Diaphanous-Related Formin 2 and Profilin I Are Required for Gastrulation Cell Movements

Abstract

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.

Formins at a glance

“…Formins also have essential roles in physiological processes ranging from cell motility in the immune system (Yayoshi-Yamamoto et al, 2000;Eisenmann et al, 2007;Shi et al, 2009), to gastrulation and neural tube closure (Habas et al, 2001;Sato et al, 2006;Lai et al, 2008), heart morphogenesis (Iskratsch et al, 2010;Li et al, 2011), kidney morphogenesis (Brown et al, 2010;Boyer et al, 2011a;Boyer et al, 2011b), and dendritic spine formation in neurons. Some of these functions depend on the actin nucleation and elongation activities of formins (Bartolini et al, 2008;Andrés-Delgado et al, 2010;Madrid et al, 2010;Andrés-Delgado et al, 2012;Ramabhadran et al, 2012;Stastna et al, 2012), which have been demonstrated by investigating point mutations that impair in these activities Lu et al, 2007;Ramabhadran et al, 2012).…”

Molecular insights on context-specific role of profilin-1 in cell migration

“…52 Similarly, gastrulation defects were observed in Xenopus and zebrafish when Pfn1 expression was downregulated through antisense morpholinos. 53,54 Relevant to mammalian cell systems, it has been shown that loss of expression or disruption of ligand interactions of Pfn1 causes impaired migration/invasion and capillary morphogenesis of human vascular endothelial cells, and defects in neurite outgrowth. 24,26,55,56 Very recently, cre/lox-based gene deletion approach has been utilized to study the effect of tissue-specific loss of Pfn1 in vivo.…”

Increased activity of Diaphanous homolog 3 ( DIAPH3 )/ diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila

“…Knockdown of the zebrafish ortholog of DIAPH3, zdia2, by antisense morpholino oligonucleotides is lethal to the embryo before the formation of the auditory organ (22).…”