Dpp and Hh signaling in the Drosophila embryonic eye field.

We have analyzed the function of the Decapentaplegic (Dpp) and Hedgehog (Hh) signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. As reported previously, Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). We discuss the similarities between vertebrate systems and Drosophila with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling.     

Specialized extraembryonic cells connect embryonic and extraembryonic epidermis in response to Dpp during dorsal closure in Drosophila

Dorsal closure in Drosophila embryogenesis involves expansion of the dorsal epidermis, followed by closure of the opposite epidermal edges. This process is driven by contractile force generated by an extraembryonic epithelium covering the yolk syncytium known as the amnioserosa. The secreted signaling molecule Dpp is expressed in the leading edge of the dorsal epidermis and is essential for dorsal closure. We found that the outermost row of amnioserosa cells (termed pAS) maintains a tight basolateral cell-cell adhesion interface with the leading edge of dorsal epidermis throughout the dorsal closure process. pAS was subject to altered cell motility in response to Dpp emanating from the dorsal epidermis, and this response was essential for dorsal closure. alphaPS3 and betaPS integrin subunits accumulated in the interface between pAS and dorsal epidermis, and were both required for dorsal closure. Looking at alphaPS3, type I Dpp receptor, and JNK mutants, we found that pAS cell motility was altered and pAS and dorsal epidermis adhesion failed under the mechanical stress of dorsal closure, suggesting that a Dpp-mediated mechanism connects the squamous pAS to the columnar dorsal epidermis to form a single coherent epithelial layer.     

Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain

The central neuroendocrine system in the Drosophila brain includes two centers, the pars intercerebralis (PI) and pars lateralis (PL). The PI and PL contain neurosecretory cells (NSCs) which project their axons to the ring gland, a complex of peripheral endocrine glands flanking the aorta. We present here a developmental and genetic study of the PI and PL. The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head. The placodes invaginate during late embryogenesis and become attached to the brain primordium. The PI placode and its derivatives express the homeobox gene Dchx1 and can be followed until the late pupal stage. NSCs labeled by the expression of Drosophila insulin-like peptide (Dilp), FMRF, and myomodulin form part of the Dchx1 expressing PI domain. NSCs of the PL can be followed throughout development by their expression of the adhesion molecule FasII. Decapentaplegic (Dpp), secreted along the dorsal midline of the early embryo, inhibits the formation of the PI and PL placodes; loss of the signal results in an unpaired, enlarged placodeal ectoderm. The other early activated signaling pathway, EGFR, is positively required for the maintenance of the PI placode. Of the dorso-medially expressed head gap genes, only tailless (tll) is required for the specification of the PI. Absence of the corpora cardiaca, the endocrine gland innervated by neurosecretory cells of the PI and PL, does not affect the formation of the PI/PL, indicating that inductive stimuli from their target tissue are not essential for early PI/PL development.     

Epidermal growth factor receptor signaling activates orthodenticle expression during Drosophila head development

The relation between signal transduction pathways and the genes that specify regional identity remains poorly understood. We investigated the interaction between the epidermal growth factor receptor (EGFR) pathway and the homeobox gene orthodenticle (otd), which specifies cell fate during head development. Previous studies of head formation in Drosophila melanogaster demonstrated that reducing either EGFR signaling or otd expression in the imaginal primordium of the dorsal head capsule eliminates the ocelli and other dorsal head structures. Here, we show that blocking EGFR signaling reduces otd expression and that activating EGFR signaling outside this primordium induces ectopic otd expression. We also demonstrate that loss of EGFR can be rescued by constitutive otd expression. Our results indicate that otd is a downstream target of the EGFR pathway during head development.     

Argos induces programmed cell death in the developing Drosophila eye by inhibition of the Ras pathway

We studied the role of Ras signaling in the regulation of cell death during Drosophila eye development. Overexpression of Argos, a diffusible inhibitor of the EGF receptor and Ras signaling, caused excessive cell death in developing eyes at pupal stages. The Argos-induced cell death was suppressed by coexpression of the anti-apoptotic genes p35, diap1, or diap2 in the eye as well as by the Df(3L)H99 chromosomal deletion that lacks three apoptosis-inducing genes, reaper, head involution defective (hid) and grim. Transient misexpression of the activated Ras1 protein (Ras1V12) later in pupal development suppressed the Argos-induced cell death. Thus, Argos-induced cell death seemed to have resulted from the suppression of the anti-apoptotic function of Ras. Conversely, cell death induced by overexpression of Hid was suppressed by gain-of-function mutations of the genes coding for MEK and ERK. These results support the idea that Ras signaling functions in two distinct processes during eye development, first triggering the recruitment of cells and later negatively regulating cell death.     

