BMP-7/OP-1, a member of the transforming growth factor-beta (TGF-beta) family of secreted growth factors, is expressed during mouse embryogenesis in a pattern suggesting potential roles in a variety of inductive tissue interactions. The present study demonstrates that mice lacking BMP-7 display severe defects confined to the developing kidney and eye. Surprisingly, the early inductive tissue interactions responsible for establishing both organs appear largely unaffected. However, the absence of BMP-7 disrupts the subsequent cellular interactions required for their continued growth and development. Consequently, homozygous mutant animals exhibit renal dysplasia and anophthalmia at birth. Overall, these findings identify BMP-7 as an essential signaling molecule during mammalian kidney and eye development.

Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors,integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf-/- mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1,Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclearβ-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.

In the olfactory epithelium (OE), generation of new neurons by neuronal progenitors is inhibited by a signal from neurons themselves. Here we provide evidence that this feedback inhibitory signal is growth and differentiation factor 11 (GDF11). Both GDF11 and its receptors are expressed by OE neurons and progenitors, and GDF11 inhibits OE neurogenesis in vitro by inducing p27(Kip1) and reversible cell cycle arrest in progenitors. Mice lacking functional GDF11 have more progenitors and neurons in the OE, whereas mice lacking follistatin, a GDF11 antagonist, show dramatically decreased neurogenesis. This negative autoregulatory action of GDF11 is strikingly like that of its homolog, GDF8/myostatin, in skeletal muscle, suggesting that similar strategies establish and maintain proper cell number during neural and muscular development.

Previous studies have demonstrated the ability of bone morphogenetic proteins (BMPs) to promote chondrogenic differentiation in vitro. However, the in vivo role of BMP signaling during chondrogenesis has been unclear. We report here that BMP signaling is essential for multiple aspects of early chondrogenesis. Whereas mice deficient in type 1 receptors Bmpr1a or Bmpr1b in cartilage are able to form intact cartilaginous elements, double mutants develop a severe generalized chondrodysplasia. The majority of skeletal elements that form through endochondral ossification are absent, and the ones that form are rudimentary. The few cartilage condensations that form in double mutants are delayed in the prechondrocytic state and never form an organized growth plate. The reduced size of mutant condensations results from increased apoptosis and decreased proliferation. Moreover, the expression of cartilage-specific extracellular matrix proteins is severely reduced in mutant elements. We demonstrate that this defect in chondrocytic differentiation can be attributed to lack of Sox9, L-Sox5, and Sox6 expression in precartilaginous condensations in double mutants. In summary, our study demonstrates that BMPR1A and BMPR1B are functionally redundant during early chondrogenesis and that BMP signaling is required for chondrocyte proliferation, survival, and differentiation in vivo.bone morphogenetic protein ͉ cartilage ͉ endochondral ossification ͉ Sox proteins ͉ skeletal development D uring endochondral ossification, mesenchymal cells condense and differentiate into chondrocytes. The chondrocytes undergo a highly organized differentiation program, forming the template for bone formation (1, 2). Bone morphogenetic proteins (BMPs) were identified by their ability to promote ectopic cartilage and bone formation (3). BMPs are members of the TGF␤ superfamily and transduce signals by binding to heteromeric complexes of type 1 and type 2 serine͞threonine kinase receptors. The binding of BMPs to the receptor complex results in the phosphorylation of intracellular Smads, which then translocate to the nucleus, where they regulate transcription (4). The differential affinities of distinct BMP ligands for the three type 1 receptors, BMPR1A (BMP receptor type 1A), BMPR1B (BMP receptor type 1B), and ActR1 (activin receptor type 1), are thought to contribute to the diversity of actions during development. Several lines of evidence suggest that Bmpr1a, Bmpr1b, and ActR1 have distinct roles during chondrogenesis. In the chick, Bmpr1a is expressed at low levels throughout the limb bud mesenchyme, whereas Bmpr1b is expressed in precartilaginous condensations, suggesting that these receptors have different roles in chondrogenesis. Furthermore, whereas constitutively active forms of either BMPR1A or BMPR1B promote chondrogenesis, only the overexpression of dominant negative (DN)-BMPR1B, and not DN-BMPR1A or DN-ActR1, blocks these events, suggesting that BMPR1B is the major transducer of BMP signals in limb condensations (5-8). However, Bmpr1b null mic...

Bone morphogenetic protein (BMP) signaling is required for endochondral bone formation. However, whether or not the effects of BMPs are mediated via canonical Smad pathways or through noncanonical pathways is unknown. In this study we have determined the role of receptor Smads 1, 5 and 8 in chondrogenesis. Deletion Development 136, 1093Development 136, -1104Development 136, (2009 signaling. Finally, we provide evidence that linker phosphorylation of Smads represents a physiologically significant mechanism regulating BMP signaling in the growth plate, but that the inhibitory effects of FGFs are likely to be mediated through different mechanisms. ). MATERIALS AND METHODS Generation of HistologySkeletal preparations were generated as described (Ivkovic et al., 2003;Yoon et al., 2006). Alcian Blue/nuclear Fast Red staining was performed as described (Luna, 1992). Von Kossa staining was performed by incubation in 1% silver nitrate under UV light for 20 minutes and counterstaining with nuclear Fast Red. Safranin O staining was performed by staining in Weigert's iron hematoxylin solution for 10 minutes, followed by Fast Green (0.001%) and Safranin O (0.1%) for 5 minutes each.For immunofluorescence, sections were boiled for 15 minutes in citrate buffer (Ivkovic et al., 2003). Sections were blocked with 5% goat or donkey serum for 1 hour and incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 hour at room temperature, then with fluorophore for 30 minutes at room temperature. Primary antibodies were as follows: phospho-Smad1/5/8 and phospho-Smad1/5 (Cell Signaling Technology); type II collagen and Pth1r (Abcam); type I collagen (Southern Biotech); type X collagen (a kind gift from Robin Poole, Shriners Hospitals for Children, Montreal, Québec, Canada); aggrecan (Developmental Studies Hybridoma Bank, Iowa City, USA); Pcna (Zymed); Fgfr1 and Stat1 (Sigma); phosphoSmad1L (a kind gift from Eddy De Robertis, University of California, Los Angeles, CA, USA). Secondary antibodies were conjugated with AlexaFluor-555 and AlexaFluor-488. Sections were counterstained with DAPI (Vectashield). For TUNEL staining, the fluorescein In Situ Cell Death Detection Kit (Roche) was used according to the manufacturer's protocol. In situ hybridization was performed as described (Song et al., 2007). Limb cultureEmbryos were harvested at 16.5 days of gestation (E16.5). Forelimbs were isolated and cultured as described (Minina et al., 2001;Minina et al., 2002). The contralateral limb was cultured in the presence of recombinant human FGF18 (10 ng/ml; Invitrogen) or the FGFR inhibitor SU5402 (10 μM; Calbiochem). In all cases, the right forelimb served as the untreated control. A total of six limbs were examined for each condition, in two separate experiments. RT-PCR and western analysis of growth plate cartilageRNA was extracted from proximal humeri using the RNeasy Kit (Qiagen). Synthesis of cDNA was performed with Superscript III (Invitrogen). Reverse transcriptase (RT)-PCR reactions compris...

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding ␣-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu؊͞؊ mice were healthy and fertile while young and could survive for 8 -12 mo. They were totally deficient in ␣-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides G M2 and GM3 in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-moold Naglu؊͞؊ mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the ␣-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

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