Zebrafish aplnra functions in epiboly

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Zebrafish <it>aplnra </it>functions in epiboly

Abstract Background The zebrafish, Danio rerio, possesses the paralogous genes aplnra and aplnrb that are duplicates of an ancestral orthologue of the human APLNR gene encoding a G-protein coupled receptor that binds the peptide ligand APELIN and is required for normal cardiovascular function. aplnrb is required for migration of cells contributing to heart development in zebrafish embryos. aplnra is transcribed in a complex pattern during early development but its function in embryogenesis is largely unknown. Findings Blockage of translation of aplnra mRNA in zebrafish embryos results in retarded or failed epiboly with the blastoderm apparently disconnected from the nuclei of the yolk syncytial layer. Gastrulation is also defective. Failure of correct tail extension is observed with ectopic structures resembling somites positioned dorsal to the spinal cord. Conclusion aplnra, unlike its duplicate aplnrb, is essential for normal epiboly, although this function appears to be independent of signalling activated by zebrafish Apelin. The defects in epiboly caused by loss of aplnra activity appear, at least partially, to be due to a requirement for aplnra activity in the yolk syncytial layer.</p

Tissue specific GFP fluorescence patterns.

<p>GFP expression diverges between the transgenic lines at later developmental time points. Expression of GFP is noted in numerous tissues over the first 96 hpf (N/A: not applicable).</p

Genomic region of the <i>tbx16</i> gene and transgenic construct.

<p>(a) <i>Eco</i>RI and <i>Bam</i>HI restriction sites in the genomic region of the <i>tbx16</i> gene surrounding the transcription initiation site. The interval marked “CLGY6" indicates the published enhancer trap insertion site resulting in GFP expression that recapitulates <i>tbx16</i> gene expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021559#pone.0021559-Ellingsen1" target="_blank">[15]</a>. (b) Boxes indicate the first three exons and filled boxes are protein-coding regions. The start of translation is shown (ATG). (c) The <i>Eco</i>RI/<i>Bam</i>HI fragment of the <i>tbx16</i> promoter used in the transgenic construct includes the translation start site, the first exon, and part of the second exon of the <i>tbx16</i> gene. (d) The promoter fragment was cloned in-frame upstream of enhanced GFP (EGFP) and a SV40 polyadenylation signal (A) in the multiple cloning site of the pEGFP-N1 vector.</p

Expression unique to individual stable transgenic lines.

<p>(a) Line 192A at 48 hpf. GFP expression is observed in regions of the midbrain (arrowhead) and in the epiphysis (e). Line 512B at 24 hpf (b) and 48 hpf (c). Expression is seen in the dorsal aorta and in ventral neurons (n) of the spinal cord. Line 812A at 24 hpf (d–e) and at 48 hpf (f). Expression can be seen in the epiphysis, the midbrain and in a banding pattern in rhombomeres (boxed). White box indicates the area enlarged in (e). Later expression is observed in the floor plate of the spinal cord (f). (g–h) Line 812C at 48 hpf. Expression is seen in the epiphysis and the midbrain. GFP is also noted in a specific subpopulation of spinal cord neurons and neurons posterior of the otic vesicle in the hindbrain (boxed). White box indicates the area enlarged in (h). Lateral views of embryos, anterior is left and dorsal is up. Scale bars in a, d, g = 250 µm, in b, c, e, f, h = 50 µm.</p

GFP expression in stable transgenic lines consistent with <i>tbx16</i> expression in wild type embryos.

<p>(a) At 12 hpf strong expression is seen in the polster (arrowhead), and the presomitic mesoderm (p). Expression persists in mesoderm that has formed somites (s). (b) At 24 hpf GFP expression is seen in the hatching gland (arrowhead) and in posterior somites, while it has faded from the earliest formed somites. (c) At 48 hpf expression remains in the hatching gland that has formed from the polster (arrowhead) and persists in the posterior somites. (d) The same embryo as in (c). GFP expression is observed throughout the notochord (n) highlighting cell extremities. Lateral views of embryos, anterior is left and dorsal is up. Scale bars in a–c = 250 µm, in d = 100 µm.</p