrated that extraembryonic ectoderm-derived BMP4 and BMP8B are both required for PGC generation. Bmp2 also plays a role in PGC generation. PGC number is significantly reduced in Bmp2 heterozygous and homozygous embryos at the N2 generation onto C57BL/6 background. Bmp2 homozygous embryos also have a short allantois and about 50% of them do not undergo normal chorioallantoic fusion. Using whole-mount in situ hybridization, it has been shown that Bmp2 is primarily expressed in the endoderm of mouse pregastrula and gastrula embryos. Using a genetic approach, it has been shown that Bmp2 and Bmp4, but not Bmp2 and Bmp8b, have an additive effect on PGC generation. These results suggest that PGC generation in the mouse embryo is regulated not only by extraembryonic ectoderm-derived BMP4 and BMP8B, but also by endoderm-derived BMP2 (Ying, 2001a).

Extraembryonic ectoderm-derived factors instruct the pluripotent epiblast cells to develop toward a restricted primordial germ cell (PGC) fate during murine gastrulation. Genes encoding Bmp4 of the Dpp class and Bmp8b of the 60A class are expressed in the extraembryonic ectoderm: targeted mutation of either results in severe defects in PGC formation. Heterodimers of DPP and 60A classes of bone morphogenetic proteins (BMPs) are more potent than each homodimer in bone and mesoderm induction in vitro, suggesting that BMP4 and BMP8B may form heterodimers to induce PGCs. To investigate how BMP4 and BMP8B interact and signal for PGC induction, epiblasts of embryonic day 6.0-6.25 embryos were cocultured with BMP4 and BMP8B proteins produced by COS cells. BMP4 or BMP8B homodimers alone cannot induce PGCs whereas they can in combination, providing evidence that two BMP pathways are simultaneously required for the generation of a given cell type in mammals and also providing a prototype method for PGC induction in vitro. Furthermore, the PGC defects of Bmp8b mutants can be rescued by BMP8B homodimers whereas BMP4 homodimers cannot mitigate the PGC defects of Bmp4 null mutants, suggesting that BMP4 proteins are also required for epiblast cells to gain germ-line competency before the synergistic action of BMP4 and BMP8B (Ying, 2001b).

Deletion of various bone morphogenetic proteins (BMPs) and their downstream Smads in mice have clearly shown that BMP signaling is essential for the formation of primordial germ cells (PGCs). However, the molecular mechanism through which this takes place is still unclear. BMP4 produced in the extraembryonic ectoderm signals through ALK2, a type I BMP receptor, in the visceral endoderm (VE) to induce formation of PGCs from the epiblast. Embryonic day 5.5-6.0 (E5.5-E6.0) embryos cultured on fibronectin formed PGCs in the presence of VE, but not in its absence. Alk2-deficient embryos completely lack PGCs and the heterozygotes have reduced numbers, resembling Bmp4-deficient phenotypes. Expression of constitutively active ALK2 in the VE, but not in the epiblast, is sufficient to rescue the PGC phenotype in Bmp4-deficient embryos. In addition, it was shown that the requirement for the VE at E5.5-E6.0 can be replaced by culturing embryos stripped of VE on STO (SIM mouse embryo-derived thioguanine and ouabain resistant feeder) cells, indicating that STO cells provide or transduce signals necessary for PGC formation that are normally transmitted by the VE. A model is proposed in which direct signaling to proximal epiblast is supplemented by an obligatory indirect BMP-dependent signal via the VE (de Sousa Lopes, 2004).

Although BMPs produced by the extraembryonic tissues are required for PGC formation, there is no evidence that BMPs signal directly or exclusively to the proximal epiblast cells to induce several of them to become PGCs. This study shows that there is an absolute requirement for ALK2 in the VE at E5.5-E6.0 for PGC formation to take place: absence of VE in explants (cultured embryos) or ALK2 in embryos results in the complete absence of PGCs, mimicking the Bmp4 mutant phenotype, whereas PGC formation in explants from Bmp4 mutant embryos can be rescued by constitutively active ALK2, but only in the presence of VE. Therefore, it is suggested that BMP4 signals through ALK2 in the VE to induce formation of PGCs from the epiblast. Furthermore, it is demonstrated that the reason for inconsistent reports in the past on whether or not VE is required for PGC formation is the consequence of different substrata used for explant cultures: STO feeder cells have the ability to induce PGCs to form in embryos stripped of VE at E5.5-E6.0, but in similar experiments using fibronectin substrata, no PGCs form, demonstrating an absolute requirement for VE at this stage (de Sousa Lopes, 2004).

