Matrix Metalloproteinase 13 Activity is Required for Normal and Hypoxia-Induced Precocious Hatching in Zebrafish Embryos

Hypoxia induces precocious hatching in zebrafish, but we do not have a clear understanding of the molecular mechanisms regulating the activation of the hatching enzyme or how these mechanisms trigger precocious hatching under unfavorable environmental conditions. Using immunohistochemistry, pharmacological inhibition of matrix metalloproteinase 13 (Mmp13), and in vivo zymography, we show that Mmp13a is present in the hatching gland just as embryos become hatching competent and that Mmp13a activity is required for both normal hatching and hypoxia-induced precocious hatching. We conclude that Mmp13a likely functions in activating the hatching enzyme zymogen and that Mmp13a activity is necessary but not sufficient for hatching in zebrafish. This study highlights the broad nature of MMP function in development and provides a non-mammalian example of extra-embryonic processes mediated by MMP activity.

Transcriptomics supports that pleuropodia of insect embryos function in degradation of the serosal cuticle to enable hatching

ABSTRACTPleuropodia are limb-derived vesicular organs that transiently appear on the first abdominal segment of embryos from the majority of insect “orders”. They are missing in the model Drosophila and little is known about them. Experiments carried out on orthopteran insects eighty years ago indicated that the pleuropodia secrete a “hatching enzyme” that at the end of embryogenesis digests the serosal cuticle to enable the larva to hatch. This hypothesis contradicts the view that insect cuticle is digested by enzymes produced by the tissue that deposited it. We studied the development of the pleuropodia in embryos of the locust Schistocerca gregaria (Orthoptera) using transmission electron microscopy. RNA-seq was applied to generate a comprehensive embryonic reference transcriptome that was used to study genome-wide gene expression of ten stages of pleuropodia development. We show that the mature and secretion releasing pleuropodia are primarily enriched in transcripts associated with transport functions. They express genes encoding enzymes capable of digesting cuticular protein and chitin. These include the potent cuticulo-lytic Chitinase 5, whose transcript rises just before hatching. The pleuropodia are also enriched in transcripts for immunity-related enzymes, including the Toll signaling pathway, melanization cascade and lysozymes. These data provide transcriptomic evidence that the pleuropodia of orthopterans produce the “hatching enzyme”, whose important component is the Chitinase 5. They also indicate that the organs facilitate epithelial immunity and may function in embryonic immune defense. Based on their gene expression the pleuropodia appear to be an essential part of insect physiology.