Very shortly after the end of gastrulation (10 hpf), the zebrafish hindbrain is already clearly subdivided into distinct molecular territories that presage the rhombomeres. Presumptive rhombomere markers include krox-20 in r3 and r5, valentino/mafB in r5 and r6, and the hox genes (Fig. 7A). The hox genes encode evolutionarily conserved homeodomain-containing transcription factors that specify segment identity, and are discussed in greater detail below. Mutational analyses in the mouse and fish have identified some of the molecules that are responsible for the finer-scale patterning that occurs within the hindbrain after its initial specification during gastrulation.
Signals From the Mid-Hindbrain Junction Pattern the Anterior Hindbrain
At the anterior end of the hindbrain lies a prominent constriction, the isthmus, which plays an important role in patterning both the midbrain and the hindbrain (Liu and Joyner, 2001). The zebrafish ace/fgf8 gene is expressed in a narrow domain within the isthmus and ace/fgf8 mutants lack this structure (Brand et al., 1996; Fig. 5D). The role of isthmic Fgf8 in patterning the adjacent midbrain of zebrafish, chick, and mouse embryos has been well described (reviewed in Rhinn and Brand, 2001; Liu and Joyner, 2001). However Fgf8 also patterns the anterior hindbrain. In ace/fgf8 mutants, cell types characteristic of the anterior part of r0, which include the neurons of the locus coeruleus, are absent (Guo et al., 1999). At the same time, the expression domain of fgfr3, a marker of r1, is expanded anteriorly (Sleptsova-Friedrich et al., 2001; Fig. 5A,B). Thus ace/fgf8 is involved in specifying r0 and in setting the boundary between r0 and r1.
Figure 5. FGF and retinoic acid signalling pattern the zebrafish hindbrain. In all panels, krox-20 expression in r3 and r5 is in pink. A,B: expression of fgfr3 marks r1 in wild-type embryos (bracket in A). This domain is expanded anteriorly in ace/fgf8 mutants (bracket in B). C,D:ace/fgf8 is expressed dynamically throughout the anterior hindbrain at 11 hours postfertilization (hpf) before becoming restricted to the isthmus by 14 hpf (arrows in C and D indicate the position of the presumptive isthmus). E,F:hoxb4 is expressed throughout the anterior spinal cord up to the r6/7 boundary in wild-type embryos (line in E). In nkl/raldh2 mutants, this domain is shortened in its anterior-posterior extent (line in F). Anterior is to the left.
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Fgf8 may in fact influence regional identity much more broadly in the hindbrain. In chick embryos in which Fgf8 signalling at the midbrain-hindbrain junction was locally disrupted by using blocking antibodies, the anterior limit of r2 was shifted anteriorly (Irving and Mason, 2000). Recently, L. Maves and C. Kimmel have shown that reducing function of both ace/fgf8 and another fgf expressed in the hindbrain, fgf3, prevents the specification of r5 and r6; however, eliminating fgf3 alone has very mild effects (personal communication). Finally, work in the chick has recently shown that eliminating fgf signalling with a pharmacological antagonist also disrupts specification of r3, r5, and r6 (Marin and Charnay, 2000).
One difficulty with interpreting the effects of loss of ace/fgf8 function on zebrafish hindbrain development is the fact that ace/fgf8 expression is not limited to the mid-hindbrain junction. Early in development, ace/fgf8 is expressed in the germ ring, and before becoming restricted to the isthmus, ace/fgf8 is expressed throughout the anterior hindbrain, including in r4 where its expression overlaps with that of fgf3 (Reifers et al., 1998; Phillips et al., 2001; Fig. 5C). Although it is likely that ace/fgf8 is required at the mid-hindbrain junction to pattern the anterior-most hindbrain, it is also likely that its broader effects on hindbrain patterning result from more short-range signalling within the hindbrain itself. Indeed, Maves and Kimmel have shown that the r5 and r6 defects caused by reducing Fgf3 and Fgf8 signalling can be rescued by wild-type cells transplanted into the hindbrain, suggesting that an Fgf signalling center(s) within the hindbrain itself acts to pattern the surrounding rhombomeres (personal communication).
The zebrafish spiel-ohne-grenzen (spg) mutant phenotype resembles that of ace/fgf8 mutants in that both lack an isthmus and have hindbrain patterning defects. In spg mutants, r1 is expanded anteriorly and r3 and r5 are reduced and abnormally shaped (Schier et al., 1996; Belting et al., 2001; Burgess et al., 2002; Hauptmann et al., in press). Spg encodes Pou2, a homeodomain transcription factor that is expressed during gastrulation in the anterior hindbrain and mid-hindbrain boundary primordia and then becomes restricted to r2 and r4 at around the time when other segment-restricted gene expression is initiated (Belting et al., 2001; Burgess et al., 2002). Ubiquitous mis-expression of spg/pou2 is sufficient to rescue the mutant hindbrain phenotype, suggesting that the spg/pou2 gene product is providing a permissive factor that is required for the embryo to respond to another, instructive determinant involved in the specification of hindbrain rhombomeres (Belting et al., 2001). That this determinant might be an Fgf is suggested by the observation that overexpression of fgf8 or implantation of Fgf8-coated beads have no effect on marker gene expression or morphogenesis in the spg/pou2 mutant neuroepithelium (Reim and Brand, 2002). Furthermore, expression of a known Fgf target, sprouty4, is strongly reduced both in the hindbrain and at the mid-hindbrain boundary of spg/pou2 mutants. Thus spg/pou2 may act as a regional competence factor for Fgf signalling in the hindbrain primordium and then, during segmentation stages, it may further limit Fgf responsiveness to specific rhombomeres (Reim and Brand, 2002).