BMP inhibition and neural induction

A dominant molecular explanation for neural induction is the 'default model', which proposes that the ectoderm is pre-programmed towards a neural fate, but is normally inhibited by endogenous BMPs. Although there is strong evidence favouring this in Xenopus, data from other organisms suggest more complexity, including an involvement of FGF and modulation of Wnt. However, it is generally believed that these additional signals also act by inhibiting BMPs. Whether BMP inhibition is necessary and/or sufficient for neural induction was investigated. In the chick, misexpression of BMP4 in the prospective neural plate inhibits the expression of definitive neural markers (Sox2 and late Sox3), but does not affect the early expression of Sox3, suggesting that BMP inhibition is required only as a late step during neural induction. Inhibition of BMP signalling by the potent antagonist Smad6, either alone or together with a dominant-negative BMP receptor, Chordin and/or Noggin in competent epiblast is not sufficient to induce expression of Sox2 directly, even in combination with FGF2, FGF3, FGF4 or FGF8 and/or antagonists of Wnt signalling. These results strongly suggest that BMP inhibition is not sufficient for neural induction in the chick embryo. To test this in Xenopus, Smad6 mRNA was injected into the A4 blastomere (which reliably contributes to epidermis but not to neural plate or its border) at the 32-cell stage: expression of neural markers (Sox3 and NCAM) is not induced. It is proposed that neural induction involves additional signalling events that remain to be identified (Linker, 2004).

The ectoderm gives rise to both neural tissue and epidermis. In vertebrates, specification of the neural plate requires repression of bone morphogenetic protein (BMP) signaling in the dorsal ectoderm. The extracellular BMP antagonist Chordin and other signals from the dorsal mesoderm play important roles in this process. Zebrafish mutant combinations that disrupt Chordin and mesoderm formation were used to reveal additional signals that contribute to the establishment of the neural domain. Fibroblast growth factor (FGF) signaling accounts for the additional activity in neural specification. Impeding FGF signaling results in a shift of ectodermal markers from neural to epidermal. However, following inhibition of FGF signaling, expression of anterior neural markers recovers in a Nodal-dependent fashion. Simultaneously blocking, Chordin, mesoderm formation, and FGF signaling together eliminates neural marker expression during gastrula stages. FGF signaling is required for chordin expression but it also acts via other mechanisms to repress BMP transcription during late blastula stages. Activation of FGF signaling is also able to repress BMP transcription in the absence of protein synthesis. These results support a model in which specification of anterior neural tissue requires early FGF-mediated repression of BMP transcript levels and later activities of Chordin and mesodermal factors (Londin, 2005).

Neural induction constitutes the first step in the generation of the vertebrate nervous system from embryonic ectoderm. Work with Xenopus ectodermal explants has suggested that epidermis is induced by BMP signals, whereas neural fates arise by default following BMP inhibition. In amniotes and ascidians, however, BMP inhibition does not appear to be sufficient for neural fate acquisition, which is initiated by FGF signalling. The roles of the BMP and FGF pathways during neural induction in Xenopus have been reevaluated. Ectopic BMP activity converts the neural plate into epidermis, confirming that this pathway must be inhibited during neural induction in vivo. Conversely, inhibition of BMP, or of its intracellular effector SMAD1 in the non-neural ectoderm leads to epidermis suppression. In no instances, however, is BMP/SMAD1 inhibition sufficient to elicit neural induction in ventral ectoderm. By contrast, neural specification occurs when weak eFGF or low ras signalling are combined with BMP inhibition. Using all available antimorphic FGF receptors (FGFR), as well as the pharmacological FGFR inhibitor SU5402, it was demonstrated that pre-gastrula FGF signalling is required in the ectoderm for the emergence of neural fates. Finally, although the FGF pathway contributes to BMP inhibition, as in other model systems, it is also essential for neural induction in vivo and in animal caps in a manner that cannot be accounted for by simple BMP inhibition. Taken together, these results reveal that in contrast to predictions from the default model, BMP inhibition is required but not sufficient for neural induction in vivo. This work contributes to the emergence of a model whereby FGF functions as a conserved initiator of neural specification among chordates (Delaune, 2005).

