Högskolan i Skövde

Abstract

Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.

Author summary

The components of the extracellular matrix are crucial for interactions and communication between cells during animal development and disease progression. One major component of the extracellular matrix is chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans, which support and modify cell functions and tissue homeostasis. The biosynthesis of CS/DS is complex and no genetic models have been developed to specifically reduce CS/DS in the zebrafish model organism. We have used CRISPR/Cas9 technology to knock out key CS/DS biosynthesis genes. We find that knocking out single genes rarely causes major effects on zebrafish morphology and viability, but by combining several knockout alleles we could observe malformations in the zebrafish craniofacial skeleton. In addition, one combination of alleles was embryonic lethal. Our findings describe the role of CS/DS in the development of the head skeleton and give insights in the regulation of genes involved in CS/DS biosynthesis. The zebrafish mutants generated in this study can be used as tools to further study human diseases caused by mutations in CS/DS biosynthesis enzymes.