Valentino Functions Cell-Autonomously to Specify r5 and r6 Identity
Confocal time-lapse imaging of the developing zebrafish hindbrain demonstrates that some segment boundaries form later than others (Moens et al., 1998). In the zebrafish, the presumptive r5-r6 domain, which expresses the bZip transcription factor Valentino (Val)/MafB initially appears as a broad domain that is subsequently subdivided into the definitive rhombomeres (Moens et al., 1998). The val/mafB mutant lacks r5 and r6 and consequently lacks rhombomere boundaries between r4 and r7 (Moens et al., 1996). Mosaic analysis of val/mafB demonstrated that this gene is required autonomously for cells to take on either r5 or r6 identity: cells lacking val/mafB function are gradually excluded from r5 and r6 of a wild-type host embryo (Moens et al., 1996, 1998; Fig. 6). Thus, val/mafB functions in a manner similar to a Drosophila gap gene, in the absence of which blocks of segments are deleted.
Figure 6. Mosaic analysis demonstrates that val/mafB is required cell-autonomously for r5 and r6 identity. Val/mafB-cells (brown) were transplanted into the presumptive hindbrain of a wild-type host embryo at the early gastrula stage. During the ensuing 12 hr, these mutant cells were excluded from precisely the domain of the wild-type host that expresses val/mafB mRNA (blue staining). Anterior is to the left. ov, otic vesicle.
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Analysis of a classic mouse mutant, Kreisler, which carries a mutation in the mouse MafB gene (Cordes and Barsh, 1994), has suggested that this gene functions differently in the mouse than in the zebrafish. In the Kr/MafB mouse, r5 is absent, whereas some markers of r6 are still expressed. Furthermore, cells from the abnormal r5/6 region of a Kr/MafB embryo transplanted into a wild-type embryo are excluded from r5 but not from r6. These experiments have led to the conclusion that r6 is normal in Kr/MafB mice (Manzanares et al., 1999b, but see McKay et al., 1994). Differences between val/mafB function in the zebrafish and Kr/MafB function in the mouse correlate with differences in both gene expression and neuroanatomy between fish and mice. For example, the mouse hoxb3 gene, which is a direct target of Kr/MafB (see below), is expressed only in r5, whereas the zebrafish hoxb3 is expressed in both r5 and r6, and in the mouse, the motor neurons of the sixth cranial nerve differentiate in r5, whereas in the zebrafish, they differentiate in r5 and r6. The putative functional differences between val/mafB and Kr/MafB do not correlate with differences in their regulation, because both genes are expressed throughout r5 and r6, but may result from differences in the expression of essential cofactors (Manzanares et al., 1997).
val/mafB and Kr/MafB both function upstream of hox genes in r5 and r6. Analysis of the regulatory sequences of the mouse Hoxb3 and Hoxa3 genes has identified essential Kr/MafB binding sites required for expression in r5 and r5 and r6, respectively (Manzanares et al., 1997, 1999a). In the zebrafish, the hoxa3 and hoxb3 genes are normally both up-regulated throughout r5 and r6, and this up-regulation fails to occur in val/mafB mutants (Prince et al., 1998). By extrapolation from the mouse work, the positive effect of val/mafB on hoxb3 expression in r5 and r6 in the zebrafish is likely to be direct. val/mafB and Kr/MafB may also negatively regulate hox gene expression in r5 and r6. Expression of hoxb1a, which is normally restricted to r4, expands posteriorly in val/mafB mutants (Prince et al., 1998). The negative effects of val/mafB and kreisler on hoxb1a expression are poorly understood, either in the fish or in the mouse where effects of Kr/MafB on hoxb1 expression remain controversial (Frohman et al., 1993; McKay et al., 1994; Manzanares et al., 1999b). It is possible that posteriorly expanded hoxb1a expression in val/mafB mutants results from loss of direct or indirect inhibition of hoxb1a expression. Alternatively, hoxb1a-expressing cells from r4 may spread posteriorly because of the absence of an r4/5 boundary. Lineage analysis of individual rhombomeres in val/mafB mutants should help to address this question.
How is val/mafB itself regulated? Recently a new player with a key role in specifying segment identity in the r5/r6 region has been identified through the isolation and cloning of an insertional mutant in the zebrafish (Sun and Hopkins, 2001). Zebrafish embryos lacking the homeobox gene vhnf1 resemble valentino mutants; however vhnf1 is genetically upstream of valentino, because mutants never express valentino in the r5/6 region. vhnf1 is expressed in the gut and pronephros where it has other essential functions, but its earliest expression is within the central nervous system with a sharp anterior limit at the r4/5 boundary. How vhnf1 itself is regulated is as yet unknown, but is clearly an important question. Further study of the relationship between vhnf1, val/mafB, and the hox genes will fill out our picture of the hierarchy of events involved in setting up pattern in the posterior hindbrain.