Bone morphogenetic protein (BMP) signaling plays a crucial role in maintaining the pluripotency of mouse embryonic stem cells (ESCs) and has negative effects on ESC neural differentiation. However, it remains unclear when and how BMP signaling executes those different functions during neural commitment. This study shows that a BMP4-sensitive window exists during ESC neural differentiation. Cells at this specific period correspond to the egg cylinder stage epiblast and can be maintained as ESC-derived epiblast stem cells (ESD-EpiSCs), which have the same characteristics as EpiSCs derived from mouse embryos. It is proposed that ESC neural differentiation occurs in two stages: first from ESCs to ESD-EpiSCs and then from ESD-EpiSCs to neural precursor cells (NPCs). It was further shown that BMP4 inhibits the conversion of ESCs into ESD-EpiSCs during the first stage, and suppresses ESD-EpiSC neural commitment and promotes non-neural lineage differentiation during the second stage. Mechanistic studies show that BMP4 inhibits FGF/ERK activity at the first stage but not at the second stage; and IDs, as important downstream genes of BMP signaling, partially substitute for BMP4 functions at both stages. It is concluded that BMP signaling has distinct functions during different stages of ESC neural commitment (Zhang, 2010).

BMP4 has been reported to suppress ESC neural differentiation by inducing ID proteins. Because ESC neural differentiation could be divided into two stages and BMP4 could induce ID gene expression in both ESCs and EpiSCs, it was asked whether ID proteins mediated BMP function at both stages. To address this question, Id1 and Id2 were overexpressed in ESCs and ESD-EpiSCs using lentiviral vectors; elevated gene expression was confirmed by Q-PCR. Th the EBs from ID2-ESCs or control-ESCs were cultured in KSR medium for 2 days; overexpression of ID2 was found to upregulate Klf4 and Rex1 expression and downregulated Fgf5 expression. Immunostaining showed that FGF5 protein levels were reduced in ID2-overexpressing cells. ESD-EpiSC colony number assay also showed that overexpression of ID2 significantly inhibited the derivation of ESD-EpiSCs from ESCs. Similar to the effect of BMP4, ID2 overexpression did not completely block the derivation of ESD-EpiSCs from ESCs. However, unlike ESD-EpiSCs derived from BMP4-treated EBs, ESD-EpiSCs derived from ID2-overexpressing ESCs could not maintained and differentiated after 1-2 passages. Reproducible results were also obtained from ID1-overexpressing ESCs. Therefore, these data suggest that IDs can partially substitute for the function of BMP4 at the first stage (Zhang, 2010).

Next analyzed was the effect of ID2 overexpression on ESD-EpiSC neural differentiation. Unfortunately, the ESD-EpiSCs with high levels of ID2 expression differentiated and could not be maintained as stable cell lines; therefore, ESD-EpiSCs could only be generated with low levels of ID2 overexpression. Relative to control ESD-EpiSCs, ID2 overexpression was associated with the downregulation of neuroectoderm marker (Sox1, Sox2) expression, and the upregulation of mesodermal (Flk1, T) and trophectodermal (Cdx2, Hand1) marker expression in day 4 ESD-EpiSC aggregates cultured in KSR medium. Similar results were also obtained from ID1-overexpressing ESD-EpiSCs. Double immunostaining of SOX and GFP proteins in the day 4 ESD-EpiSC aggregates showed that most of the ID2-overexpressing cells were SOX-negative. Statistical analyses showed that the percentage of SOX+ and TUJ1+ cells decreased significantly among ID2-overexpressing cells, relative to negative control GFP-expressing cells, suggesting that ID2 overexpression inhibits ESD-EpiSC neural determination. Taken together, these results suggest that IDs also partially mediate BMP4 function at the second stage of ESC neural differentiation (Zhang, 2010).

