Microvillar and ciliary defects in zebrafish lacking an actin-binding bioactive peptide amidating enzyme

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 8 (2018): 4547, doi:10.1038/s41598-018-22732-9.The assembly of membranous extensions such as microvilli and cilia in polarized cells is a tightly regulated, yet poorly understood, process. Peptidylglycine α-amidating monooxygenase (PAM), a membrane enzyme essential for the synthesis of amidated bioactive peptides, was recently identified in motile and non-motile (primary) cilia and has an essential role in ciliogenesis in Chlamydomonas, Schmidtea and mouse. In mammalian cells, changes in PAM levels alter secretion and organization of the actin cytoskeleton. Here we show that lack of Pam in zebrafish recapitulates the lethal edematous phenotype observed in Pam−/− mice and reveals additional defects. The pam−/− zebrafish embryos display an initial striking loss of microvilli and subsequently impaired ciliogenesis in the pronephros. In multiciliated mouse tracheal epithelial cells, vesicular PAM staining colocalizes with apical actin, below the microvilli. In PAM-deficient Chlamydomonas, the actin cytoskeleton is dramatically reorganized, and expression of an actin paralogue is upregulated. Biochemical assays reveal that the cytosolic PAM C-terminal domain interacts directly with filamentous actin but does not alter the rate of actin polymerization or disassembly. Our results point to a critical role for PAM in organizing the actin cytoskeleton during development, which could in turn impact both microvillus formation and ciliogenesis.This study was supported by grants DK032949 (to BAE and REM), DK044464 (to JDG) and GM051293 (to SMK) from the National Institutes of Health

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Metal levels in oocytes and embryonic and larval stages of zebrafish.

<p>ICP-MS measurements for copper (Cu) (A, D), manganese (Mn) (B, E), and zinc (Zn) (C, F) were performed on oocytes, embryos (2-cell, 512-cell, blastula, 30% epiboly, 50% epiboly, 6, 12, 24, 36, 48, 60, 72 hours post-fertilization (hpf)) (A-C) and larval fish (5, 7, 10, 15, 30 days post-fertilization (dpf)) (D-F). Each bar represents the average and standard deviation of four to five independently collected replicates for a specific developmental stage. Each replicate represents a pooled group of 20 oocytes, 10–20 embryos, or 5–10 larval fish. Metal levels are expressed per oocyte, embryo, or larva. Horizontal brackets above bars indicate a statistically significant difference between bracketed values. (For data in all panels, the Shapiro-Wilk normality test failed, therefore Kruskal-Wallis one-way ANOVA on ranks was performed, with Dunn’s method used for multiple comparison and P<0.05 as cut-off for significance.)</p

Distribution of metals in bodies and yolk sacs of embryos.

<p>(A-C) ICP-MS measurements were collected for copper (Cu) (A), manganese (Mn) (B), and zinc (Zn) (C) in whole embryos, bodies only, and yolk sacs only of embryos at 48 hpf, reared in presence or absence of 5 μM neocuproine, a Cu chelator. Each bar represents the average and standard deviation of three independently collected replicates, with each replicate consisting of a pooled group of 47–50 embryos. Horizontal brackets above bars indicate statistically significant difference between bracketed values. (Data in all panels passed Shapiro-Wilk normality and equal variance tests. One-way ANOVA indicated significant difference between groups, with the Holm-Sidak method used for multiple comparisons and P<0.05 as cut-off for significance.) There was no significant difference in metal levels between neocuproine-treated and–untreated samples of the same sample type. (D, E) Images were taken of zebrafish embryos at 48 hpf, reared in presence or absence of 5 μM neocuproine.</p

The genome of the sea urchin Strongylocentrotus purpuratus.

International audienceWe report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes