The vertebrate hindbrain develops from a series of segments (rhombomeres) distributed along the anteroposterior axis. We are studying the roles of Wnt and Delta-Notch signaling in maintaining rhombomere boundaries as organizing centers in the zebrafish hindbrain. Several wnt genes (wnt1, wnt3a, wnt8b, and wnt10b) show elevated expression at rhombomere boundaries, whereas several delta genes (dlA, dlB, and dlD) are expressed in transverse stripes flanking rhombomere boundaries. Partial disruption of Wnt signaling by knockdown of multiple wnt genes, or the Wnt mediator tcf3b, ablates boundaries and associated cell types. Expression of dlA is chaotic, and cell types associated with rhombomere centers are disorganized. Similar patterning defects are observed in segmentation mutants spiel-ohne-grenzen (spg) and valentino (val), which fail to form rhombomere boundaries due to faulty interactions between adjacent rhombomeres. Stripes of wnt expression are variably disrupted, with corresponding disturbances in metameric patterning. Mutations in dlA or mind bomb (mib) disrupt Delta-Notch signaling and cause a wide range of patterning defects in the hindbrain. Stripes of wnt1 are initially normal but subsequently dissipate, and metameric patterning becomes increasingly disorganized. Driving wnt1 expression using a heat-shock construct partially rescues metameric patterning in mib mutants. Thus, rhombomere boundaries act as Wnt signaling centers required for precise metameric patterning, and Delta signals from flanking cells provide feedback to maintain wnt expression at boundaries. Similar feedback mechanisms operate in the Drosophila wing disc and vertebrate limb bud, suggesting coaptation of a conserved signaling module that spatially organizes cells in complex organ systems.

We have characterized mutations in the early arrest gene, harpy (hrp), and show that they introduce premature stops in the coding region of early mitotic inhibitor1 (Rca1/emi1). In harpy mutants, cells stop dividing during early gastrulation. Lineage analysis confirms that there is little change in cell number after approximately cycle-14. Gross patterning occurs relatively normally, and many organ primordia are produced on time but with smaller numbers of cells. Despite the lack of cell division, some organ systems continue to increase in cell number, suggesting recruitment from surrounding areas. Analysis of BrdU incorporation shows that endoreduplication continues in many cells well past the first day of development, but cells cease endoreduplication once they begin to differentiate and express cell-type markers. Despite relatively normal gross patterning, harpy mutants show a number of defects in morphogenesis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division.

The valentino (val) mutation in zebrafish perturbs hindbrain patterning and, as a secondary consequence, also alters development of the inner ear. We have examined the relationship between these defects and expression of fgf3 and fgf8 in the hindbrain. The otic vesicle in val/val mutants is smaller than normal, yet produces nearly twice the normal number of hair cells, and some hair cells are produced ectopically between the anterior and posterior maculae. Anterior markerspax5 and nkx5.1 are expressed in expanded domains that include the entire otic epithelium juxtaposed to the hindbrain, and the posterior marker zp23 is not expressed. In the mutant hindbrain,expression of fgf8 is normal, whereas the domain of fgf3expression expands to include rhombomere 4 through rhombomere X (an aberrant segment that forms in lieu of rhombomeres 5 and 6). Depletion of fgf3by injection of antisense morpholino (fgf3-MO) suppresses the ear patterning defects in val/val embryos: Excess and ectopic hair cells are eliminated, expression of anterior otic markers is reduced or ablated, andzp23 is expressed throughout the medial wall of the otic vesicle. By contrast, disruption of fgf8 does not suppress the val/valphenotype but instead interacts additively, indicating that these genes affect distinct developmental pathways. Thus, the inner ear defects observed inval/val mutants appear to result from ectopic expression offgf3 in the hindbrain. These data also indicate that valnormally represses fgf3 expression in r5 and r6, an interpretation further supported by the effects of misexpressing val in wild-type embryos. This is in sharp contrast to the mouse, in which fgf3 is normally expressed in r5 and r6 because of positive regulation by kreisler, the mouse ortholog of val. Implications for co-evolution of the hindbrain and inner ear are discussed.

Recent studies of inner ear development suggest that hair cells and support cells arise within a common equivalence group by cell-cell interactions mediated by Delta and Notch proteins. We have extended these studies by analyzing the effects of a mutant allele of the zebrafish deltaA gene, deltaA(dx2), which encodes a dominant-negative protein. deltaA(dx2/dx2)homozygous mutants develop with a 5- to 6-fold excess of hair cells and a severe deficiency of support cells. In addition, deltaA(dx2/dx2) mutants show an increased number of cells expressing pax2.1 in regions where hair cells are normally produced. Immunohistological analysis of wild-type and deltaA(dx2/dx2) mutant embryos confirmed that pax2.1 is expressed during the initial stages of hair cell differentiation and is later maintained at high levels in mature hair cells. In contrast, pax2.1 is not expressed in support cells. To address the function of pax2.1, we analyzed hair cell differentiation in no isthmus mutant embryos, which are deficient for pax2.1 function. no isthmus mutant embryos develop with approximately twice the normal number of hair cells. This neurogenic defect correlates with reduced levels of expression of deltaA and deltaD in the hair cells in no isthmus mutants. Analysis of deltaA(dx2/dx2); no isthmus double mutants showed that no isthmus suppresses the deltaA(dx2) phenotype, probably by reducing levels of the dominant-negative mutant protein. This interpretation was supported by analysis of T(msxB)(b220), a deletion that removes the deltaA locus. Reducing the dose of deltaA(dx2) by generating deltaA(dx2)/T(msxB)(b220)trans-heterozygotes weakens the neurogenic effects of deltaA(dx2), whereas T(msxB)(b220) enhances the neurogenic defects of no isthmus. mind bomb, another strong neurogenic mutation that may disrupt reception of Delta signals, causes a 10-fold increase in hair cell production and is epistatic to both no isthmus and deltaA(dx2). These data indicate that deltaA expressed by hair cells normally prevents adjacent cells from adopting the same cell fate, and that pax2.1 is required for normal levels of Delta-mediated lateral inhibition.