Genetic interaction of Lobe with its modifiers in dorsoventral patterning and growth of the Drosophila eye

Dorsoventral (DV) patterning is essential for growth of the Drosophila eye. Recent studies suggest that ventral is the default state of the early eye, which depends on Lobe (L) function, and that the dorsal fate is established later by the expression of the dorsal selector gene pannier (pnr). However, the mechanisms of regulatory interactions between L and dorsal genes are not well understood. For studying the mechanisms of DV patterning in the early eye disc, we performed a dominant modifier screen to identify additional genes that interact with L. The criterion of the dominant interaction was either enhancement or suppression of the L ventral eye loss phenotype. We identified 48 modifiers that correspond to 16 genes, which include fringe (fng), a gene involved in ventral eye patterning, and members of both Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways, which promote L function in the ventral eye. Interestingly, 29% of the modifiers (6 enhancers and 9 suppressors) identified either are known to interact genetically with pnr or are members of the Wingless (Wg) pathway, which acts downstream from pnr. The detailed analysis of genetic interactions revealed that pnr and L mutually antagonize each other during second instar of larval development to restrict their functional domains in the eye. This time window coincides with the emergence of pnr expression in the eye. Our results suggest that L function is regulated by multiple signaling pathways and that the mutual antagonism between L and dorsal genes is crucial for balanced eye growth.     

Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression

Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signalling from an organising centre (the isthmus) located at the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by it being the only hindbrain segment in which no Hox genes are expressed. In this study we show that ectopic FGF8 protein, a candidate for the isthmic organising activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy we demonstrate that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the rl territory. Previous studies have suggested that a retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. We propose a model whereby activation by retinoids is antagonised by inhibition by FGF8 in the anterior hindbrain to set aside the territory from which the cerebellum will develop.     

Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

Dorso-ventral asymmetric functions of teashirt in Drosophila eye development depend on spatial cues provided by early DV patterning genes

The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral by Wingless mediated Homothorax (HTH) induction [Development 129 (2002) 4271]. We looked for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. We propose that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye.     

Temporal and spatial effects of Sonic hedgehog signaling in chick eye morphogenesis

Proper dorsal--ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal--distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal--ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium.     

Allocation and specification of the genital disc precursor cells in Drosophila

The adult structures of Drosophila melanogaster are derived from larval imaginal discs, which originate as clusters of cells within the embryonic ectoderm. The genital imaginal disc is composed of three primordia (female genital, male genital, and anal primordia) that originate from the embryonic tail segments A8, A9, and A10, respectively, and produce the sexually dimorphic genitalia and analia. We show that the genital disc precursor cells (GDPCs) are first detectable during mid-embryogenesis as a 22-cell cluster in the ventral epidermis. Analysis of mutant and double mutant phenotypes of embryonic patterning genes in the GDPCs, together with their expression patterns in these cells, revealed the following with respect to the origins and specification of the GDPCs. The allocation of the GDPCs from the ventral epidermis requires the function of ventral patterning genes, including the EGF receptor and the spitz group of genes. The ventral localization of the GDPCs is further restricted by the action of dorsal patterning genes. Along the anterior-posterior axis, several segment polarity genes (wingless, engrailed, hedgehog, and patched) are required for the proper allocation of the GDPCs. These segment polarity genes are expressed in some, but not all of the GDPCs, indicating that anterior and posterior compartments are not fully established in the GDPCs. In addition, we found that the three primordia of the larval genital disc have already been specified in the GDPCs by the coordinated actions of the homeotic (Hox) genes, abdominal-A, Abdominal-B, and caudal. By identifying how these different patterning networks regulate the allocation and primordial organization of the 22 embryonic precursors of the compound genital disc, we demonstrate that at least some of the organization of the larval disc originates as positional information in the embryo, thus providing a context for further studies on the development of the genital disc.     

