In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth

The apical ectodermal ridge (AER) in the vertebrate limb is required for limb outgrowth and patterning. To investigate the role BMP ligands expressed in the AER play in limb development we selectively inactivated both Bmp2 and Bmp4 in this tissue. The autopods of mice lacking both of these genes contained extra digits, digit bifurcations and interdigital webbing due to a decrease in programmed cell death and an increase in cell proliferation in the underlying mesoderm. Upon removal of Bmp2 and Bmp4 in the AER, no defects in proximal-distal patterning were observed. At the molecular level, removal of Bmp2 and Bmp4 in the AER caused an increase in Fgf expression, which correlated with an increase in both the width and length of the AER. Investigation of Engrailed-1 (En1) expression in the AER of limb buds in which Bmp2 and Bmp4 had been removed indicated that En1 expression was absent from this tissue. Our data suggests that AER expression of Bmp2 and Bmp4 is required for digit and dorsal-ventral patterning but surprisingly not for limb outgrowth.     

Bmp signaling in molar cusp formation

Tooth cusp is a crucial structure, since the shape of the molar tooth is determined by number, shape, and size of the cusp. Bone morphogenetic protein (Bmp) signaling is known to play a critical role in tooth development, including in initiation. However, it remains unclear whether Bmp signaling is also involved in cusp formation. To address this question, we examined cusp in two different transgenic mouse lines: mice with overexpression of Bmp4 (K14-Bmp4), and those with Bmp inhibitor, Noggin, (K14-Noggin) under keratin14 (K14) promoter. K14-Noggin mice demonstrated extra cusps, whereas reduced number of cusps was observed in K14-Bmp4 mice. To further understand how Bmps are expressed during cusp formation, we performed whole-mount in situ hybridisation analysis of three major Bmps (Bmp2, Bmp4, and Bmp7) in murine maxillary and mandibular molars from E14.5 to P3. The linear expressions of Bmp2 and Bmp4 were observed in both maxillary and mandibular molars at E14.5. The expression patterns of Bmp2 and Bmp4 became significantly different between the maxillary and mandibular molars at E16.5. At P3, all Bmps were expressed in all the cusp regions of the maxillary molar; however, the patterns differed. All Bmps thus exhibited dynamic temporo-spatial expression during the cusp formation. It could therefore be inferred that Bmp signaling is involved in regulating cusp formation.     

Bmp4 and Noggin expression during early thymus and parathyroid organogenesis

The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have previously shown that thymus and parathyroid-specific organ domains are established within the primordium prior to formation of the organs proper: Gcm2 expression defines the prospective parathyroid cells in the dorsal pouch from E9.5, while Foxn1 is expressed in the thymus domain from E11.25. Bmp (bone morphogenetic protein) signaling has been implicated in thymic epithelial cell differentiation and thymus organogenesis. In the present study, we report expression patterns of Bmp4 and Noggin, a Bmp4 antagonist, in the third pharyngeal pouch using two lacZ transgenic mouse strains. Results from this gene expression study revealed localization of Bmp4 expression to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5, in those cells that will express Foxn1 and form the thymus. Conversely, the expression of Noggin was confined to the dorsal region of the pouch and primordium at these stages, and thus appeared to be co-expressed with Gcm2 in the parathyroid domain. This represents the first detailed study of Bmp4 and Noggin expression during the early stages of thymus and parathyroid organogenesis.     

Dissection of promoter control modules that direct Bmp4 expression in the epithelium-derived components of hair follicles

Bone morphogenetic protein 4 (Bmp4) plays a significant role in development. Using transgenic approaches, we studied the mechanisms controlling Bmp4 expression during primordial and mature tissue development, as well as in epithelium- and mesenchyme-derived components with hair follicles as a model. In this report, we demonstrated that the promoter region between the -0.26 and the -1.14 kb, highly conserved between human and mouse, controls Bmp4 expression in the epithelium-derived tissues but not in mesenchyme-derived tissues of hair follicles, suggesting that control modules for Bmp4 expression in epithelium-derived tissues and mesenchyme originated tissues are in separate regions. Using live embryos and mice, we demonstrated the temporal and spatial activities of these modules. We also demonstrated that control regions for Bmp4 expression in primordial and differentiated hair follicle tissues are separated. Therefore we present a model to explain a mechanism controlling expression of the Bmp4 gene in different tissue types, as well as different development stages as related to hair development.     

Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis

Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the anterior foregut where the tracheal primordium originates and targeted ablation of Bmp4 (Bmp4^cko) resulted in a loss-of-trachea phenotype that closely resembles the Floyd type II pathology, the most common form of TA in humans. In Bmp4^cko embryos, tracheal specification was not affected; however, its outgrowth was severely impaired due to reduced epithelial and mesenchymal proliferation. In agreement, we also observed significant reduction in the expression of Cyclin D1, a key cell cycle regulator associated with cellular proliferation. However, the proliferative effect of Bmp signaling appears to be independent of Wnt signaling. Interestingly, we found significantly reduced expression of activated extracellular signal-regulated kinase (Erk) in the Bmp4^cko ventral foregut, suggesting that Bmp signaling promotes Erk phosphorylation which has been associated with cellular proliferation. This study provides the first evidence linking Bmp signaling to tracheal formation by regulating the proliferative response of the anterior ventral foregut. Our finding sheds light on human tracheal malformations by providing a novel mouse model implicating Bmp signaling, non-canonical Erk activation and cellular proliferation.     

Developmental expression and function of Bmp4 in spermatogenesis and in maintaining epididymal integrity

Bone morphogenetic proteins (BMPs) play essential roles in many aspects of developmental biology. We have previously shown that Bmp7, Bmp8a, and Bmp8b of the 60A class of Bmp genes have additive effects in spermatogenesis and in maintaining the epididymal integrity of the caput and caudal regions. Here we report that Bmp4 of the Dpp class has a unique expression pattern in the developing testis and epididymis. Bmp4 heterozygous males on a largely C57BL/6 background show compromised fertility due to degeneration of germ cells, reduced sperm counts, and decreased sperm motility. More interestingly, some of these males show extensive degeneration of the epididymal epithelium in the corpus region, rather than in the caput and cauda regions as for Bmp7 and Bmp8 mutants. Thus, these genetic data reveal a region-specific requirement of different classes of BMPs for epididymal epithelium to survive and have significant implications on male reproductive health and perhaps birth control.     

Zebrafish Bmp4 regulates left-right asymmetry at two distinct developmental time points

Left-right (LR) asymmetry is regulated by early asymmetric signals within the embryo. Even though the role of the bone morphogenetic protein (BMP) pathway in this process has been reported extensively in various model organisms, opposing models for the mechanism by which BMP signaling operates still prevail. Here we show that in zebrafish embryos there are two distinct phases during LR patterning in which BMP signaling is required. Using transgenic lines that ectopically express either noggin3 or bmp2b, we show a requirement for BMP signaling during early segmentation to repress southpaw expression in the right lateral plate mesoderm and regulate both visceral and heart laterality. A second phase was identified during late segmentation, when BMP signaling is required in the left lateral plate mesoderm to regulate left-sided gene expression and heart laterality. Using morpholino knock down experiments, we identified Bmp4 as the ligand responsible for both phases of BMP signaling. In addition, we detected bmp4 expression in Kupffer's vesicle and show that restricted knock down of bmp4 in this structure results in LR patterning defects. The identification of these two distinct and opposing activities of BMP signaling provides new insight into how BMP signaling can regulate LR patterning.     

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung‐to‐gas bladder evolutionary transition in ray‐finned fishes

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray‐finned fishes is the gas bladder, an air‐filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe‐finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral‐to‐dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray‐finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.     

Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.     

Deltex/Dtx mediates NOTCH signaling in regulation of Bmp4 expression in cranial neural crest formation during avian development

NOTCH signaling plays a key role in cell fate determination in both vertebrates and invertebrates. It is well known that Su(H)/RBP-J is a major mediator of NOTCH signaling. In a previous study, it was shown that NOTCH signaling was involved in cranial neural crest formation in avian embryos. However, Su(H)/RBP-J activity did not appear to be required in this process. In this study, the Deltex/Dtx gene was focussed on as a potential mediator of NOTCH signaling in neural crest formation. At the time of neural crest formation, quail Deltex2 was expressed throughout the ectoderm. Misexpression of a dominant-negative form of Deltex in the ectoderm caused reduced expression of Slug, a neural crest marker. Dominant-negative Deltex expression reduced the expression of Bmp4, a neural crest inducer, whereas co-transfection of Bmp4 with dominant-negative Deltex rescued Slug expression. In parallel, Hairy2 expression in the epidermis was regulated by a Su(H)-dependent pathway. These results indicate that NOTCH signaling has dual functions mediated by either Su(H) or Deltex in the avian embryonic ectoderm.     

