Phenotypic spectrum of TGFB3 disease-causing variants in a Dutch-French cohort and first report of a homozygous patient.

Disease-causing variants in TGFB3 cause an autosomal dominant connective tissue disorder which is hard to phenotypically delineate because of the small number of identified cases. The purpose of this retrospective cross-sectional multicenter study is to elucidate the genotype and phenotype in an international cohort of TGFB3 patients. Eleven (eight novel) TGFB3 disease-causing variants were identified in 32 patients (17 families). Aortic root dilatation and mitral valve disease represented the most common cardiovascular findings, reported in 29% and 32% of patients, respectively. Dissection involving distal aortic segments occurred in two patients at age 50 and 52 years. A high frequency of systemic features (65% high-arched palate, 63% arachnodactyly, 57% pectus deformity, 52% joint hypermobility) was observed. In familial cases, incomplete penetrance and variable clinical expressivity were noted. Our cohort included the first described homozygous patient, who presented with a more severe phenotype compared to her heterozygous relatives. In conclusion, TGFB3 variants were associated with a high percentage of systemic features and aortic disease (dilatation/dissection) in 35% of patients. No deaths occurred from cardiovascular events or pregnancy-related complications. Nevertheless, homozygosity may be driving a more severe phenotype.

Systematic analysis of palatal transcriptome to identify cleft palate genes within TGFß3-knockout mice alleles: RNA-Seq analysis of TGFß3 Mice.

BACKGROUND: In humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGFß3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGFß3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGFß3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGFß3 knockout mice to identify TGFß3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGFß3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5). RESULTS: The overall transcriptome analysis of TGFß3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGFß3+/- and TGFß3-/- were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGFß3-/- mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGFß3-/- mice. When the significantly altered CP genes were overlaid with TGFß signaling, all of these genes followed the Smad-dependent pathway. CONCLUSIONS: Our study represents the first analysis of the palatal transcriptome of the mouse, as well as TGFß3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGFß3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.

Tgfbeta3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest.

Zebrafish tgfbeta3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfbeta3 in head skeletal formation, we knocked down tgfbeta3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfbeta3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfbeta3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfbeta3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfbeta3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfbeta3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfbeta3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfbeta3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfbeta3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfbeta3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfbeta3 protein levels in zebrafish.

Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation.

Tendons and ligaments mediate the attachment of muscle to bone and of bone to bone to provide connectivity and structural integrity in the musculoskeletal system. We show that TGFbeta signaling plays a major role in the formation of these tissues. TGFbeta signaling is a potent inducer of the tendon progenitor (TNP) marker scleraxis both in organ culture and in cultured cells, and disruption of TGFbeta signaling in Tgfb2(-/-);Tgfb3(-/-) double mutant embryos or through inactivation of the type II TGFbeta receptor (TGFBR2; also known as TbetaRII) results in the loss of most tendons and ligaments in the limbs, trunk, tail and head. The induction of scleraxis-expressing TNPs is not affected in mutant embryos and the tendon phenotype is first manifested at E12.5, a developmental stage in which TNPs are positioned between the differentiating muscles and cartilage, and in which Tgfb2 or Tgfb3 is expressed both in TNPs and in the differentiating muscles and cartilage. TGFbeta signaling is thus essential for maintenance of TNPs, and we propose that it also mediates the recruitment of new tendon cells by differentiating muscles and cartilage to establish the connections between tendon primordia and their respective musculoskeletal counterparts, leading to the formation of an interconnected and functionally integrated musculoskeletal system.

Transforming growth factor beta (TGFbeta1, TGFbeta2 and TGFbeta3) null-mutant phenotypes in embryonic gonadal development.

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice.

Tgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants.

Although TGF-beta isoforms (TGF-beta1-3) display very similar biochemical characteristics in vitro, it has been determined that they demonstrate different or even opposing effects in vivo. During embryogenesis, TGF-betas play important roles in several developmental processes. Tgfb3 is strongly expressed in the prefusion palatal epithelium, and mice lacking Tgfb3 display a cleft of the secondary palate. To test whether the effect of TGF-beta3 in palatogenesis is isoform-specific in vivo, we generated a knockin mouse by replacing the coding region of exon1 in the Tgfb3 gene with the full-length Tgfb1 cDNA, which resulted in the expression of Tgfb1 in the Tgfb3 expressing domain. The homozygote knockin mice display a complete fusion at the mid-portion of the secondary palate, while the most anterior and posterior regions fail to fuse appropriately indicating that in vivo replacement of TGF-beta3 with TGF-beta1 can only partially correct the epithelial fusion defect of Tgfb3 knockout embryos. Palatal shelves of Tgfb1 knockin homozygote mice adhere, intercalate, and form characteristic epithelial triangles. However, decreased apoptosis in the midline epithelium, slower breakdown of the basement membrane and a general delay in epithelial fusion were observed when compared to control littermates. These results demonstrate an isoform-specific role for TGF-beta3 in the palatal epithelium during palate formation, which cannot be fully substituted with TGF-beta1.