Identification of Retinal Transformation Hot Spots in Developing Drosophila Epithelia

Background: The retinal determination (RD) network is an evolutionarily conserved regulatory circuit that governs early events in the development of eyes throughout the animal kingdom. Ectopic expression of many members of this network leads to the transformation of non-retinal epithelia into eye tissue. An often-overlooked observation is that only particular cell-populations within a handful of tissues are capable of having their primary developmental instructions superseded and overruled. Methodology/Preliminary Findings: Here we confirm that indeed, only a discrete number of cell populations within the imaginal discs that give rise to the head, antenna, legs, wings and halteres have the cellular plasticity to have their developmental fates altered. In contrast to previous reports, we find that all transformable cell populations do not lie within the TGFb or Hedgehog signaling domains. Additionally neither signaling cascade alone is sufficient for non-retinal cell types to be converted into retinal tissue. The transformation ‘‘hot spots’ ’ that we have identified appear to coincide with several previously defined transdetermination ‘‘weak spots’’, suggesting that ectopic eye formation is less the result of one network overriding the orders of another, as previously thought, but rather is the physical manifestation of redirecting cell populations of enormous cellular plasticity. We also demonstrate that the initiation of eye formation in non-retinal tissues occurs asynchronously compared to that of the normal eye suggesting that retinal development is not under the control o

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. Conclusions Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain

Transcriptome analysis of haploid male gametophyte development in Arabidopsis

BACKGROUND: The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development. RESULTS: We have developed staged spore isolation procedures for Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth. CONCLUSIONS: The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development

Role of Kinesin Heavy Chain in Crumbs Localization along the Rhabdomere Elongation in Drosophila Photoreceptor

BACKGROUND:Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS:Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc's gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors. CONCLUSIONS/SIGNIFICANCE:In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development

Dcas Supports Cell Polarization and Cell-Cell Adhesion Complexes in Development

Mammalian Cas proteins regulate cell migration, division and survival, and are often deregulated in cancer. However, the presence of four paralogous Cas family members in mammals (BCAR1/p130Cas, EFS/Sin1, NEDD9/HEF1/Cas-L, and CASS4/HEPL) has limited their analysis in development. We deleted the single Drosophila Cas gene, Dcas, to probe the developmental function of Dcas. Loss of Dcas had limited effect on embryonal development. However, we found that Dcas is an important modulator of the severity of the developmental phenotypes of mutations affecting integrins (If and mew) and their downstream effectors Fak56D or Src42A. Strikingly, embryonic lethal Fak56D-Dcas double mutant embryos had extensive cell polarity defects, including mislocalization and reduced expression of E-cadherin. Further genetic analysis established that loss of Dcas modified the embryonal lethal phenotypes of embryos with mutations in E-cadherin (Shg) or its signaling partners p120- and β-catenin (Arm). These results support an important role for Cas proteins in cell-cell adhesion signaling in development

Microarray comparison of anterior and posterior drosophila wing imaginal disc cells identifies novel wing genes

Signaling between cells in the anterior (A) and posterior (P) compartments directs Drosophila wing disc development and is dependent on expression of the homeodomain transcription factor Engrailed (En) in P cells. Downstream of en, posteriorly expressed H

The winged-helix transcription factor JUMU regulates development, nucleolus morphology and function, and chromatin organization of Drosophila melanogaster

The PEV-modifying winged-helix/forkhead domain transcription factor JUMU of Drosophila is an essential protein of pleiotropic function. The correct gene dose of jumu is required for nucleolar integrity and correct nucleolus function. Overexpression of jumu results in bloating of euchromatic chromosome arms, displacement of the JUMU protein from the chromocenter and the nucleolus, fragile weak points, and disrupted chromocenter of polytene chromosomes. Overexpression of the acidic C terminus of JUMU alone causes nucleolus disorganization. In addition, euchromatic genes are overexpressed and HP1, which normally accumulates in the pericentric heterochromatin and spreads into euchromatic chromosome arms, although H3-K9 di-methylation remains restricted to the pericentric heterochromatin. The human winged-helix nude gene shows similarities to jumu and its overexpression in Drosophila causes bristle mutations

CRB1 has a cytoplasmic domain that is functionally conserved between human and Drosophila.

Item does not contain fulltextMutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The CRB1 gene is expressed specifically in human retina and brain and encodes a protein homologous to the Drosophila Crumbs protein. In crumbs mutant embryos apico-basal polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be partially rescued by expression of just the membrane-bound cytoplasmic domain, and overexpression of this domain in a wild-type background results in a multilayered epidermis. A striking difference between CRB1 and Crumbs was that the latter contains a transmembrane region and a 37 amino acid cytoplasmic domain. Here we describe an alternative splice variant of human CRB1 that encodes a cytoplasmic domain 72% similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary for function in Drosophila are absolutely conserved. Rescuing and overexpression studies in Drosophila show that the cytoplasmic domains are functionally related between these distant species. This suggests that CRB1 organizes an intracellular protein scaffold in the human retina. Human homologues of proteins binding to Crumbs may be part of this complex and represent candidate genes for retinal dystrophies