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.

Characterization of biklf/klf17-deficient zebrafish in posterior lateral line neuromast and hatching gland development

Krüpple-like factors (Klfs) are highly conserved zinc-finger transcription factors that regulate various developmental processes, such as haematopoiesis and cardiovascular development. In zebrafish, transient knockdown analysis of biklf/klf17 using antisense morpholino suggests the involvement of biklf/klf17 in primitive erythropoiesis and hatching gland development; however, the continuous physiological importance of klf17 remains uncharacterized under the genetic ablation of the klf17 gene among vertebrates. We established the klf17-disrupted zebrafish lines using the CRISPR/Cas9 technology and performed phenotypic analysis throughout early embryogenesis. We found that the klf17-deficient embryos exhibited abnormal lateral line neuromast deposition, whereas the production of primitive erythrocytes and haemoglobin production were observed in the klf17-deficient embryos. The expression of lateral line neuromast genes, klf17 and s100t, in the klf17-deficient embryos was detected in posterior lateral line neuromasts abnormally positioned at short intervals. Furthermore, the klf17-deficient embryos failed to hatch and died without hatching around 15 days post-fertilization (dpf), whereas the dechorionated klf17-deficient embryos and wild-type embryos were alive at 15 dpf. The klf17-deficient embryos abolished hatching gland cells and Ctsl1b protein expression, and eliminated the expression of polster and hatching gland marker genes, he1.1, ctsl1b and cd63. Thus, the klf17 gene plays important roles in posterior lateral line neuromast and hatching gland development.

Oviposition and development in the glass frog Hyalinobatrachium orientale (Anura: Centrolenidae)

Oviposition and development in the glass frog Hyalinobatrachium orientale (Anura: Centrolenidae). Oviposition and external embryonic developmental features are described in the Tobago glass frog, Hyalinobatrachium orientale. Egg clutches are nearly always laid on the undersides of leaves (one exception); usually leaves of Heliconia sp. are used, but Philodendron and palms may be used in the absence of Heliconia. Clutches contain 28.0 ± 5.3 eggs (mean ± SD) and eggs are 1.86 ± 0.11 mm in diameter. The behavior of one amplectant pair was followed for more than five hours; the pair rotated several times around a small area of the leaf depositing eggs in a tight spiral formation. External embryonic features were observed by scanning electron microscopy. Surface ciliation is extensive up to the time of hatching when it is lost; external gills are short and a cement gland is absent. Hatching gland cells were detectable on the anterodorsal surface of the head from Day 4 after deposition and persisted until at least Day 10, and hatching occurred between Days 9 and 16. During this period, progressive development in tail length, surface pigmentation, intestinal coiling, and oral disc features was observed. Post-hatching larvae reared for six weeks grew 37% in length and tripled in weight, but remained at Gosner Stage 25.