Characterization of harpy/Rca1/emi1 mutants: patterning in the absence of cell division.

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 bromodeoxyuridine 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 several defects in morphogenesis, cell migration and differentiation resulting directly or indirectly from the arrest of cell division.

Rhombomere boundaries are Wnt signaling centers that regulate metameric patterning in the zebrafish hindbrain.

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.

An expanded domain of fgf3 expression in the hindbrain of zebrafish valentino mutants results in mis-patterning of the otic vesicle.

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 markers pax5 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 fgf3 expression expands to include rhombomere 4 through rhombomere X (an aberrant segment that forms in lieu of rhombomeres 5 and 6). Depletion of fgf3 by 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, and zp23 is expressed throughout the medial wall of the otic vesicle. By contrast, disruption of fgf8 does not suppress the val/val phenotype but instead interacts additively, indicating that these genes affect distinct developmental pathways. Thus, the inner ear defects observed in val/val mutants appear to result from ectopic expression of fgf3 in the hindbrain. These data also indicate that val normally 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.