Notch signaling is a central mechanism for controlling embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and specifically, in bone homeostasis remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis due to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by up-regulating Cyclin D, Cyclin E, and Osterix. Notch also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all physiologic Notch signaling in osteoblasts, generated by deletion of Presenilin 1 and 2 in bone, is associated with late onset, age-related osteoporosis resulting from increased osteoblast-dependent osteoclastic activity due to decreased production of Osteoprotegerin. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.Evolutionarily conserved Notch signaling plays a critical role in cell fate determination, and various developmental processes by translating cell-cell interactions into specific transcriptional programs 1, 2 . Temporal and spatial modulation of this pathway can significantly affect proliferation, differentiation and apoptotic events 3 . Moreover, the timing of Notch signaling can lead to diverse effects within the same cell lineage 4, 5 . In mammals, activation of up to four Notch receptors by membrane-bound ligands initiates a process leading to presenilin-mediated cleavage and release of the Notch intracellular domain (NICD) from the membrane that then traffics to the nucleus. NICD subsequently regulates the expression of genes in cooperation with the transcription factor RBP-Jκ and Mastermind-like proteins.The observation that mutations in the Notch ligand Delta homologue-3 (Dll-3) and γ-secretase Presenilin1 both cause axial skeletal phenotypes originally linked Notch signaling with skeletal development 6, 7 . Recently, several in vitro studies with conflicting results implicated the Notch pathway in the regulation of osteoblast differentiation, but the in vivo role of Notch signaling in bone homeostasis still remains unknown 8-12 .Corresponding Author: Brendan Lee, M.D., Ph.D., One Baylor Plaza, Rm 635E, Houston, Tx 77030,, Email E-mail: blee@bcm.tmc.edu. In this study, we investigate the tissue, cellular, and molecular consequences of both gain and loss of function of Notch signaling in committed osteoblasts. NIH Public Access RESULTS Gain of function of Notch signaling results in severe osteosclerosisTo determine the pathological consequences of in vivo gain of Notch function during bone formation and homeostasis, we generated transgenic mice expressing the Notch1 intracellular domain (N1ICD) under the control of the type I collagen (Col1a1) promoter (Suppl. Fig. 1a,b). Here, gain of Notch function would occur in committed osteoblastic ce...

The α1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte–specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive β-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2 +/− background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

The LIM-homeodomain protein Lmx1b plays a central role in dorso-ventral patterning of the vertebrate limb. Targeted disruption of Lmx1b results in skeletal defects including hypoplastic nails, absent patellae and a unique form of renal dysplasia (see accompanying manuscript by H. Chen et al.; ref. 2). These features are reminiscent of the dominantly inherited skeletal malformation nail patella syndrome (NPS). We show that LMX1B maps to the NPS locus and that three independent NPS patients carry de novo heterozygous mutations in this gene. Functional studies show that one of these mutations disrupts sequence-specific DNA binding, while the other two mutations result in premature termination of translation. These data demonstrate a unique role for LMX1B in renal development and in patterning of the skeletal system, and suggest that alteration of Lmx1b/LMX1B function in mice and humans results in similar phenotypes. Furthermore, we provide evidence for the first described mutations in a LIM-homeodomain protein which account for an inherited form of abnormal skeletal patterning and renal failure.

Mesenchymal stem cell-derived osteochondroprogenitors express two master transcription factors, SOX9 and RUNX2, during condensation of the skeletal anlagen. They are essential for chondrogenesis and osteogenesis, respectively, and their haploinsufficiency causes human skeletal dysplasias. We show that SOX9 directly interacts with RUNX2 and represses its activity via their evolutionarily conserved high-mobility-group and runt domains. Ectopic expression of full-length SOX9 or its RUNX2-interacting domain in mouse osteoblasts results in an osteodysplasia characterized by severe osteopenia and down-regulation of osteoblast differentiation markers. Thus, SOX9 can inhibit RUNX2 function in vivo even in established osteoblastic lineage. Finally, we demonstrate that this dominant inhibitory function of SOX9 is physiologically relevant in human campomelic dysplasia. In campomelic dysplasia, haploinsufficiency of SOX9 results in up-regulation of the RUNX2 transcriptional target COL10A1 as well as all three members of RUNX gene family. In summary, SOX9 is dominant over RUNX2 function in mesenchymal precursors that are destined for a chondrogenic lineage during endochondral ossification.differentiation ͉ mesenchymal ͉ skeletal dysplasias ͉ osteoblasts ͉ transcriptional repressor D uring embryogenesis, the majority of bones are formed via endochondral ossification; mesenchymal progenitor cells differentiate into chondrocytes that are eventually replaced by osteoblasts (1, 2). It is a well coordinated process regulated by a complex transcriptional network in which the transcription factors Runx2 and Sox9 play essential roles. Runx2 is required for osteoblast differentiation and chondrocyte maturation both in vivo and in vitro (3). We and others have shown that mutations in RUNX2 cause cleidocranial dysplasia, a dominantly inherited skeletal dysplasia characterized by hypoplastic clavicles, large fontanels, dental anomalies, and delayed skeletal development (4, 5). Sox9 is a potent transcriptional activator for chondrocyte-specific genes such as Col2a1 and Col11a1, and mouse genetic studies demonstrate that it is required for the successive steps of chondrocyte differentiation and cartilage formation (6-8). Mutations in human SOX9 result in campomelic dysplasia (CMD1), a disorder characterized by generalized hypoplasia of endochondral bones (9, 10).Although Runx2 is a strong transcriptional activator for osteoblast-specific and hypertrophic chondrocyte-specific genes, its embryonic expression is present in osteochondroprogenitor cells during mesenchymal condensations as early as embryonic day 10 (E10), before overt chondrocyte differentiation or osteoblast differentiation (11, 12). Hence, a strong context-dependent inhibition of Runx2 must occur before cell fate commitment to the chondrogenic lineage. Because Sox9 is also highly expressed in all osteochondroprogenitor cells and in proliferating (prehypertrophic) chondrocytes (6), we hypothesize that, in addition to its well established role as transcriptional activator for ch...