DPP homologs and spermatogenesis

The specificity of bone morphogenetic proteins (BMPs) to their putative heteromeric receptor complexes in vivo is largely unclear. Closely related BMPs may use the same or different receptor complexes for signaling in a time- and space-dependent manner during development and differentiation. Bmp7 expression in epididymal epithelium is developmentally regulated. Bmp7 expression is also developmentally regulated in male germ cells. Bmp7 transcripts are detected in spermatogonia and early primary spermatocytes during early puberty and in stage-7 to -15 spermatids of the adult mice. Since Bmp7 homozygous mutants die perinatally and heterozygotes do not show obvious defects in the testis and the epididymis, the role of Bmp7 in spermatogenesis and epididymal function cannot be revealed by simply examining these mutants. Therefore, a genetic approach was used by creating Bmp7/Bmp8a double mutants to investigate the role of Bmp7 in spermatogenesis and epididymal function. Removal of one allele of Bmp7 exacerbates the phenotype of Bmp8a null mutants in spermatogenesis and epididymis of the adult. These indicate that, similar to Bmp8a, Bmp7 plays a role in both the maintenance of spermatogenesis and epididymal function and it further suggests that BMP8 and BMP7 signal through the same or similar receptors in these two systems (Zhao, 2001).

DPP homologs and genitourinary development

In humans and mice, mutations in Hoxa13 cause malformation of limb and genitourinary (GU) regions. In males, one of the most common GU malformations associated with loss of Hoxa13 function is hypospadia, a condition defined by the poor growth and closure of the urethra and glans penis. By examining early signaling in the developing mouse genital tubercle, Hoxa13 has been found to be essential for normal expression of Fgf8 and Bmp7 in the urethral plate epithelium. In Hoxa13GFP-mutant mice, hypospadias occur as a result of the combined loss of Fgf8 and Bmp7 expression in the urethral plate epithelium, as well as the ectopic expression of noggin (Nog) in the flanking mesenchyme. In vitro supplementation with Fgf8 restores proliferation in homozygous mutants to wild-type levels, suggesting that Fgf8 is sufficient to direct early proliferation of the developing genital tubercle. However, the closure defects of the distal urethra and glans can be attributed to a loss of apoptosis in the urethra, which is consistent with reduced Bmp7 expression in this region. Mice mutant for Hoxa13 also exhibit changes in androgen receptor expression, providing a developmental link between Hoxa13-associated hypospadias and those produced by antagonists to androgen signaling. Finally, a novel role for Hoxa13 in the vascularization of the glans penis is also identified (Morgan, 2003).

Urinary tract morphogenesis requires the sub-division of the ureteric bud (UB) into the intra-renal collecting system and ureter, two tissues with unique structural and functional properties. This report investigated the cellular and molecular mechanisms that mediate their differentiation. Fate mapping experiments in the developing chick indicate that the UB is surrounded by two distinct mesenchymal populations: nephrogenic mesenchyme derived from the intermediate mesoderm and tailbud-derived mesoderm, which is selectively associated with the domain of the UB that differentiates into the ureter. Functional experiments utilizing murine metanephric kidney explants show that BMP4, a paracrine factor secreted by tailbud-derived mesenchyme, is required for ureter morphogenesis. Conversely, ectopic BMP4 signaling is sufficient to induce ureter morphogenesis in domains of the UB normally fated to differentiate into the intra-renal collecting system. Collectively, these results indicate that the border between the kidney and ureter forms where mesenchymal tissues originating in two different areas of the early embryo meet. These data raise the possibility that the susceptibility of this junction to congenital defects in humans, such as ureteral-pelvic obstructions, may be related to the complex morphogenetic movements that are required to integrate cells from these different lineages into a single functional structure (Brenner-Anantharam, 2007).

DPP homologs and visceral endoderm differentiation

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo has been examined. Analysis of the expression patterns of BMP-2, -4 and -7 has shown that these BMPs are present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, is restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression results in an increase in the number of glands formed. Moreover, ectopic expression of Noggin in the proventricular mesenchyme or epithelium, antagonizing the effect of BMPs, leads to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands (Narita, 2000).

In Xenopus, XHex, coding for a homeodomain transcription factor, and cerberus, coding for a secreted head inducing factor, are early marker genes of the anterior endomesoderm (AE), a subset of endoderm cells fated to form the liver and foregut and implicated in head induction. Using XHex and cerberus as markers, the signals underlying AE induction have been examined. The AE is specified by the early blastula in the absence of mesodermal signals but cell-cell contact between presumptive AE cells is required. In overexpression experiments maternal Wnt/beta-catenin and TGF-beta signals (Vg1, Xnr1-2) can induce ectopic XHex and cerberus. Inhibiting these pathways with dominant interfering signaling components block endogenous XHex and cerberus expression. The role of signals from the organizer has been assessed. The BMP antagonists noggin and chordin are shown to be important for maintaining XHex and cerberus expression. Ventral injection of XHex mRNA can induce ectopic cerberus. These results indicate that endodermal and mesodermal patterning are closely coordinated and that the AE is likely to be specified by the combined action of dorsal Wnt/beta-catenin signals and endoderm-specific factors mediated by TGF-beta signaling. These results provide a starting point for understanding the molecular events underlying the progressive determination of endodermally derived organs, such as the liver and foregut (Zorn, 1999).

