Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish.

During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO). Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL) but not in the enveloping layer (EVL) and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.

Diaphanous-related formin 2 and profilin I are required for gastrulation cell movements.

Intensive cellular movements occur during gastrulation. These cellular movements rely heavily on dynamic actin assembly. Rho with its associated proteins, including the Rho-activated formin, Diaphanous, are key regulators of actin assembly in cellular protrusion and migration. However, the function of Diaphanous in gastrulation cell movements remains unclear. To study the role of Diaphanous in gastrulation, we isolated a partial zebrafish diaphanous-related formin 2 (zdia2) clone with its N-terminal regulatory domains. The GTPase binding domain of zDia2 is highly conserved compared to its mammalian homologues. Using a yeast two-hybrid assay, we showed that zDia2 interacts with constitutively-active RhoA and Cdc42. The zdia2 mRNAs were ubiquitously expressed during early embryonic development in zebrafish as determined by RT-PCR and whole-mount in situ hybridization analyses. Knockdown of zdia2 by antisense morpholino oligonucleotides (MOs) blocked epiboly formation and convergent extension in a dose-dependent manner, whereas ectopic expression of a human mdia gene partially rescued these defects. Time-lapse recording further showed that bleb-like cellular processes of blastoderm marginal deep marginal cells and pseudopod-/filopod-like processes of prechordal plate cells and lateral cells were abolished in the zdia2 morphants. Furthermore, zDia2 acts cell-autonomously since transplanted zdia2-knockdown cells exhibited low protrusive activity with aberrant migration in wild type host embryos. Lastly, co-injection of antisense MOs of zdia2 and zebrafish profilin I (zpfn 1), but not zebrafish profilin II, resulted in a synergistic inhibition of gastrulation cell movements. These results suggest that zDia2 in conjunction with zPfn 1 are required for gastrulation cell movements in zebrafish.

LPA1 is essential for lymphatic vessel development in zebrafish.

Lysophosphatidic acid (LPA) has long been implicated in regulating vascular development via endothelial cell-expressed G protein-coupled receptors. However, because of a lack of notable vascular defects reported in LPA receptor knockout mouse studies, the regulation of vasculature by LPA receptors in vivo is still uncertain. Using zebrafish as a model, we studied the gene expression patterns and functions of an LPA receptor, LPA(1), during embryonic development, in particular, vascular formation. Whole-mount in situ hybridization experiments revealed that zebrafish lpa(1) (zlpa(1)) was ubiquitously expressed early in development, and its expression domains were later localized to the head region and the vicinity of the dorsal aorta. The expression of zlpa(1) surrounding the dorsal aorta suggests its role in vasculature development. Knocking down of zLPA(1) by injecting morpholino (MO) oligonucleotides at 0.625-1.25 ng per embryo resulted in the absence of thoracic duct and edema in pericardial sac and trunk in a dose-dependent manner. These zlpa(1)-MO-resulted defects could be specifically rescued by ectopic expression of zlpa(1). In addition, overexpression of vegf-c, a well-known lymphangiogenic factor, also partially ameliorated the inhibition of thoracic duct development. Taken together, these results demonstrate that LPA(1) is necessary for lymphatic vessel formation during embryonic development in zebrafish.

Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus.

The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.