Rachel Macdonald

Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain

During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors, but not rod precursors, in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration.

floating head and masterblind Regulate Neuronal Patterning in the Roof of the Forebrain

The epiphysial region of the dorsal diencephalon is the first site at which neurogenesis occurs in the roof of the zebrafish forebrain. We show that the homeobox containing gene floating head (flh) is required for neurogenesis to proceed in the epiphysis. In flh- embryos, the first few epiphysial neurons are generated, but beyond the 18 somite stage, neuronal production ceases. In contrast, in masterblind- (mbl-) embryos, epiphysial neurons are generated throughout the dorsal forebrain. We show that mbl is required to prevent the expression of flh in dorsal forebrain cells rostral to the epiphysis. Furthermore, epiphysial neurons are not ectopically induced in mbl-/flh- embryos, demonstrating that the epiphysial phenotype of mbl- embryos is mediated by ectopic Flh activity. We propose a role for Flh in linking the signaling pathways that regulate regional patterning to the signaling pathways that regulate neurogenesis.

Pax proteins and eye development

Homologous members of the Pax gene family are required for eye development in Drosophila and vertebrates. Despite superficial similarities in the phenotypes of vertebrates with mutations in pax-6 and Drosophila eyeless mutants, it remains uncertain whether the two proteins encoded by these genes have comparable functions. The genetic cascade triggered by eyeless leads to eye formation, whereas pax-6 is not necessary for optic vesicle formation, but is required at other stages of eye development. A second vertebrate Pax gene, pax-2, is also required during eye development and appears to play a role during closure of the choroid fissure.

Distribution of Pax6 protein during eye development suggests discrete roles in proliferative and differentiated visual cells.

Abstract Although Pax6 is required during eye development in rodents and humans, little is known about the precise role of the protein in this process. To aid in the interpretation of functional studies, we have determined the precise spatial and temporal distributions of the Pax6 protein in the eye. We find that Pax6 is initially distributed contiguously throughout a large domain of the anterior neural plate of zebrafish, including the presumptive eye fields and the dorsal diencephalon. After evagination of the optic vesicle, Pax6 becomes restricted to all proliferating cells of the pigment epithelial and neural layers of the retina. Pax6 is downregulated in most cells concomitant with differentiation. However, it remains present in several mature cell types of the eye including amacrine cells and the lens and corneal epithelia. This expression is conserved across diverse vertebrate species and suggests that Pax6 has additional conserved functions in the mature eye.

ANALYSIS OF AXON TRACT FORMATION IN THE ZEBRAFISH BRAIN : THE ROLE OF TERRITORIES OF GENE EXPRESSION AND THEIR BOUNDARIES

Abstract. Mutant analysis in the zebrafish is revealing the genes that are expressed in the early neuroepithelium and that regulate factors responsible for the guidance of commissural axons. We review work on the developing zebrafish brain illustrating the way in which territories of regulatory gene expression influence the formation and positioning of axon pathways.

58 Genes involved in neuronal specification at boundary zones in the embyronic zebrafish forebrain

Development of the chick midbrain, isthmus and cerebellum involves morphogenetic signals produced at the mes-metencephalic boundary. FGFB is a candidate for a secreted protein involved in signaling from this boundary. To test the effect of FGFB on brain development we implanted beads soaked in recombinant FGFS protein into the embryonic chick brain caudal to the zona limitans (ZL). We found that FGFS beads induce ectopic midbrain, isthmic. and cerebellar development and that the type of tissue induced depends on the proximity of the inducer to the ZL. Cerebellum was not induced when the beads were. placed near the ZL (in p2) but was induced when the beads were placed in more caudal tissue, suggesting that the ZL may have signaling properties that suppress ectopic cerebellum induction. In addition we found that whereas beads placed in p2 affect both p2 and the more. caudal prosomere, pl. beads placed m pl oc the mesencephaloo did not affect more rostra1 prosomeres. These observations suggest that the pl/p2 and mes/pl boundaries may act as barriers that prevent rhe rostra1 spread of FGFS induced effects. In summary OUT results suggest thal FGFB plays a central role in the signaling activity of the mes/metencephalic boundary, that prosomere boundaries block the rostra1 spread of FGFB induced effects, and that the ZL inhibits FGFS mediated ectopic cerebellum induction.