Explant culture data have suggested that the liver and pancreas originate from common progenitors. Single-cell-lineage tracing in zebrafish was used to investigate this question in vivo as well as to analyze the hepatic versus pancreatic fate decision. At early somite stages, endodermal cells located at least two cells away from the midline can give rise to both liver and pancreas. In contrast, endodermal cells closer to the midline give rise to pancreas and intestine, but not liver. Loss- and gain-of-function analyses show that Bmp2b, expressed in the lateral plate mesoderm, signals through Alk8 to induce endodermal cells to become liver. When Bmp2b was overexpressed, medially located endodermal cells, fated to become pancreas and intestine, contributed to the liver. These data provide in vivo evidence for the existence of bipotential hepatopancreatic progenitors and indicate that their fate is regulated by the medio-lateral patterning of the endodermal sheet, a process controlled by Bmp2b (Chung, 2008).

DPP homologs and ectodermal development

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously — although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype (Ma, 2003).

What factors reside upstream of Msx2 in the matrix and precortex region? During hair differentiation, Bmp4 is expressed in hair matrix cells and in hair shaft cells in contact with the IRS. Bmp2 is specifically expressed in the precortex cells, while noggin is expressed in the dermal papilla. Ectopic expression of noggin in the hair matrix under a minimal Msx2 promoter disrupts hair differentiation, with the cells remaining in a highly proliferative state in the precortex and hair shaft regions. These results provides strong evidence that BMPs are required during hair differentiation. Msx2 expression is markedly reduced in these mice in which noggin expression is driven by Msx2 promoter. Conversely, Bmp4 expression is preserved in Msx2 mutant skin. In addition, since the defects in Msx2-deficient hair follicles are mainly restricted to the hair shaft and are less severe than those associated with abolition of Bmp signaling (which involves both IRS and hair shaft), Msx2 is likely to function downstream of Bmp genes during hair differentiation (Ma, 2003).

Morphogen-dependent epidermal-specific transacting factors have not been defined in vertebrates. A member of the grainyhead transcription factor family, Grainyhead-like 1 (XGrhl1) has been identified that is essential for ectodermal ontogeny in Xenopus laevis. Expression of this factor is restricted to epidermal cells. Moreover, XGrhl1 is regulated by the BMP4 signaling cascade. Disruption of XGrhl1 activity in vivo results in a severe defect in terminal epidermal differentiation, with inhibition of XK81A1 epidermal keratin gene expression, a key target of BMP4 signaling. Furthermore, transcription of the XK81A1 gene is modulated directly by binding of XGRHL1 to a promoter-localized binding motif that is essential for high-level expression. These results establish a novel developmental role for XGrhl1 as a crucial tissue-specific regulator of vertebrate epidermal differentiation (Tao, 2005).

These studies demonstrate that XGrhl1 is a downstream epidermal-specific target of the BMP signaling. This observation varies significantly from hat reported in Drosophila, in which grh expression modulates BMP4 activity. This evolutionary divergence raises three questions: (1) is XGrhl1 necessary for BMP4-dependent epidermal specification; (2) if not, what is the role of XGrhl1 in the pathway; (3) is XGrhl1 involved in other epidermal-specific signaling events (Tao, 2005)?

Ectopic expression of BMP4 or of immediate early response (IER) factors, such as Xmad1, result in epidermal re-specification in cellular progeny of blastomeres with a neural fate. Similarly, co-expression of IER factors and BMP antagonists/inhibitors induces epidermal specification in injected ectodermal cells, with coincident repression of neural gene expression. Given the temporal pattern of endogenous expression, a similar outcome is expected with enforced expression of XGrhl1. Differing sharply from the effects of IER factors, ectopic expression of XGrhl1 fails to induce epidermal specification. These observations suggest that XGrhl1 activity is dispensable for this process, a conclusion supported by the inability of injection of Delta227XGrhl1-encoding transcripts (227XGRHL1 lacks a N-terminal activation domain encoding the first 227 amino acids, dimerizes with wild-type XGRHL1 and has comparable binding affinity to XGRHLl for a consensus Grh-binding motif) or XGrhl1-specific MOs to affect germ layer specification (Tao, 2005).

