The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.     

Dcas is required for importin-alpha3 nuclear export and mechano-sensory organ cell fate specification in Drosophila

We have studied the in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin beta-like export receptor for importin alpha. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-alpha3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. We interpret the specificity of the dcas phenotype as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. We propose that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders.     

numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos

Neurons and support cells of each sensory organ in Drosophila embryos are most likely derived from a single precursor cell. This cell lineage is affected in numb mutants. Morphological alterations of sensory structures, as well as changes in the number of cells expressing cell type-specific markers, indicate that sensory neurons in numb mutant embryos are transformed into lineage-related nonneuronal support cells. Thus the numb gene controls the fate of progeny derived from sensory organ precursors. The numb gene has been isolated by the plasmid rescue method. The structure of its predicted product is discussed.     

Retinoic acid signalling is required for specification of pronephric cell fate

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.     

Crumbs and the apical spectrin cytoskeleton regulate R8 cell fate in the Drosophila eye

The Hippo pathway is an important regulator of organ growth and cell fate. In the R8 photoreceptor cells of the Drosophila melanogaster eye, the Hippo pathway controls the fate choice between one of two subtypes that express either the blue light-sensitive Rhodopsin 5 (Hippo inactive R8 subtype) or the green light-sensitive Rhodopsin 6 (Hippo active R8 subtype). The degree to which the mechanism of Hippo signal transduction and the proteins that mediate it are conserved in organ growth and R8 cell fate choice is currently unclear. Here, we identify Crumbs and the apical spectrin cytoskeleton as regulators of R8 cell fate. By contrast, other proteins that influence Hippo-dependent organ growth, such as the basolateral spectrin cytoskeleton and Ajuba, are dispensable for the R8 cell fate choice. Surprisingly, Crumbs promotes the Rhodopsin 5 cell fate, which is driven by Yorkie, rather than the Rhodopsin 6 cell fate, which is driven by Warts and the Hippo pathway, which contrasts with its impact on Hippo activity in organ growth. Furthermore, neither the apical spectrin cytoskeleton nor Crumbs appear to regulate the Hippo pathway through mechanisms that have been observed in growing organs. Together, these results show that only a subset of Hippo pathway proteins regulate the R8 binary cell fate decision and that aspects of Hippo signalling differ between growing organs and post-mitotic R8 cells.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling

Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.     

The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye.

The argos gene encodes a protein that is required for viability and that regulates the determination of cells in the Drosophila eye. A developmental analysis of argos mutant eyes indicates that the mystery cells, which are usually nonneuronal, are transformed into extra photoreceptors, and that supernumerary cone cells and pigment cells are also recruited. Clonal analysis indicates that argos acts nonautonomously and can diffuse over the range of several cell diameters. Conceptual translation of the argos gene suggests that it encodes a secreted protein.     

Specification of cell fate in the developing eye of Drosophila.

Determination of cell fate in the developing eye of Drosophila depends on a precise sequence of cellular interactions which generate the stereotypic array of ommatidia. In the eye imaginal disc, an initially unpatterned epithelial sheath of cells, the first step in this process may be the specification of R8 photoreceptor cells at regular intervals. Genes such as Notch and scabrous, known to be involved in bristle development, also participate in this process, suggesting that the specification of ommatidial founder cells and the formation of sensory organs in the adult epidermis may involve a similar mechanism, that of lateral inhibition. The subsequent steps of ommatidial assembly, following R8 assignment, involve a different mechanism: Undetermined cells read their position based on the contacts they make with neighbors that have already begun to differentiate. The development of the R7 photoreceptor cell, one of the eight photoreceptor cells in the ommatidium, is best understood. An important role seems to be played by sevenless, a receptor tyrosine kinase on the surface of the R7 precursor. It transmits the positional information--most likely encoded by the boss protein on the neighboring R8 cell membrane--into the cell via its tyrosine kinase, which activates a signal transduction cascade. Constitutive activation of the sevenless kinase by overexpression of an N-terminally truncated form results in the diversion of other ommatidial cells into the R7 pathway suggesting that activation of the sevenless signalling pathway is sufficient to specify R7 development. Genetic dissection of this pathway should therefore identify components of a signalling cascade activated by a tyrosine kinase.     

Characterization of Drosophila mini-me, a gene required for cell proliferation and survival

In the developing Drosophila eye, the morphogenetic furrow is a developmental organizing center for patterning and cell proliferation. The furrow acts both to limit eye size and to coordinate the number of cells to the number of facets. Here we report the molecular and functional characterization of Drosophila mini-me (mnm), a potential regulator of cell proliferation and survival in the developing eye. We first identified mnm as a dominant modifier of hedgehog loss-of-function in the developing eye. We report that mnm encodes a conserved protein with zinc knuckle and RING finger domains. We show that mnm is dispensable for patterning of the eye disc, but required in the eye for normal cell proliferation and survival. We also show that mnm null mutant cells exhibit altered cell cycle profiles and contain excess nucleic acid. Moreover, mnm overexpression can induce cells to proliferate and incorporate BrdU. Thus, our data implicate mnm as a regulator of mitotic progression during the proliferative phase of eye development, possibly through the control of nucleic acid metabolism.     

The RING domain of Siaz, the zebrafish homologue of Drosophila seven in absentia, is essential for cellular growth arrest

Siah is a mammalian homologue of Drosophila seven in absentia (sina) that is required for R7 photoreceptor development. Both the SINA and Siah family interact with ubiquitin-conjugating enzymes via an N-terminal RING domain and the C-terminal domain of SINA/ Siahs interacts with proteins targeted for degradation. Siah induces cell growth arrest by promoting beta-catenin degradation in a phosphorylation-independent manner as a result of indirect binding to beta-catenin. We previously cloned a zebrafish homologue (Siaz) of Siah. Siaz shares high sequence homology with vertebrate Siah-2. We have now examined the role of Siaz in growth regulation using the trypan blue exclusion assay and flow cytometry and found that Siaz induces cellular growth arrest by inhibiting the G2/M transition. The C-terminal domain of Siaz that interacts with target proteins is not required for growth inhibition. We conclude that the N-terminal RING and central domain of Siaz are sufficient to block the G2/M phase transition.     

Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes.

In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. We propose that the spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth.     

beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis

The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that beta-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for beta-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that beta-catenin is one of the essential components of this specification.