Analysis of the expression pattern of Mysidium columbiae wingless provides evidence for conserved mesodermal and retinal patterning processes among insects and crustaceans

The Wnt family includes a number of genes, such as wingless ( wg), which encode secreted glycoproteins that function in numerous developmental patterning processes. In order to gain a better understanding of crustacean pattern formation, a wg orthologue was cloned from the malacostracan crustacean Mysidium columbiae(mysid), and the expression pattern of this gene was compared with that of Drosophila wg. Although Drosophila wg is expressed in many developing tissues, such as the ventral neuroectoderm, M. columbiae wg (mcowg)expression is detected within only a subset of these tissues. mcowg is expressed in the dorsal part of each developing segment and within the developing eye, but not within the ventral neuroectoderm. Dorsal wg expression in Drosophila is required for heart and muscle development, and conservation of this dorsal wgexpression pattern suggests that mcowgmay function to pattern these tissues in mysids. Consistent with this, expression of Even-skipped (Eve) protein in heart precursor and muscle cells, which is dependent on Wg signaling in Drosophila, is also conserved in mysids. Within the developing mysid eye, mcowg is expressed in a pattern that is similar to the expression pattern of Drosophila wg in the fly eye disc. In Drosophila,Wg inhibits neural differentiation at the anterior margin of the eye disc and patterns the dorsal/ventral axis of the eye. These data indicate that mcowg may function similarly during mysid eye development. Analysis of mcowgexpression provides molecular evidence suggesting that the processes of heart, muscle, and eye patterning are likely to be conserved among insects and crustaceans.     

Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm

The successful organization of the vertebrate body requires that local information in the embryo be translated into a functional, global pattern. Somite cells form the bulk of the musculoskeletal system. Heterotopic transplants of segmental plate along the axis from quail to chick were performed to test the correlation between autonomous morphological patterning and Hox gene expression in somite subpopulations. The data presented strengthen the correlation of Hox gene expression with axial specification and focus on the significance of Hox genes in specific derivatives of the somites. We have defined two anatomical compartments of the body based on the embryonic origin of the cells making up contributing structures: the dorsal compartment, formed from purely somitic cell populations; and the ventral compartment comprising cells from somites and lateral plate. The boundary between these anatomical compartments is termed the somitic frontier. Somitic tissue transplanted between axial levels retains both original Hox expression and morphological identity in the dorsal compartment. In contrast, migrating lateral somitic cells crossing the somitic frontier do not maintain donor Hox expression but apparently adopt the Hox expression of the lateral plate and participate in the morphology appropriate to the host level. Dorsal and ventral compartments, as defined here, have relevance for experimental manipulations that influence somite cell behavior. The correlation of Hox expression profiles and patterning behavior of cells in these two compartments supports the hypothesis of independent Hox codes in paraxial and lateral plate mesoderm.     

The Iroquois homeobox genes function as dorsal selectors in the Drosophila head

The Iroquois complex (Iro-C) genes are expressed in the dorsal compartment of the Drosophila eye/antenna imaginal disc. Previous work has shown that the Iro-C homeoproteins are essential for establishing a dorsoventral pattern organizing center necessary for eye development. Here we show that, in addition, the Iro-C products are required for the specification of dorsal head structures. In mosaic animals, the removal of the Iro-C transforms the dorsal head capsule into ventral structures, namely, ptilinum, prefrons and suborbital bristles. Moreover, the Iro-C(-) cells can give rise to an ectopic antenna and maxillary palpus, the main derivatives of the antenna part of the imaginal disc. These transformations are cell-autonomous, which indicates that the descendants of a dorsal Iro-C(-) cell can give rise to essentially all the ventral derivatives of the eye/antenna disc. These results support a role of the Iro-C as a dorsal selector in the eye and head capsule. Moreover, they reinforce the idea that developmental cues inherited from the distinct embryonic segments from which the eye/antenna disc originates play a minimal role in the patterning of this disc.     

