Abstract
Decorin, an archetypal member of the small leucine-rich proteoglycan gene family, regulates collagen fibrillogenesis and cell growth. To further explore its biological function, we examined the role of Decorin during zebrafish development. Zebrafish Decorin is a chondroitin sulfate proteoglycan that exhibits a high degree of conservation with its mammalian counterpart and displays a unique spatiotemporal expression pattern. Morpholino-mediated knockdown of zebrafish decorin identified a developmental role during medial-lateral convergence and anterior-posterior extension of the body plan, as well as in craniofacial cartilage formation. decorin morphants displayed a pronounced shortening of the head-to-tail axis as well as compression, flattening, and extension of the jaw cartilages. The morphant phenotype was efficiently rescued by zebrafish decorin mRNA. Unexpectedly, microinjection of excess zebrafish decorin mRNA or proteoglycan/protein core into one-cell stage embryos caused cyclopia. The morphant and overexpression phenotype represent a convergent extension defect. Our results indicate a central function for Decorin during early embryogenesis.
Highlights
Characterization and Analysis of Zebrafish decorin—Our analysis of the zebrafish genome revealed the presence of one single gene encoding decorin (Gene ID: 64698)
The zebrafish decorin mRNA sequence (ϳ1.1 kb) is composed of seven exons interspersed by six introns and encodes a protein of ϳ373 amino acids, with a predicted molecular mass of ϳ38 kDa
We found that co-injection of Dcn-MOSPLICE along with zebrafish decorin mRNA was capable of rescuing the decorin morphant phenotype (Fig. 7)
Summary
Analysis, Synthesis, and Purification of Zebrafish decorin mRNA and Protein—Zebrafish decorin was PCR-amplified from a zebrafish cDNA template with the following primer pair, 5Ј-GGCGCGCCTTATGAAATCGGCCTGTCTCTCCCTG-3Ј and 5Ј-CTCGAGCTTCTTCCTGTAGTTGCCGAGCT-3Ј (Operon). The day cells were incubated with zebrafish Decorin (30 g/ml) for 2 h, washed twice with Dulbecco’s PBS, followed by extraction with RIPA buffer. Sections were washed in 1% FBS three times for 5 min each, followed by incubation with secondary antibody, goat anti-rabbit rhodamine (Santa Cruz Biotechnology), at a 1:300 dilution in 1% FBS for 2 h at room temperature. Embryos were cleared in PBS containing Tween 20 (0.2%), followed by cartilage staining in 0.1% Alcian blue (Chroma-Gesellschaft) according to the procedure described previously [43]with the following modifications: 4 h of staining in sterilefiltered Alcian blue at 24 °C, followed by dehydration in ethanol series, washing in PBST, and incubation at 37 °C with 0.05% trypsin, 2.21 mM EDTA in Hanks’ balanced salt solution (Cellgro) for 2 h to digest excess tissue for clear visualization. For zebrafish embryo load control, a portion of the gel was stained with the colloidal blue staining kit (Invitrogen, LC6025)
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