Cloning, expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling

Abstract

The organizer at the midbrain-hindbrain boundary (MHB) forms at the interface between Otx2 and Gbx2 expressing cell populations, but how these gene expression domains are set up and integrated with the remaining machinery controlling MHB development is unclear. Here we report the isolation, mapping, chromosomal synteny and spatiotemporal expression of gbx1 and gbx2 in zebrafish. We focus in particular on the expression of these genes during development of the midbrain-hindbrain territory. Our results suggest that these genes function in this area in a complex fashion, as evidenced by their highly dynamic expression patterns and relation to Fgf signaling. Analysis of gbx1 and gbx2 expression during formation of the MHB in mutant embryos for pax2.1, fgf8 and pou2 (noi, ace, spg), as well as Fgf-inhibition experiments, show that gbx1 acts upstream of these genes in MHB development. In contrast, gbx2 activation requires ace (fgf8) function, and in the hindbrain primordium, also spg (pou2). We propose that in zebrafish, gbx genes act repeatedly in MHB development, with gbx1 acting during the positioning period of the MHB at gastrula stages, and gbx2 functioning after initial formation of the MHB, from late gastrulation stages onwards. Transplantation studies furthermore reveal that at the gastrula stage, Fgf8 signals from the hindbrain primordium into the underlying mesendoderm. Apart from the general involvement of gbx genes in MHB development reported also in other vertebrates, these results emphasize that early MHB development can be divided into multiple steps with different genetic requirements with respect to gbx gene function and Fgf signaling. Moreover, our results provide an example for switching of a specific gene function of gbx1 versus gbx2 between orthologous genes in zebrafish and mammals.

Evolutionarily conserved function of Gbx2 in anterior hindbrain development

“…In contrast to mouse Gbx2, zebrafish gbx2 appears to be only involved in the maintenance of the anterior hindbrain (our results) and not in the establishment of the MHB (Kikuta et al, 2003;Rhinn et al, 2003Rhinn et al, , 2009). Therefore, it is possible that gbx2 is behaving as a survival factor in maintaining anterior hindbrain development.…”

“…However, this expression is significantly down-regulated at 26 hpf, suggesting a maintenance role for gbx2 at the MHB (Kikuta et al, 2003). We tested whether gbx1 expression (Kikuta et al, 2003;Rhinn et al, 2003) is altered in our gbx2 morphants. Our analysis revealed that between 80% epiboly and the 20-somite stage, the pattern of gbx1 expression in the MHB, hindbrain, and anterior spinal cord was very similar between control and gbx2 morphant embryos ( Fig.…”

“…Later it becomes restricted to a sharp transverse band at the isthmic region and is maintained in the anterior hindbrain in the developing neuraxis (Bouillet et al, 1995;Li and Joyner, 2001;Martinez-Barbera et al, 2001). Studies in zebrafish suggest that establishment of the MHB and positioning of the isthmic organizer is not a conserved role for gbx2 among vertebrate species (Kikuta et al, 2003;Rhinn et al, 2003Rhinn et al, , 2009. These studies show that gbx2 is not expressed early enough during development of the midbrain/hindbrain territory to accomplish the functions of Gbx2 in mouse.…”

“…This analysis revealed a significant truncation in gbx2 morphants (n ¼ 15, mean ¼ 61.33 mm) compared to wild-type siblings (n ¼ 8, mean ¼ 74.50 mm) (P < 0.0001) at 10 somites, persisting to the 20-somite stage. Since gbx2 expression extends caudally up to r5 during early somite stages (Rhinn et al, 2003), we next determined which rhombomere(s) are being affected by a loss of functional gbx2. We examined the width of the r1 domain by performing pax2.1, hoxa2 double in situ hybridization.…”

“…Interestingly, despite many studies in mice showing the necessity of Gbx2 for normal hindbrain development (Wassarman et al, 1997;Millet et al, 1999;Liu and Joyner, 2001b;Li et al, 2002;Waters and Lewandoski, 2006), few studies have focused on this role of Gbx2 in other vertebrate species. Since expression of Gbx2 during segmentation of the anterior hindbrain is similar among vertebrates (von Bubnoff et al, 1996;Wassarman et al, 1997;Rhinn et al, 2003), we hypothesized that unlike in the establishment of the MHB, the role of Gbx2 is functionally conserved during anterior hindbrain development in zebrafish.…”