tgfβ3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest

Abstract

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.

Synergistic effects of overexpression of BMP-2 and TGF-β3 on osteogenic differentiation of bone marrow mesenchymal stem cells

“…Scaffolds infused with BMP-2 and TGF-β3 enhanced bone formation in vivo and improved treatment of the orthotopic defect region, which was consistent with the data from Oest et al (48). A previous study also suggested that craniofacial osteogenesis relied on tight modulation of TGF-β3 levels in zebrafish embryos (49). These previous studies indicate TGF-β3 may serve as a promoter to accelerate and induce bone formation.…”

Examination of a palatogenic gene program in zebrafish

“…Tgfb signaling is critical for palatogenesis in mouse and loss of either Tgfb2 or Tgfb2 causes cleft palate. Morpholino knockdown of tgfb3 in zebrafish has been shown to cause craniofacial defects, including disruption of the palate (Cheah et al, 2010). In our hands, injection of a different tgfb3 morpholino, even at very low doses, caused severe edema that precluded analysis of the palatal skeleton (data not shown).…”

“…Tgfb2 and Tgfb3 play important roles in fusion of the palatal shelves in mice, with loss of gene function resulting in cleft palate with low penetrance in Tgfb2 mutants and almost complete penetrance in Tgfb3 mutants. Others have implicated tgfb3 signaling in zebrafish craniofacial development (Cheah et al, 2010). However, we were unable to test the role of tgfb3 in zebrafish palatogenesis because the morpholino‐injected zebrafish embryos died from what appeared to be cardiac defects.…”

Transforming Growth Factor β Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells

“…Antisense MOs (GeneTools) were used to target runx1 (Gering and Patient, 2005), tgfβ3 ( tgfb3 MO2 ) (Cheah et al., 2010), kdr  + kdrl (Bahary et al., 2007), and jag1a (Yamamoto et al., 2010) at the amounts specified. The MOs selected for this study were tgfbR2 MO1 , tgfb1a MO2 + tgfb1b MO2 (referred to as tgfb1 MO2 ), tgfb2 MO2 , and tgfb3 MO2 at the amounts indicated (see Supplemental Experimental Procedures).…”