Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed.

The Drosophila sprouty gene encodes an antagonist of FGF and EGF signaling whose expression is induced by the signaling pathways that it inhibits. Here we describe a family of vertebrate Sprouty homologs and demonstrate that the regulatory relationship with FGF pathways has been conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there was a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3.     

Hearts and bones: shared regulatory mechanisms in heart valve, cartilage, tendon, and bone development

The atrioventricular heart valve leaflets and chordae tendineae are composed of diverse cell lineages and highly organized extracellular matrices that share characteristics with cartilage and tendon cell types in the limb buds and somites. During embryonic chicken valvulogenesis, aggrecan and sox9, characteristic of cartilage cells, are observed in the AV valve leaflets, in contrast to tendon-associated genes scleraxis and tenascin, present in the chordae tendineae. In the limb buds and somites, cartilage cell lineage differentiation is regulated by BMP2, while FGF4 controls tendon cell fate. The ability of BMP2 and FGF4 to induce similar patterns of gene expression in heart valve precursor cells was examined. In multiple assays of cells from prefused endocardial cushions, BMP2 is sufficient to activate Smad1/5/8 phosphorylation and induce sox9 and aggrecan expression, while FGF4 treatment increases phosphorylated MAPK (dpERK) signaling and promotes expression of scleraxis and tenascin. However, these treatments do not alter differentiated lineage gene expression in valve progenitors from fused cushions of older embryos. Together, these studies define regulatory pathways of AV valve progenitor cell diversification into leaflets and chordae tendineae that share inductive interactions and differentiation phenotypes with cartilage and tendon cell lineages.     

EGR1基因在牛骨骼肌卫星细胞分化不同时期的表达及定位

目的:研究EGR1基因在牛骨骼肌卫星细胞(MDSCs)分化过程的表达、定位及入核机制。方法:以牛的MDSCs为实验材料,在分化培养基中分别分化培养1 d、3 d和5 d,每组3个重复,检测不同分化时间的MDSCs中EGR1基因的表达和EGR1蛋白的定位情况;采用 CRISPRi方法干扰内源EGR1的表达,结合定点突变和激光共聚焦方法初步探索了EGR1蛋白入核的机制。结果:qRT-PCR和Western blot检测结果显示随着分化时间的进行,EGR1 基因在转录水平和蛋白水平的表达都显著高于未分化的细胞,并随时间的延长而表达逐渐升高,分化第3日时表达量最高,随后开始下降。免疫荧光检测到EGR1蛋白主要在分化的MDSCs中表达,并随肌管数量增多而表达量增加。共聚焦结果显示随着细胞分化的进行,部分EGR1蛋白转移进入细胞核。定点突变EGR1蛋白S533A后,分化的MDSCs细胞核内没有检测到EGR1蛋白。结论:在牛骨骼肌卫星细胞分化过程中,EGR1基因转录表达水平升高,部分EGR1蛋白转移入细胞核,且EGR1蛋白C端第533位丝氨酸磷酸化是入核所必需的。     

Requirements for FGF3 and FGF10 during inner ear formation

Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.     

SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.

The identification of mutations in the SRY-related SOX9 gene in patients with campomelic dysplasia, a severe skeletal malformation syndrome, and the abundant expression of Sox9 in mouse chondroprogenitor cells and fully differentiated chondrocytes during embryonic development have suggested the hypothesis that SOX9 might play a role in chondrogenesis. Our previous experiments with the gene (Col2a1) for collagen II, an early and abundant marker of chondrocyte differentiation, identified a minimal DNA element in intron 1 which directs chondrocyte-specific expression in transgenic mice. This element is also a strong chondrocyte-specific enhancer in transient transfection experiments. We show here that Col2a1 expression is closely correlated with high levels of SOX9 RNA and protein in chondrocytes. Our experiments indicate that the minimal Col2a1 enhancer is a direct target for Sox9. Indeed, SOX9 binds to a sequence of the minimal Col2a1 enhancer that is essential for activity in chondrocytes, and SOX9 acts as a potent activator of this enhancer in cotransfection experiments in nonchondrocytic cells. Mutations in the enhancer that prevent binding of SOX9 abolish enhancer activity in chondrocytes and suppress enhancer activation by SOX9 in nonchondrocytic cells. Other SOX family members are ineffective. Expression of a truncated SOX9 protein lacking the transactivation domain but retaining DNA-binding activity interferes with enhancer activation by full-length SOX9 in fibroblasts and inhibits enhancer activity in chondrocytes. Our results strongly suggest a model whereby SOX9 is involved in the control of the cell-specific activation of COL2A1 in chondrocytes, an essential component of the differentiation program of these cells. We speculate that in campomelic dysplasia a decrease in SOX9 activity would inhibit production of collagen II, and eventually other cartilage matrix proteins, leading to major skeletal anomalies.     

Perichondrial expression of Wdr5 regulates chondrocyte proliferation and differentiation

Wdr5 is developmentally expressed in osteoblasts and is required for osteoblast differentiation. Mice overexpressing Wdr5 under the control of the mouse α(1)I collagen promoter (Col I-Wdr5) display accelerated osteoblast differentiation as well as accelerated chondrocyte differentiation, suggesting that overexpression of Wdr5 in osteoblasts affects chondrocyte differentiation. To elucidate the molecular mechanism by which overexpression of Wdr5 in the perichondrium regulates chondrocyte differentiation, studies were undertaken using skeletal elements and cultured metatarsals isolated from wild-type and Col I-Wdr5 embryos. FGF18 mRNA levels were decreased in Col I-Wdr5 humeri. Furthermore, local delivery of FGF18 to the bone collar of ex vivo cultures of metatarsals attenuated the chondrocyte phenotype of the Col I-Wdr5 metatarsals. Impairing local FGF action in wild-type metatarsals resulted in a chondrocyte phenotype analogous to that of Col I-Wdr5 metatarsals implicating impaired FGF action as the cause of the phenotype observed. The expression of Twist-1, which regulates chondrocyte differentiation, was increased in Col I-Wdr5 humeri. Chromatin immunoprecipitation analyses demonstrated that Wdr5 is recruited to the Twist-1 promoter. These findings support a model in which overexpression of Wdr5 in the perichondrium promotes chondrocyte differentiation by modulating the expression of Twist-1 and FGF18.     

Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

Recent evidences have suggested that humoral factors released from the appropriate co-cultured cells influenced the expansion and differentiation of mesenchymal stem cells (MSCs). However, little is known about the proliferation and differentiation of MSCs subjected to co-culture condition with tenocytes. In this study, we aimed to establish a co-culture system of MSCs and tenocytes and investigate the proliferation and tendon/ligament related gene expression of MSCs. MTT assay was used to detect the expansion of MSCs. Semi-quantitative RT-PCR was performed to investigate the expression of proliferation associated c-fos gene and tendon/ligament related genes, including type I collagen (Col I), type III collagen (Col III), tenascin C and scleraxis. Significant increase in MSCs expansion was observed after 3 days of co-culture with tenocytes. The c-fos gene expression was found distinctly higher than for control group on day 4 and day 7 of co-culture. The mRNA expression of four tendon/ligament related genes was significantly up-regulated after 14 days of co-culture with tenocytes. Thus, our research indicates that indirect co-culture with tenocytes promotes the proliferation and mRNA expression of tendon/ligament related genes in MSCs, which suggests a directed differentiation of MSCs into tendon/ligament.     

Six1 is not involved in limb tendon development, but is expressed in limb connective tissue under Shh regulation

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3-/- mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1-/- mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.