Retinal determination genes as targets and possible effectors of extracellular signals

Retinal determination genes are sufficient to specify eyes in ectopic locations, raising the question of how these master regulatory genes define an eye developmental field. Genetic mosaic studies establish that expression of the retinal determination genes eyeless, teashirt, homothorax, eyes absent, sine oculis, and dachshund are each regulated by combinations of Dpp, Hh, N, Wg, and Ras signals in Drosophila. Dpp and Hh control eyeless, teashirt, sine oculis, and dachshund expression, Dpp and Ras control homothorax, and all the signaling pathways affect eyes absent expression. These results suggest that eye-specific development uses retinal determination gene expression to relay positional information to eye target genes, because the distinct, overlapping patterns of retinal determination gene expression reflect the activities of the extracellular signaling pathways.     

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.     

Dpp represses eagle expression at short-range, but can repress its expression at a long-range via EGFR signal repression

Nervous system development takes place after positional information has been established along the dorsal-ventral (D/V) axis. The initial subdivision provided by a gradient of nuclear dorsal protein is maintained by the zygotic genes expressed along the D/V axis. In this study, an investigation was conducted to determine the range of Dpp function in repressing the expression of eagle (eg) that is present in intermediate neuroblasts defective (ind) and muscle specific homeobox (msh) gene domain. eg is expressed in neuroblast (NB) 2-4, 3-3 and 6-4 of the msh domain, and NB7-3 of the ind domain at the embryonic stage 11. In decapentaplegic (dpp) loss-of-function mutant embryos, eg was ectopically expressed in the dorsal region, while in dpp gain-of-function mutants produced by sog or sca-GAL4/UAS-dpp, eg was repressed by Dpp. It is worthy of note that Dpp produced from sim;;dpp embryos showed that Dpp could function at long range. However, Dpp produced from en-GAL4/UAS-dpp or wg-GAL4/UAS-dpp primarily acted at short-range. This result demonstrated that this discrepancy seems to be due to the repression of Dpp to EGFR signaling in sim;;dpp embryos. Taken together, these results suggest that Dpp signaling works at short-range, but can function indirectly at long-range by way of repression of EGFR signaling during embryonic neurogenesis.     

Local inhibition of Drosophila homeobox-containing neural dorsoventral patterning genes by Dpp

An important step in Drosophila neurogenesis is to establish the neural dorsoventral (DV) patterning. Here we describe how dpp loss-of- and gain-of-function mutation affects the homeobox-containing neural DV patterning genes expressed in the ventral neuroectoderm. Ventral nervous system defective (vnd), intermediate neuroblast defective (ind), muscle-specific homeobox (msh), and orthodenticle (otd) genes participate in development of the central nervous system and peripheral nervous system, and encode homeodomain proteins. otd and msh genes were ectopically expressed in dpp loss-of-function mutation, but vnd and ind were not affected. However, when dpp was ectopically expressed in the ventral neuroectoderm by rho-GAL4/UAS-dpp system, it caused the repression of vnd, and msh expressions in ventral and dorsal columns of the neuroectoderm, respectively, but not that of ind. The later expression pattern of otd was also restricted by Dpp. The expression pattern of msh, vnd and otd in dpp loss-of-function and gain-of-function mutation indicates that Dpp activity does not reach to the ventral midline and it works locally to establish the dorsal boundary of the ventral neuroectoderm.     

Dpp and Notch specify the fusion cell fate in the dorsal branches of the Drosophila trachea.

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. We report that Dpp signaling is also required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling is Delta. In conditional Notch loss of function mutants additional tracheal cells adopt the fusion cell fate and ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch(ts1) mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches.     

Wingless eliminates ommatidia from the edge of the developing eye through activation of apoptosis

The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye, which often lack the full complement of photoreceptors and support cells, undergo apoptosis during mid-pupation. We have found that this cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective, grim and reaper, which are required for ommatidial elimination. We estimate that approximately 6-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, we show that the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. We consider the implications of these findings for eye development and function in Drosophila and other organisms.