Altered Chondrocyte Differentiation and Extracellular Matrix Homeostasis in a Zebrafish Model for Mucolipidosis II

Abstract

Mucolipidosis II (ML-II) is a pediatric disorder causedby defects in the biosynthesis of mannose 6-phosphate, the carbohydrate recognition signal responsible for targeting certain acid hydrolases to lysosomes. The mechanisms underlying the developmental defects of ML-II are largely unknown due in part to the lack of suitable animal models. To overcome these limitations, we developed a model for ML-II in zebrafish by inhibiting the expression of N-acetylglucosamine-1-phosphotransferase, the enzyme that initiates mannose 6-phosphate biosynthesis. Morphant embryos manifest craniofacial defects , impaired motility , and abnormal otolith and pectoral fin development. Decreased mannose phosphorylation of several lysosomal glycosidases was observed in morphant lysates, consistent with the reduction in phosphotransferase activity. Investigation of the craniofacial defects in the morphants uncovered striking changes in the timing and localization of both type II collagen and Sox9 expression , suggestive of an accelerated chondrocyte differentiation program. Accumulation of type II collagen was also noted within misshapen cartilage elements at later stages of development. Furthermore, we observed abnormal matrix formation and calcium deposition in morphant otoliths. Collectively, these data provide new insight into the developmental pathology of ML-II and suggest that altered production and/or homeostasis of extracellular matrix proteins are integral to the disease process. These findings highlight the potential of the zebrafish system in studying lysosomal disease pathogenesis.

The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase is a substrate recognition module

“…Expression of GlcNAc-1-phosphotransferase (α/β subunit, gnptab) was inhibited by injection of morpholino oligonucleotides, as previously described (14). Experiments involving mRNA rescue in the morphant background were performed on embryos sequentially injected with 8 ng of the gnptab splice blocking morpholino oligonucleotides, which was previously validated to yield maximal specific inhibition, and 300 pg of the appropriate mRNA.…”

“…Cathepsin K activity assays were performed using fluorogenic substrates, as previously described (15). CI-MPR affinity chromatography was performed as described previously (14). Activity of β-galactosidase was measured in each column fraction, using 4-methylumbelliferyl-β-galactoside.…”

“…Embryos were stained with Alcian blue, as described previously (14). Analysis of craniofacial structures was performed using the morphometric parameters outlined in Results.…”

“…The Message Machine kit (Ambion) was used to synthesize all fulllength mRNAs from PCSII+ constructs containing either a chimera of mouse DMAP and zebrafish gnptab (referred to as WT in text) or the gnptab/DMAP chimera containing the K732N mutation (referred to as K732N in text). The PCSII+WT zebrafish gnptab construct was generated as previously described (14) and used to generate both the WT DMAP chimera and the K732N mutant.…”

“…Recently, we generated a morpholino-based model for MLII in the vertebrate organism zebrafish (Danio rerio) (14). Embryos depleted of GlcNAc-1-phosphotransferase exhibit decreased mannose phosphorylation of lysosomal acid hydrolases, craniofacial and cardiac defects, and altered development of pectoral fins and otic vesicles.…”