Eph-ephrin signaling maintains the boundary of dorsal forerunner cell cluster during morphogenesis of the zebrafish embryonic left-right organizer

Abstract

The Kupffer's vesicle (KV) is the so-called left-right organizer in teleost fishes. KV is formed from dorsal forerunner cells (DFCs) and generates asymmetrical signals for breaking symmetry of embryos. It is unclear how DFCs or KV cells are prevented from intermingling with adjacent cells. In this study, we show that the Eph receptor gene ephb4b is highly expressed in DFCs whereas ephrin ligand genes, including efnb2b, are expressed in cells next to the DFC cluster during zebrafish gastrulation. ephb4b knockdown or mutation and efnb2b knockdown cause dispersal of DFCs, a smaller KV and randomization of laterality organs. DFCs often dynamically form lamellipodium-like, bleb-like and filopodium-like membrane protrusions at the interface, which attempt to invade but are bounced back by adjacent non-DFC cells during gastrulation. Upon inhibition of Eph/ephrin signaling, however, the repulsion between DFCs and non-DFC cells is weakened or lost, allowing DFCs to migrate away. Ephb4b/Efnb2b signaling by activating RhoA activity mediates contact and repulsion between DFCs and neighboring cells during gastrulation, preventing intermingling of different cell populations. Therefore, our data uncover an important role of Eph/ephrin signaling in maintaining DFC cluster boundary and KV boundary for normal left-right asymmetrical development.

Epigenetic regulation of left–right asymmetry by DNA methylation

“…Disaggregation of DFCs suggests a possible involvement of cell adhesion in DFC clustering (Matsui et al, 2011(Matsui et al, , 2015. Previous studies have shown that a tight and stable cluster of DFCs is essential for KV ciliogenesis and LR patterning in zebrafish (Oteiza et al, 2010;Matsui et al, 2011;Zhang et al, 2016). Cdh1-mediated cell adhesion between adjacent DFCs facilitates cell cluster formation, while DFC-specific knockdown of cdh1 leads to the broken-up DFC phenotype (Matsui et al, 2011), similar to what we observed in dnmt3bb.1-deficient embryos.…”

“…Moreover, overexpression of dnmt3bb.1 mis-mRNA partially restored the disrupted DFC clustering in dnmt3bb.1 morphants (Fig 6C). Previous studies have shown that a tight and stable cluster of DFCs is essential for KV ciliogenesis and LR patterning in zebrafish (Oteiza et al, 2010;Matsui et al, 2011;Zhang et al, 2016). Disaggregation of DFCs suggests a possible involvement of cell adhesion in DFC clustering (Matsui et al, 2011(Matsui et al, , 2015.…”

Probing Cadherin Interactions in Zebrafish with E- and N-Cadherin Missense Mutants

“…In any case, the combined effect of two cadherin missense mutations is synergistic, suggesting that cells in the forerunner cluster perhaps utilize a combination of homotypic and heterotypic cellular interactions, which experimental and theoretical studies demonstrate have markedly different adhesive properties (Katsamba et al 2009). However, there are other adhesion molecules that control forerunner cell behavior (Ablooglu et al 2010;Oteiza et al 2010;Zhang et al 2016), whose interactions in the missense mutants might also be impaired.…”

An Ephrin-Eph Tug and Push in Left-Right Organ Placement

“…A number of these molecules, including Ephrin-Eph, Slit-Robo and semaphorins, have also been found to be essential in multiple developmental contexts outside of the nervous system (Branchfield, et al, 2016; Ochsenbein, et al, 2016; Lewis, et al, 2015). While Ephrin/Eph signaling has recently been implicated in left-right pattern establishment in the zebrafish organizer, its role in later stages of left-right asymmetry had not been explored (Zhang, et al, 2016). Cayuso and colleagues (2016) found that the EphrinB1 ligand is one of the first genes expressed in hepatic progenitors, whereas its receptor, EphB3b , is subsequently restricted to the lateral plate mesoderm.…”