Year: 2017
Type: article
Abstract: Fibulin-4 is an extracellular matrix protein required for the formation of the elastic fibers. Human mutations in the fibulin-4 gene cause autosomal recessive cutis laxa with widespread systemic involvement and congenital presentation. To study the developmental functions of fibulin-4, I studied one of the two fibulin-4 genes, fbln4a. The mRNA for fbln4a RNA was expressed in the adaxial cells around the notochord during somite formation, and in the myosepta, head, and heart at later stages. Fbln4a protein was localized to the notochord sheath starting from somite formation suggesting differential expression of fbln4a RNA and protein in the midline structures during development. To inactivate fbln4a in vivo, we used a retroviral mutant and an antisense morpholino oligonucleotide (MO) for transient knockdown. Embryos homozygous for the fbln4a mutation showed a complete loss of the Fbln4a protein, but presented no obvious gross abnormalities. However, fbln4a knockdown yielded cardiovascular and musculoskeletal defects at 2 days post fertilization including pooling of blood at the caudal vein plexus, vascular hemorrhage in the head, reduced circulation and heart rate, bent notochord, rounded somites and reduced embryo length. All of these features were rescued by co-injection of fbln4a mRNA with the MO. Early cardiac and vascular progenitor markers showed an expansion of the heart field and reduction of the vascular fields in knockdown embryos. Homozygous mutants were resistant, whereas heterozygotes were sensitized to the effect of fbln4a MO, protective compensatory mechanisms as a possible reason for phenotypic discrepancy between fbln4a mutant and knockdown animals. Inhibition of transforming growth factor beta (Tgfb) signaling with a small molecule rescued both the cardiovascular and connective tissue anomalies in knockdown embryos. By varying the period of treatment, I identified late gastrulation and early segmentation as the critical periods. I conclude that Fbln4a inhibits Tgfb signals emanating from the notochord and regulates cardiac and vascular progenitor pools. The public health significance of this work is the identification of Tgfb inhibition as a candidate approach for treating of fibulin-4-related cutis laxa, an orphan disease for which no treatment has been available to date.
Author: Sandeep Khatri
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2746088136