Cleidocranial dysplasia (CCD) is a dominantly inherited skeletal dysplasia caused by mutations in the osteoblast-specific transcription factor CBFA1. To correlate CBFA1 mutations in different functional domains with the CCD clinical spectrum, we studied 26 independent cases of CCD and a total of 16 new mutations were identified in 17 families. The majority of mutations were de novo missense mutations that affected conserved residues in the runt domain and completely abolished both DNA binding and transactivation of a reporter gene. These, and mutations which result in premature termination in the runt domain, produced a classic CCD phenotype by abolishing transactivation of the mutant protein with consequent haploinsufficiency. We further identified three putative hypomorphic mutations (R391X, T200A and 90insC) which result in a clinical spectrum including classic and mild CCD, as well as an isolated dental phenotype characterized by delayed eruption of permanent teeth. Functional studies show that two of the three mutations were hypomorphic in nature and two were associated with significant intrafamilial variable expressivity, including isolated dental anomalies without the skeletal features of CCD. Together these data show that variable loss of function due to alterations in the runt and PST domains of CBFA1 may give rise to clinical variability, including classic CCD, mild CCD and isolated primary dental anomalies.

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-␣1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1-3) that were essential for its chondrocytespecific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bp Col2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as larger Col2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocytespecific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.Acquisition of the chondrocytic phenotype occurs along a major pathway of differentiation of mesenchymal cells (1, 2). With the goal of identifying transcription factors that control chondrocyte-specific gene expression, we used the gene for collagen type II (Col2a1), 1 an early and abundant marker of chondrocytes (3-5), to delineate minimal sequences in this gene that control chondrocyte-specific expression in transgenic mice. Elucidation of the transcriptional mechanisms that control the chondrocyte-specific expression of the Col2a1 gene should provide important insights into the molecular specifications of chondrocytes.We previously identified a 48-bp element in intron 1 of the mouse Col2a1 gene that, when present as four tandem copies, conferred chondrocyte-specific expression both in transgenic mice and in transient expression experiments in tissue culture cells (6). A multimerized 18-bp element located at the 3Ј end of the 48-bp sequence also acted as a powerful chondrocyte-specific enhancer in transient transfection assays of rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not of fibroblasts (6).SOX9 is a member of a family of transcription factors with a DNA-binding domain that shows more than 50% similarity with the high mobility group HMG DNA-binding domain of SRY, the testis-determi...

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the pro␣1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3 repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465-and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.Acquisition of the chondrocyte phenotype by mesenchymal cells is one of the major pathways of differentiation of these cells. Chondrocytes form several types of cartilages including the growth plate cartilages essential to skeletal formation and cartilages that have supporting roles and persist throughout adult life such as the articular cartilages and the cartilages of the nose, ear, and trachea. Chondrocyte differentiation presumably involves first the commitment of undifferentiated mesenchymal cells to the chondrocyte lineage (1). Cell condensation and further maturation lead to a fully differentiated phenotype characterized by the synthesis of cartilage extracellular matrix proteins, including collagen types II, IX, and XI, the large proteoglycan aggrecan, the link protein, and the cartilage oligomeric protein (24). Recent molecular and biochemical studies with cell culture, gene inactivation experiments with mice, and the identification of genes responsible for mouse and human skeletal abnormalities have documented the importance of growth and differentiation factors, extracellular matrix proteins, signaling mediators, and tr...