These studies suggest an alternate model, XGrhl1 functioning downstream of the IER factors in the BMP-signaling cascade. In this context, AP2 and Dlx-like factors have been shown to be essential for appropriate epidermal differentiation. However, it remains unclear how these factors achieve tissue specificity given their wider pattern of gene expression. Induction of epidermal keratin gene XK81A1 expression is dependent on appropriate XGrhl1 function. Like Dlx3, expression of XGrhl1 does not induce expression of the epidermal structural gene XK81A1 in the absence of a functional BMP4 pathway, suggesting that morphogen-induced expression of other factors is necessary. One candidate may be AP2, given its ability to rescue the epidermal defect induced by dominant-negative truncated BMP4-specific receptor (tBR) expression in a similar manner to IER regulatory factors. Furthermore, like XGrhl1, AP2 fails to repress expression of pan-neural gene markers, a divergence from the effects of IER factor expression. These observations, together with studies of the XK81A1 promoter, indicate that XGrhl1 functions predominantly downstream of the IER factors in the BMP4 signaling cascade. Furthermore, studies of the XK81A1 promoter demonstrate that both AP2 and XGRHL1 are required for XK81A1 expression (see below). Thus, it is suggested that characterization of the expression of XGrhl1 and its mechanism of action represents a significant new insight into the regulation of BMP4-responsive epidermal-specific targets, this tissue-specific factor modulating structural gene expression in concert with the more widely expressed regulator AP2 during terminal differentiation (Tao, 2005).

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). The head mesoderm is identified as the source of PPR inducing signals, reinforced by factors from the neural plate. Several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in naive ectoderm. WNT signalling plays a crucial role in restricting placode formation to the head. Finally, the decision of multipotent cells to become placode or neural crest precursors is demonstrated to be mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify (Litsiou, 2005).

This study finds that FGF signalling cooperates with WNT and BMP antagonists to impart generic placode character to uncommitted ectoderm. In the chick, activation of the FGF pathway in naive ectoderm leads to rapid expression of pre-neural markers such as Sox3 and Erni, both of which are later co-expressed at the border of the neural plate. However, activation of the FGF pathway is not sufficient to specify cells (neural crest and placode precursors) that arise from this border. The observation that continued FGF signalling is not required for pre-placodal Six4 expression, but can directly induce Eya2, suggests that FGFs may play a dual role. Early FGF signalling may confer 'border character' to ectodermal cells to make them responsive to PPR and crest inducing signals. The finding that ectopic PPR induction occurs only in the presence of active FGF signalling supports this notion. Later, FGFs from the head mesoderm, probably FGF4, initiate the expression of Eya2 in the placode territory as a crucial step to activate downstream target genes (Litsiou, 2005).

Simultaneously, the head mesoderm provides both BMP and WNT antagonists, most likely DAN and Cerberus, to counteract the inhibitory effect of both factors on the generation of placode precursors. The results show that attenuation of either the BMP or WNT pathway leads to an expansion of the PPR into the adjacent ectoderm. However, while the expansion in response to BMP inhibition is limited to the head ectoderm, WNT antagonism also results in the expression of PPR specific genes in the trunk. This is in agreement with recent findings in Xenopus reporting that simultaneous overexpression of BMP and WNT antagonist expands Six1 expression posteriorly along the induced secondary axis. In the chick, Wnt8c is expressed in trunk mesoderm and the mesoderm lateral to the heart primordium, whereas Wnt6 is found in trunk ectoderm. It is proposed that WNT activity from surrounding tissues is essential to restrict the placode territory to the head ectoderm next to the neural plate and thus ensure that sensory placodes are confined to the head. To allow placode formation, WNT antagonists in cooperation with FGF and anti-BMPs from the head mesoderm protect placode precursors from this inhibitory influence (Litsiou, 2005).

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Two signalling outcomes of the Eda pathway are reported in this study: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracts Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restores primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity is compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor NF-kappaB, but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, it is shown, in cultured embryonic skin, that Eda induces the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, the data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh is unable to rescue primary hair placode formation in Eda-deficient skin explants (Pummila, 2007).

Table of contents

decapentaplegic: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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