The role of Xmsx-2 in the anterior-posterior patterning of the mesoderm in Xenopus laevis

Many molecules are involved in defining mesodermal patterning of the Xenopus embryo. In this paper, evidence is provided that a member of the msx family of genes, the Xmsx-2 gene, is involved in anterior-posterior patterning of the mesoderm. A comparison of its sequence to another previously cloned msx-2 Xenopus homolog, Xhox-7.1' [45] showed that they are closely related. The Xmsx-2 gene is first expressed at midgastrulation predominantly in the dorsal part of the embryo. It showed a complex pattern of spatial expression, consistent with a role in patterning of the anterior-posterior axis. This inference is confirmed by gain-of-function experiments in which overexpressed msx-2 mRNA in developing Xenopus embryos resulted in embryos lacking anterior structures. Analysis of markers in mutant embryos showed that genes involved in ventral-posterior patterning such as Xhox-3, Xwnt-8, and Xvent-1 were upregulated, confirming the posteriorized nature of the embryos. We believe that the Xmsx-2 gene is involved in refining the patterning of the anterior-posterior part of the dorsal mesoderm after the initial signals determining the dorsal or ventral nature of the mesoderm have been specified.     

Differential Activity of Ras1 during Patterning of the Drosophila Dorsoventral Axis

In Drosophila, the Ras1 gene is required downstream of receptor tyrosine kinases for correct eye development, embryonic patterning, wing vein formation, and border cell migration. Here we characterize a P-element allele of Ras1, Ras1(5703), that affects viability, eye morphogenesis, and early and late stages of oogenesis. Flies transheterozgyous for Ras1(5703) and existing EMS-induced Ras1 alleles are viable and exhibit a range of eye and eggshell defects. Differences in the severity of these phenotypes in different tissues suggest that there are allele-specific effects of Ras1 in development. Analysis of rescue constructs demonstrates that these differential phenotypes are due to loss of function in Ras1 alone and not due to effects on neighboring genes. Females mutant at the Ras1 locus lay eggs with reduced or missing dorsal eggshell structures. We observe dominant interactions between Ras1 mutants and other dorsoventral pathway mutants, including Egfr(top) and gurken. Ras1 is also epistatic to K10. Unlike Egfr(top) and gurken mutants, however, Ras1 females are moderately fertile, laying eggs with ventralized eggshells that can hatch normal larvae. These results suggest that Ras1 may have a different requirement in the patterning of the eggshell axis than in the patterning of the embryonic axis during oogenesis.     

Domain specific genetic mosaic system in the Drosophila eye

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss‐of‐function as well as gain‐of‐function clones on the dorsal and ventral eye margins. We used the bifid‐Gal4 driver to drive expression of UAS‐FLP. This reagent can have multiple applications in (i) studying spatio‐temporal function of a gene during dorso‐ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner. genesis 51:68–74, 2013. © 2012 Wiley Periodicals, Inc.     

On the Development of Ectopic Eyes in DROSOPHILA MELANOGASTER Produced by the Mutation Extra Eye (EE)

The mutation ee often produces an ectopic eye on the vertex that is a mirror image partial duplication of the normal eye on the ipsilateral side of the head. The pattern of the duplication and a clonal analysis by mitotic recombination indicate that the duplications are of dorsal eye and orbital structures. Large ectopic eyes (more than 100 ommatidia) and their surrounding bristles may be produced without cuticular deficiencies. The penetrance of ee is temperature dependent with penetrance higher (72%) at 25 degrees and 29 degrees than at 19 degrees (43%). Temperature shift experiments show two temperature-sensitive periods: one at midembryogenesis, the other at mid-first larval instar. Microscopic examination of ee late-second and third instar imaginal cephalic discs show no indication of growth of the extra tissue needed to produce the duplication until after mid-third instar. This was confirmed by cell counts of ee and wild-type discs. There is no evidence of differential cell death in the two types of discs at this stage, although much earlier cell death is postulated. Tests for cell autonomy of the mutation by the production of morphogenetic clones suggest nonautonomy. Formation of pattern duplications by mutant genes is discussed in terms of cell death that eliminates whole developmental compartments, restricted cell death that occurs within a compartment, extensive cell death within a compartment and proliferative growth unassociated with cell lethality.