Temporal expression of ZF-RNases-1, -2 and -3 during zebrafish embryos development. A, zebrafish embryos at different stages were analyzed by reverse transcriptase followed by PCR (RT-PCR) for the expression of ZF-RNases-1, -2 and -3 genes. The -RT lane is a negative control for the reverse transcription reaction. ZF-Odc is a loading control. B, quantitative analysis of ZF-RNases-1gene was performed by real-time PCR. Quantitative real-time PCR correlated with the above results. Both analysis show that during embryonic development ZF-RNases-1 is the only gene expressed. Quantified mRNA values were normalized by the amounts of ZF-Odc mRNA, the house keeping gene, and results are given as fold induction. Abbreviations used: (hpf), hours post-fertilization, (ZF-Odc), ornithine decarboxylase.

Temporal expression of ZF-RNases-1, -2 and -3 genes in zebrafish late embryos and larvae. Zebrafish late embryos and larvae stages were analyzed by reverse transcriptase followed by PCR (RT-PCR). A, Only ZF-RNases-1 is detected in late embryos and all larvae stages analyzed. In contrast, neither ZF-RNases-2, nor ZF-RNases-3 gene expression is observed at these stages. The -RT lane is a negative control for the reverse transcription reaction. ZF-Odc is a loading control. B, Quantitative analysis of ZF-RNases-1 gene performed by real-time PCR. These data correlate with the above results showing the highest expression level of ZF-RNases-1 at 24 hpf. C, Quantitative real-time PCR (40 cycles) performed on late embryos and larvae stages of ZF-RNase-2 at 13 dpf stage. D, Quantitative real-time PCR (40 cycles) on late embryos and larvae stages for ZF-RNase-3 expression in 13 dpf larvae. Quantified mRNA values were normalized by the amounts of ZF-Odc mRNA, the house keeping gene, and results are given as fold induction.

Spatial expression pattern of ZF-RNases-1. Whole-mount in situ hybridization experiments were carried out on early and late embryo stages. A, views of embryos showing the expression of ZF-RNases-1 at shield stage. The arrow indicates the anterior part of embryo and the arrowhead the posterior part. B, expression of ZF-RNases-1 at 3-somite stage; the arrow indicates the presumptive head. C, expression of ZF-RNases-1 at 24 hpf; D is a magnification (20x) of the boxed area in C. E, expression of ZF-RNases-1 at 48 hpf stage; the arrow indicates the heart. Abbreviations used: T, telencephalon; D, diencephalon; M, mesencephalon; H, hearth. Scale bar = 250 μm.

Temporal expression of ZF-RNases-1, -2 and -3 genes in adult zebrafish. A, 4-month-old zebrafish dissected into anterior (A), mid (M) and posterior (P) part toward the body plan axis and analyzed by reverse transcriptase followed by PCR (RT-PCR) for the expression of ZF-RNases-1, -2 and -3 genes. The -RT lane is a negative control for the reverse transcription reaction. RNAs ZF-Odc is a loading control. B, quantitative analysis of ZF-RNases-1, -2 and -3 genes performed by real-time PCR. ZF-RNases-1, -2 and -3 genes expression is mainly detected in the anterior (A) and mid- (M) parts of the body. Note, only a slight expression of ZF-RNases-1, -2 and -3 genes is observed in the posterior part (P). Quantified mRNA values were normalized by the amounts of ZF-Odc mRNA, the house keeping gene, and results are given as fold induction.

Spatial expression of ZF-RNase-1 in adult zebrafish. A, (upper panel) in situ hybridization analyses performed on liver frozen sections obtained from 4 mpf specimens Abbreviation used: (C) liver capsule, (H) cords of hepatocytes, (S) sinusoids containing red blood cells, (BD) bile duct. A sense riboprobe was used as negative (lower panel). B, (left panel) ZF-RNase-1 transcripts localize in the gut at level of intestine's loops. A sense riboprobe was used as negative control (right panel). C, (upper panel) ZF-RNase-1 gene expression is also detected in the exocrine (ExP) and endocrine (EnP) pancreas A sense riboprobe was used as negative (lower panel). D, (upper panel) ZF-RNase-1 gene expression in the heart. Atrium (At), ventricle (Vc). A sense riboprobe was used as negative (lower panel).

E, (upper panel) ZF-RNase-1 transcript also localizes in the swim bladder. A sense riboprobe was used as negative control (lower panel).

Spatial expression of ZF-RNase-2 in adult zebrafish. A, in situ hybridization analyses performed on frozen sections obtained from a 4 mpf specimens show low levels of ZF-RNase-2 transcript in the liver. B, High and broad expression of ZF-RNase-2 is observed in the gut.

Spatial expression of ZF-RNase-3 in adult zebrafish. A, in situ hybridization analyses performed on frozen sections obtained from 4 mpf specimens reveal low levels of ZF-RNase-3 transcripts in the liver (arrow). B, higher levels of ZF-RNase-3 transcripts are present in the heart. C, ZF-RNase-3 gene expression is also elevated in the gut. D, Immunohistochemistry analysis of ZF-RNase-3 protein in the liver (arrow) and gut.

E, ZF-RNase-3 expression is also detected in the swim bladder at transcript and protein levels.

A, ZF-RNases-1, -2 and -3 genes expression analysis in the adult brain reveals that ZF-RNases-1 is the only genes expressed in the adult brain. B, ZF-RNases-1 expression is also confirmed by in situ hybridization analysis.

A, Immunoblotting of ZF-Rnase-1, -2 and -3 proteins using anti-ZF-RNase-3 antibody diluted 1:200 B, Immunohistochemistry analysis of ZF-RNase-3 protein on swim bladder sections using as negative control a non immune (NI) serum.

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