Chordacentrum mineralization is delayed in zebrafish betaglycan‐null mutants

Abstract

Background The Transforming Growth Factor β (TGFβ) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFβ family that participates in heart, liver and kidney development as revealed by the tgfbr3‐null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish. Results Here, we present CRISPR/Cas9‐derived zebrafish Tgfbr3‐null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3‐null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFβ signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process. Conclusion Betaglycan's “ligand presentation” function contributes to the optimal TGFβ signaling required for zebrafish chordacentra mineralization.

Betaglycan promoter activity is differentially regulated during myogenesis in zebrafish embryo somites

“…We have demonstrated that in the null tgfbr3 zebrafish there is a delay in the mineralization of the chordocentra, the vertebrae primordia, and have proposed that this delay is due to the absence of TGFβ signaling enhancement, a function that has been well documented for this co-receptor. 19 However, given the many biochemicals activities that have been found in vitro for Tgfbr3, it is hard to propose a specific role for its selective expression in the adaxial cells and the SSM, particularly, given its A-P gradient expression documented in Figure 7. It is tempting to hypothesize that tgfbr3 expression is not only responding to a morphogen gradient but may help creating such a gradient.…”

“…Importantly, this time of initial mCherry expression matches with the time of appearance of tgfbr3 mRNA as detected by RT-PCR. 19 By 14 hpf, mCherry is observed in the eye and mesoderm, including the somites (Figure 4D). At 24 hpf, the reporter expression starts to be confined to specific structures like heart, pronephric duct, eyes, and the somites where it adopts a chevron like arrangement (Figure 4E) which becomes very evident at later stages (Figure 4H).…”

“…Recent work from our laboratory indicates that tgfbr3 downregulation in zebrafish embryos using morpholino oligonucleotides causes non‐cell autonomous angiogenesis defects and improper arrangement of actin fibers in the posterior somites which exhibit a rounder appearance 18 . Nonetheless, the tgfbr3 null zebrafish does not phenocopy the drastic defects observed in the morphant, adding to the complexity of Tgfbr3 biology and suggesting some type of genetic compensation in the null fish or an off target morpholino effect 19 . To gain deeper insights into these findings we report the identification of the zebrafish tgfbr3 promoter and its activity during the development of somitic skeletal muscle.…”

“…18 Nonetheless, the tgfbr3 null zebrafish does not phenocopy the drastic defects observed in the morphant, adding to the complexity of Tgfbr3 biology and suggesting some type of genetic compensation in the null fish or an off target morpholino effect. 19 To gain deeper insights into these findings we report the identification of the zebrafish tgfbr3 promoter and its activity during the development of somitic skeletal muscle.…”

“…(C-H) immunofluorescent staining of mCherry (yellow) and F59 (cyan). Dorsal view (panel C) of an embryo at 19.5 hpf (somites[16][17][18][19]. Adaxial cells were labeled with F59 (cyan), mCherry is observed with higher fluorescence in the nuclei of adaxial cells from the somitic mesoderm (white arrow) and adaxial cells from presomitic mesoderm (double white arrows).…”