贵州白山羊Bmp15和Gdf9基因多态性及其编码蛋白的进化分析(英文)

BMP15和GDF9是转化生长因子β（TGFβ）超家族的成员,对绵羊的繁殖性状有直接的调节作用,从中发现的多个高产突变位点直接提高了排卵数和产羔数。在之前的研究中,作者从贵州白山羊中找到了一个高产突变位点。为了进一步揭示Bmp15和Gdf9基因突变与繁殖性状之间的关系,对贵州白山羊Bmp15和Gdf9基因编码区进行了克隆,以人BMP7的晶体结构为模板构建了贵州白山羊BMP15和GDF9成熟肽的三维模型。贵州白山羊Bmp15和Gdf9基因分别编码394和453个氨基酸的蛋白前体。对BMP15和GDF9成熟肽序列进行分析发现,除了之前确认的BMP15中的FecX^B 突变（S99I）和GDF9中的V79I突变之外,还从贵州白山羊的BMP15和GDF9成熟肽分别发现7个和3个位点突变。其中,BMP15成熟肽的S32G、N66H、S99I/P99I和G107R突变可能影响二聚体与受体的结合;GDF9成熟肽的P78Q和V79I影响二聚体与I型受体的亲和力,将值得进一步深入研究。对Bmp15和Gdf9基因编码的蛋白前体序列进行聚类分析,结果显示在鱼类到哺乳类的进化过程中,BMP15出现长度逐渐增加的现象,以BMP15成熟肽N端长度增加为主。这种演变可能使BMP15对低排卵哺乳动物繁殖力的控制更为灵敏。该文的研究结果为贵州白山羊Bmp15和Gdf9基因变异与繁殖力的关系提出了合理的解释,并支持这两个因子是贵州白山羊高产性状重要调节因子的观点。     

Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cell-autonomous and translation-dependent manner

The intermediate mesoderm lies between the somites and the lateral plate and is the source of all kidney tissue in the developing vertebrate embryo. While bone morphogenetic protein (Bmp) signaling is known to regulate mesodermal cell type determination along the medio-lateral axis, its role in intermediate mesoderm formation has not been well characterized. The current study finds that low and high levels of Bmp ligand are both necessary and sufficient to activate intermediate and lateral mesodermal gene expression, respectively, both in vivo and in vitro. Dose-dependent activation of intermediate and lateral mesodermal genes by Bmp signaling is cell-autonomous, as demonstrated by electroporation of the avian embryo with constitutively active Bmp receptors driven by promoters of varying strengths. In explant cultures, Bmp activation of Odd-skipped related 1 (Odd-1), the earliest known gene expressed in the intermediate mesoderm, is blocked by cyclohexamide, indicating that the activation of Odd-1 by Bmp signaling is translation-dependent. The data from this study are integrated with that of other studies to generate a model for the role of Bmp signaling in trunk mesodermal patterning in which low levels of Bmp activate intermediate mesoderm gene expression by inhibition of repressors present in medial mesoderm, whereas high levels of Bmp repress both medial and intermediate mesoderm gene expression and activate lateral plate genes.     

Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos

To address the patterning function of the Bmp2, Bmp4 and Bmp7 growth factors, we designed antisense morpholino oligomers (MO) that block their activity in Xenopus laevis. Bmp4 knockdown was sufficient to rescue the ventralizing effects caused by loss of Chordin activity. Double Bmp4 and Bmp7 knockdown inhibited tail development. Triple Bmp2/Bmp4/Bmp7 depletion further compromised trunk development but did not eliminate dorsoventral patterning. Unexpectedly, we found that blocking Spemann organizer formation by UV treatment or beta-Catenin depletion caused BMP inhibition to have much more potent effects, abolishing all ventral development and resulting in embryos having radial central nervous system (CNS) structures. Surprisingly, dorsal signaling molecules such as Chordin, Noggin, Xnr6 and Cerberus were not re-expressed in these embryos. We conclude that BMP inhibition is sufficient for neural induction in vivo, and that in the absence of ventral BMPs, Spemann organizer signals are not required for brain formation.     

