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Introduction Translocations are chromosomal rearrangements that can be inherited and are often associated with cancers and congenital disorders. One of the great difficulties in determining mechanistic causes is the fact [...], Translocations are chromosomal rearrangements that are frequently associated with a variety of disease states and developmental disorders. We identified 2 families with brachydactyly type E (BDE) resulting from different translocations affecting chromosome 12p. Both translocations caused downregulation of the parathyroid hormone-like hormone (PTHLH) gene by disrupting the cis-regulatory landscape. Using chromosome conformation capturing, we identified a regulator on chromosome 12q that interacts in cis with PTHLH over a 24.4-megabase distance and in trans with the sex-determining region Y-box 9 (SOX9) gene on chromosome 17q. The element also harbored a long noncoding RNA (IncRNA). Silencing of the IncRNA, PTHLH, or SOX9 revealed a feedback mechanism involving an expression-dependent network in humans. In the BDE patients, the human lncRNA was upregulated by the disrupted chromosomal association. Moreover, the lncRNA occupancy at the PTHLH locus was reduced. Our results document what we believe to be a novel in cis- and in trans-acting DNA and lncRNA regulatory feedback element that is reciprocally regulated by coding genes. Furthermore, our findings provide a systematic and combinatorial view of how enhancers encoding IncRNAs may affect gene expression in normal development.

The study reports on an investigation into two unrelated families with hair and teeth abnormalities associated with ectodermal dysplasia-syndactyly syndrome (EDSS) and identifies a homozygous mutation in the PVRL4 gene causing the disease. In addition, it demonstrates high nectin-4 expression in hair follicle structures and in the separating digits of murine embryos, the tissues that are mainly affected by the EDSS phenotype.

Elongation of the digit rays resulting in the formation of a defined number of phalanges is a process poorly understood in mammals, whereas in the chicken distal mesenchymal bone morphogenetic protein (BMP) signaling in the so-called phalanx-forming region (PFR) or digit crescent (DC) seems to be involved. The human brachydactylies (BDs) are inheritable conditions characterized by variable degrees of digit shortening, thus providing an ideal model to analyze the development and elongation of phalanges. We used a mouse model for BDB1 ([Ror2.sup.W749X/W749X]) lacking middle phalanges and show that a signaling center corresponding to the chick PFR exists in the mouse, which is diminished in BDB1 mice. This resulted in a strongly impaired elongation of the digit condensations due to reduced chondrogenic commitment of undifferentiated distal mesenchymal cells. We further show that a similar BMP-based mechanism accounts for digit shortening in a mouse model for the closely related condition BDA1 ([Ihh.sup.E95K/E95K]), altogether indicating the functional significance of the PFR in mammals. Genetic interaction experiments as well as pathway analysis in BDB1 mice suggest that Indian hedgehog and WNT/[beta]-catenin signaling, which we show is inhibited by receptor tyrosine kinase-like orphan receptor 2 (ROR2) in distal limb mesenchyme, are acting upstream of BMP signaling in the PFR. bone morphogenetic protein signaling | brachydactyly | cartilage | limb development | Wnt signaling doi/ 10.1073/pnas.1009314107

Mutations in human SHH and IHH cause developmental defects such as holoprosencephaly (6), acrocapitofemoral dysplasia (7) and BDA1 (refs 1, 2). However, the molecular basis of these mutations is not [...], Brachydactyly type A1 (BDA1) was the first recorded disorder of the autosomal dominant Mendelian trait in humans, characterized by shortened or absent middle phalanges in digits. It is associated with heterozygous missense mutations in indian hedgehog (IHH) (1,2). Hedgehog proteins are important morphogens for a wide range of developmental processes (3,4). The capacity and range of signalling is thought to be regulated by its interaction with the receptor PTCH1 and antagonist HIPI. Here we show that a BDA1 mutation (E95K) in Ihh impairs the interaction of IHH with PTCH1 and HIP1. This is consistent with a recent paper showing that BDA1 mutations cluster in a calcium-binding site essential for the interaction with its receptor and cell-surface partners (5). Furthermore, we show that in a mouse model that recapitulates the E95K mutation, there is a change in the potency and range of signalling. The mice have digit abnormalities consistent with the human disorder.

The pathway mutations associated with the acetylcholine receptor (AChR) are studied to explain the various fetal akinesia deformation sequence (FADs) disorders. Severe alterations in the AChR lead to lethal multiple pterygium syndrome, whereas minor changes lead to fetal hypokinesia.

Linkage studies are performed in families with Escobar syndrome and eight mutations are identified within the gamma-subunit gene (CHRNG) of the acetylcholine receptor (AChR). The results have shown that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis and as gamma expression is restricted to early development, patients have no myasthenic symptoms later in life.

Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening and lateral deviation of the index fingers and, to a variable degree, shortening and deviation of the first and second toes. We performed linkage analysis in two unrelated German families and mapped a locus for BD type A2 to 4q21-q25. This interval includes the gene bone morphogenetic protein receptor 1B (BMPR1B), a type I transmembrane serinethreonine kinase. In one family, we identified a T599 [right arrow] A mutation changing an isoleucine into a lysine residue (1200K) within the glycine/serine (GS) domain of BMPR1B, a region involved in phosphorylation of the receptor. In the other family we identified a C1456 [right arrow] T mutation leading to an arginine-to-tryptophan amino acid change (R486W) in a highly conserved region C-terminal of the BMPR1B kinase domain. An in vitro kinase assay showed that the 1200K mutation is kinase-deficient, whereas the R486W mutation has normal kinase activity, indicating a different pathogenic mechanism. Functional analyses with a micromass culture system revealed a strong inhibition of chondrogenesis by both mutant receptors. Overexpression of mutant chBmpR1b in vivo in chick embryos by using a retroviral system resulted either in a BD phenotype with shortening and/or missing phalanges similar to the human phenotype or in severe hypoplasia of the entire limb. These findings imply that both mutations identified in human BMPR1B affect cartilage formation in a dominant-negative manner.

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs. Key Words: Runx2; Cbfa1; Runx3; endochondral ossification; chondrocyte differentiation; RCAS; joint development.