Eph signalling functions downstream of Val to regulate cell sorting and boundary formation in the caudal hindbrain

Abstract

Rhombomeres are segmental units of the developing vertebrate hindbrain that underlie the reiterated organisation of cranial neural crest migration and neuronal differentiation. valentino (val), a zebrafish homologue of the mouse bzip transcription factor-encoding gene, kreisler, is required for segment boundary formation caudal to rhombomere 4 (r4). val is normally expressed in r5/6 and is required for cells to contribute to this region. In val(−) mutants, rX, a region one rhombomere in length and of mixed identity, lies between r4 and r7. While a number of genes involved in establishing rhombomeric identity are known, it is still largely unclear how segmental integrity is established and boundaries are formed. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are candidates for functioning in rhombomere boundary formation. Indeed, expression of the receptor ephB4a coincides with val in r5/6, whilst ephrin-B2a, which encodes a ligand for EphB4a, is expressed in r4 and r7, complementary to the domain of val expression. Here we show that in val(−) embryos, ephB4a expression is downregulated and ephrin-B2a expression is upregulated between r4 and r7, indicating that Val is normally required to establish the mutually exclusive expression domains of these two genes. We show that juxtaposition of ephB4a-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formation. Loss of the normal spatial regulation of eph/ephrin expression in val mutants correlates not only with absence of boundaries but also with the inability of mutant cells to contribute to wild-type r5/6. Using a genetic mosaic approach, we show that spatially inappropriate Eph signalling underlies the repulsion of val(−) cells from r5/6. We propose that Val controls eph expression and that interactions between EphB4a and Ephrin-B2a mediate cell sorting and boundary formation in the segmenting caudal hindbrain.

Segmentation and patterning of the vertebrate hindbrain

“…This progressive establishment of cell segregation mechanisms during hindbrain segmentation reflects the fact that Eph receptors and ephrins, which restrict intermingling, are regulated downstream of TFs that underlie segmentation. For example, Epha4 is a direct target of Krox20 (Theil et al, 1998) and is repressed by Hoxa3 and Hoxb4 (Prin et al, 2014); Ephb4 is regulated downstream of Kreisler (Cooke et al, 2001), and ephrin B2 (Efnb2) is regulated by Hoxb4 and Hoxd4 (Prin et al, 2014). Consistent with this, some isolated Krox20-expressing cells are observed in r2+r4+r6 during the period of border sharpening (Cooke and Moens, 2002;Irving et al, 1996) (Fig.…”

“…Eph receptors and ephrins that have a high affinity are expressed in complementary hindbrain segments, and consequently bidirectional signalling occurs at segment borders. For example, in zebrafish, this is seen for Epha4 and Efnb3, and for Ephb4 and Efnb2a (Chan et al, 2001;Cooke et al, 2001;Xu et al, 1995) (Fig. 5A).…”

“…Initial evidence for how cell intermingling is restricted between rhombomeres came from transplantation experiments in chick, which found that it involved cell affinity properties that distinguish r3+r5 from r2+r4+r6 (Guthrie et al, 1993). The identification of segmentally expressed Eph receptors (Becker et al, 1994;Nieto et al, 1992) led to functional analyses (Calzolari et al, 2014;Cayuso et al, 2019;Cooke et al, 2001Cooke et al, , 2005Xu et al, 1995Xu et al, , 1999 that revealed key roles in cell segregation that establish and maintain sharp borders in the hindbrain. Eph receptor tyrosine kinases are clustered and activated upon interacting with membrane-bound ephrins, which also mediate signal transduction, leading to 'forward' and 'reverse' signalling, in the Eph-expressing cell and ephrin-expressing cell, respectively (Kania and Klein, 2016;Pasquale, 2008).…”

Interplay of Eph-Ephrin Signalling and Cadherin Function in Cell Segregation and Boundary Formation

“…For example, in the zebrafish hindbrain, expression of EphB4 in r2, r5, and r6 is complementary to ephrinB2 in r1, r4, and r7, and expression of EphA4 in r3 and r5 is complementary to ephrinB3 in r2, r4, and r6, as well as to ephrinB2 ( Figure 1B ). There is extensive evidence that signalling through these Eph receptors and ephrins underlies the sharpening of segment borders in the hindbrain ( Xu et al, 1995 ; Xu et al, 1999 ; Cooke et al, 2001 ; Cooke et al, 2005 ; Kemp et al, 2009 ; Sela-Donenfeld et al, 2009 ; Calzolari et al, 2014 ; Cayuso et al, 2019 ). Studies of cell segregation and border formation in vivo have found a dominant role of Eph receptor forward signalling through kinase-dependent pathways ( Cayuso et al, 2019 ; O'Neill et al, 2016 ; Rohani et al, 2014 ), although reverse signalling through ephrinBs could also contribute ( Wu et al, 2019 ).…”

“…A model for how cells segregate to form sharp segment borders in the hindbrain can be proposed based on analyses of Eph-ephrin function in other tissues, summarised above, together with studies in the hindbrain itself. The initial ragged border of hindbrain segments is transformed into a sharp and straight border through a combination of cell identity regulation ( Zhang et al, 2012 ; Addison et al, 2018 ) and Eph-ephrin-mediated cell segregation ( Xu et al, 1995 ; Xu et al, 1999 ; Cooke et al, 2001 ; Cooke et al, 2005 ; Kemp et al, 2009 ; Calzolari et al, 2014 ; Cayuso et al, 2019 ). Cell segregation is likely driven by increased cortical tension at the heterotypic interface, principally through Eph receptor forward signaling ( Cayuso et al, 2019 ) that acts through ROCK and MLCK to increase actomyosin contraction.…”