过表达Msx1、Pax9和Bmp4对牙髓干细胞牙向分化的影响

在组织工程研究领域中,利用干细胞进行牙齿再生是一种途径。目前,研究认为牙齿的发育过程是上皮与间充质相互诱导的结果,利用干细胞进行再生牙齿时也需要有上皮源性和间充质源性干细胞的参与。牙髓干细胞是牙齿自体的干细胞,具有多向分化潜能,在牙齿再生中是一种理想的间充质源性干细胞。该研究通过慢病毒介导在牙髓干细胞中分别过表达人Msx1、Pax9和Bmp4基因,研究其对牙向分化的诱导潜能。过表达这三个基因均能显著提高牙髓干细胞碱性磷酸酶的水平,并且促使牙髓干细胞表达成牙本质细胞标志蛋白——牙本质涎磷蛋白、骨钙素、骨桥素和形成钙化组织。但在诱导牙向分化的能力上,三个基因有一定的区别。过表达Msx1基因对牙髓干细胞体外诱导牙向分化能力最为明显,其次是Bmp4基因,过表达Pax9在促进牙髓干细胞表达骨桥素和钙质形成上不是很显著。     

Anterior–posterior patterning of neural differentiated embryonic stem cells by canonical Wnts,Fgfs, Bmp4 and their respective antagonists

Embryonic stem (ES) cells are pluripotent and can differentiate into every cell type of the body. Next to their potential in regenerative medicine, they are excellent tools to study embryonic development. In this work the processes of neural induction and neural patterning along the antero‐posterior (A/P) body axis are studied and evidence suggests a two step mechanism for these events. First, neural induction occurs by default in the primitive ectoderm, forming anterior neural tissue and thereafter, a series of factors can posteriorize this anterior neurectoderm. In a gain‐of‐function/loss‐of‐function approach using mouse ES cells, we show that Fgf2 has the strongest caudalizing potential of all Fgfs tested. Furthermore, Bmp4 and Wnt3a, but not Wnt1, can caudalize the neurectodermal cells. The effect of the antagonists of these factors was also examined and though Dkk1 and Noggin clearly have an effect that opposes that of Wnt3a and Bmp4 respectively, they fail to anteriorize the neurectoderm. The patterning effect of SU5402, an Fgf receptor inhibitor, was rather limited. These data confirm that in the mouse, two steps are involved in neural patterning and we show that while Fgf4, Fgf8 and Wnt1 have no strong patterning effect, Fgf2, Wnt3a and Bmp4 are strong posteriorizing factors.     

Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.     

Abnormal glucose metabolism in heterozygous mutant mice for a type I receptor required for BMP signaling

BMPRIA and its high‐affinity ligand BMP4 have recently been shown to be expressed in the β‐cells of the pancreas. Here, we report the abnormalities of heterozygous mice for Bmpr1a in glucose metabolism during the course of intraperitoneal glucose tolerance test. The heterozygous mice had increased blood glucose levels throughout the first 2.5 h after the administration of glucose. Analysis of glucose‐stimulated insulin secretion (GSIS) indicates that insulin secretion in the heterozygous mice is compromised, and induction of secreted insulin by stimulation is substantially lower compared with the wild‐type controls. No apparent abnormalities in pancreas, thyroid, and liver were seen upon histological examination. Real‐time PCR results of selected genes showed an increase in the mRNA level of Ins1 and Ins2 in the heterozygous group. These results indicate that the glucose‐sensing pathway in these heterozygous mice is altered because of the heterozygosity in Bmpr1a. Together, our data suggest that BMP signaling through BMPRIA plays an important role in glucose metabolism and possibly working through the GSIS pathway. genesis 47:385–391, 2009. © 2009 Wiley‐Liss, Inc.     

Mypt1 regulates Bmp signaling to promote embryonic exocrine pancreas growth in zebrafish

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 ^sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 ^sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss‐of‐function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.     

Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.