Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis.
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ABSTRACT:
SUBMITTER: Bandyopadhyay A
PROVIDER: S-EPMC1713256 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:BMP7/BMP2 or BMP7/BMP4 heterodimers are more active than homodimers in vitro, but it is not known whether these heterodimers signal in vivo. To test this, we generated knock in mice carrying a mutation (Bmp7R-GFlag) that prevents proteolytic activation of the dimerized BMP7 precursor protein. This mutation eliminates the function of BMP7 homodimers and all other BMPs that normally heterodimerize with BMP7. While Bmp7 null homozygotes are live born, Bmp7R-GFlag homozygotes are embryonic lethal and have broadly reduced BMP activity. Furthermore, compound heterozygotes carrying the Bmp7R-G allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis with defects in ventral body wall closure and/or the heart. Co-immunoprecipitation assays confirm that endogenous BMP4/7 heterodimers exist. Thus, BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian development, which may explain why mutations in either Bmp4 or Bmp7 lead to a similar spectrum of congenital defects in humans.
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Project description:Feathers, like hairs, first appear as primordia consisting of an epidermal placode associated with a dermal condensation that is necessary for the continuation of their differentiation. Previously, the BMPs have been proposed to inhibit skin appendage formation. We show that the function of specific BMPs during feather development is more complex. BMP2 and BMP7, which are expressed in both the epidermis and the dermis, are involved in an antagonistic fashion in regulating the formation of dermal condensations, and thus are both necessary for subsequent feather morphogenesis. BMP7 is expressed earlier and functions as a chemoattractant that recruits cells into the condensation, whereas BMP2 is expressed later, and leads to an arrest of cell migration, likely via its modulation of the EIIIA fibronectin domain and alpha4 integrin expression. Based on the observed cell proliferation, chemotaxis and the timing of BMP2 and BMP7 expression, we propose a mathematical model, a reaction-diffusion system, which not only simulates feather patterning, but which also can account for the negative effects of excess BMP2 or BMP7 on feather formation.
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Project description:The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense agrin research very little is known about the function of the other isoforms and the function of the larger, N-terminal part of agrins that is common to all isoforms. Since the N-terminal part of agrins contains several follistatin-domains, a domain type that is frequently implicated in binding TGFbetas, we have explored the interaction of the N-terminal part of rat agrin (Agrin-Nterm) with members of the TGFbeta family using surface plasmon resonance spectroscopy and reporter assays. Here we show that agrin binds BMP2, BMP4 and TGFbeta1 with relatively high affinity, the K(D) values of the interactions calculated from SPR experiments fall in the 10(-8) M-10(-7) M range. In reporter assays Agrin-Nterm inhibited the activities of BMP2 and BMP4, half maximal inhibition being achieved at approximately 5x10(-7) M. Paradoxically, in the case of TGFbeta1 Agrin N-term caused a slight increase in activity in reporter assays. Our finding that agrin binds members of the TGFbeta family may have important implications for the role of these growth factors in the regulation of synaptogenesis as well as for the role of agrin isoforms that are unable to induce clustering of acetylcholine receptors. We suggest that binding of these TGFbeta family members to agrin may have a dual function: agrin may serve as a reservoir for these growth factors and may also inhibit their growth promoting activity. Based on analysis of the evolutionary history of agrin we suggest that agrin's growth factor binding function is more ancient than its involvement in acetylcholine receptor clustering.
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Project description:Bardet-Biedl syndrome (BBS) is a pleiotropic, genetically heterogeneous disorder characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies, as well as hypertension and diabetes. Multiple genes are known to independently cause BBS. These genes do not appear to code for the same functional category of proteins; yet, mutation of each results in a similar phenotype. Gene knockdown of different BBS genes in zebrafish shows strikingly overlapping phenotypes including defective melanosome transport and disruption of the ciliated Kupffer's vesicle. Here, we demonstrate that individual knockdown of bbs1 and bbs3 results in the same prototypical phenotypes as reported previously for other BBS genes. We utilize the zebrafish system to comprehensively determine whether simultaneous pair-wise knockdown of BBS genes reveals genetic interactions between BBS genes. Using this approach, we demonstrate eight genetic interactions between a subset of BBS genes. The synergistic relationships between distinct combinations are not due to functional redundancy but indicate specific interactions within a multi-subunit BBS complex. In addition, we utilize the zebrafish model system to investigate limb development. Human polydactyly is a cardinal feature of BBS not reproduced in BBS-mouse models. We evaluated zebrafish fin bud patterning and observed altered Sonic hedgehog (shh) expression and subsequent changes to fin skeletal elements. The SHH fin bud phenotype was also used to confirm specific genetic interactions between BBS genes. This study reveals an in vivo requirement for BBS function in limb bud patterning. Our results provide important new insights into the mechanism and biological significance of BBS.
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Project description:HypothesisGenetic variation in BMP2 and BMP4 found in otosclerosis patients result in altered Smad signaling.BackgroundOtosclerosis is a common form of adult-onset conductive hearing loss resulting from abnormal bone remodeling of the bony labyrinth that surrounds the inner ear. Both genetic and environmental factors are implicated in the disease, yet very little is known about its pathogenesis. The evidence for a genetic component has been established through family-based linkage and population-based association studies. Previously, members of the TGF-β superfamily of genes have been associated with otosclerosis.MethodsSequencing of BMP2 and BMP4 coding regions was performed to identify common and rare variation in German otosclerosis patients compared with controls. Functional analyses of rare variation in the patient cohort were conducted by exposing an osteosarcoma cell line to conditioned media containing either wild type or variant forms of BMP2 or BMP4 and analyzing Smad1/5/8 phosphorylation.ResultsAlthough no significant association with common variation in these 2 genes was detected, there were 8 singleton variants identified in the German population. Of the 4 coding variants found solely in otosclerosis patients, two--BMP4(N150K) and BMP2(K357-R396del)--were found to decrease Smad1/5/8 signaling.ConclusionRare variants in BMP2 and BMP4 are not a major genetic component in the otosclerosis population. However, those with functional affect showed decreased Smad signaling. Further analysis of Smad signaling molecules should be performed to determine if these pathways in combination are a major contributor to otosclerosis, which could lead to additional treatment options for otosclerosis patients.
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Project description:Half a century ago, the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal-distal (P-D) axis, but how these signals influence limb patterning is still much debated. Fibroblast growth factor (FGF) gene family members are key AER-derived signals, with Fgf4, Fgf8, Fgf9 and Fgf17 expressed specifically in the mouse AER. Here we demonstrate that mouse limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P-D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P-D patterning can be integrated with the concept of early specification to explain the genetic data presented here.
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