Specification of Primary Pigment Cell and Outer Photoreceptor Fates byBarH1Homeobox Gene in the DevelopingDrosophilaEye

In the developingDrosophilaeye,BarH1andBarH2, paired homeobox genes expressed in R1/R6 outer photoreceptors and primary pigment cells, are essential for normal eye morphogenesis. Here, we show evidence thatBarH1ectopically expressed under the control of thesevenlessenhancer (sev-BarH1) causes two types of cone cell transformation: transformation of anterior/posterior cone cells into outer photoreceptors and transformation of equatorial/polar cone cells into primary pigment cells.sev-BarH1repressed the endogenous expression of theroughhomeobox gene in R3/R4 photoreceptors, while theBarH2homeobox gene was activated bysev-BarH1in an appreciable fraction of extra outer photoreceptors. In primary pigment cells generated by cone cell transformation, the expression ofcut,a homeobox gene specific to cone cells, was completely replaced with that ofBarhomeobox genes. Extra outer photoreceptor formation was suppressed and enhanced, respectively, by reducing the activity of Ras/MAPK signaling and by dosage reduction ofyan,a negative regulator of the pathway, suggesting interactions betweenBarhomeobox genes (cell fate determinants) and Ras/MAPK signaling in eye development.     

A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Previously, we have shown that ectopic expression of a constitutively active protein (yan(ACT)) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, we have performed a genetic screen to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan(ACT). Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes we recovered are four RTK pathway components, rolled (MAPK), son-of-sevenless, Star, and pointed, and two genes, eyes absent and string, that have not been implicated previously in RTK signaling events. We also isolated mutations in five previously uncharacterized genes, one of which, split ends, we have characterized molecularly and have shown to encode a member of the RRM family of RNA-binding proteins.     

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.     

Modifiers of muscle and heart cell fate specification identified by gain-of-function screen in Drosophila

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors and are both implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, we have performed a gain-of-function (GOF) screen for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Amongst the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of our screen. Observed GOF phenotypes have led us to identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Amongst them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Our analysis of rac2 phenotypes clearly demonstrates that the altered mesodermal level of Rho-GTPase Rac2 can influence specification of a number of cardiac and muscular cell types including those expressing ladybird. Finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicate a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinases (RTKs) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types.     

ten-a overexpression causes abnormal pattern in the Drosophila compound eye

Ten-a is one of the two Drosophila proteins that belong to the Ten M protein family. This protein is a type Ⅱ transmembrane protein and is expressed mainly in the embryonic CNS, in the larval eye imaginal disc and in the compound eye of the pupa. Here, we investigate the role of ten-α during development of the compound eye by using the Gal4/ UAS system to induce ten-α overexpression in the developing eye. We found that overexpression of ten-α can perturb eye development during all stages examined. In an early stage, overexpression of ten-α in eye primordial cells caused small and rough eyes and interfered with photoreceptor cell recruitment, resulting in some ommatidia having fewer or extra photoreceptor cells. Conversely, ten-α overexpression daring ommatidial formation caused severe eye defects due to absence of many cellular components. Interestingly, overexpression of ten-α in the late stage developing ommatidial cluster affected the number of pigment cells, caused cone cells proliferation in many ommatidia, and caused some photoreceptor cell defects. These results suggest that ten-α may be a novel gene required for normal eye morphogenesis.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates.

The regulatory gene hairy is expressed and required during early embryogenesis to control segmentation gene expression properly and during larval and pupal development to control the pattern of certain adult sensory structures. We have found the hairy protein to be expressed transiently during two stages of eye imaginal disc development, including all cells immediately anterior to the morphogenetic furrow that traverses the developing eye disc, and again in the presumptive R7 photoreceptor cells of the developing ommatidia. This pattern is conserved in a significantly diverged Drosophila species. We show that, surprisingly, ommatidia formed by homozygous hairy- mutant clones are apparently normal, indicating that hairy function in the eye is dispensable. However, we do find that ectopic expression of hairy causes numerous structural abnormalities and the alteration of cell fates. Thus, proper regulation of hairy is still essential for normal eye development. We suggest that the loss of hairy function may be compensated by other regulatory proteins, as has been observed previously for several structurally and functionally related genes involved in sensory organ development. The effects of ectopic hairy expression may result from interactions with proneural genes involved in the development of the eye and other sensory organs.     

Rx-Cre, a tool for inactivation of gene expression in the developing retina

Rx is a homeobox-containing gene that is critical for vertebrate eye development. Its expression domain delineates a field of cells from which the retina and the ventral hypothalamus develop. The 5' upstream regulatory sequences of the medaka fish Rx gene are functionally conserved during evolution to a degree that they direct gene expression into the Rx-expressing field of cells in mice. Using these sequences, we made a Cre line that can be used for inactivation of gene expression in the developing retina.     

Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma

Vax2 is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralisation of the retina, thus suggesting its role in the molecular pathway that underlies eye development. We describe the generation and characterisation of a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalisation of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.