The Interactive Fly
Genes involved in tissue and organ development
A major part of the adult insect protocerebrum is the optic lobe, consisting of the first, second and third optic ganglion (lamina, medulla and lobula/lobula plate, respectively). In Dipterans, the optic lobe develops from an epithelial placode which invaginates from the procephalic ectoderm, posteriorly adjacent to (and partially overlapping with) the posterior protocerebral neuroblasts. The optic lobe placode (not to be confused with the eye-antennal disc which has an independent origin) invaginates during stages 12 and 13. Its cells constrict apically, leading to the formation of a vertically elongated groove in the center of the optic lobe placode. By the end of stage 12 the optic lobe invagination forms a deep pouch containing about 85 cells. During the invagination process, the placode adopts a V-like shape, with a conspicous anterior and posterior lip. It is thought that the posterior lip gives rise to the outer optic anlage, which in turn produces the lamina and medulla, and that the anterior lip forms the inner optic anlage which mainly produces the lobula/lobula plate complex (Younossi-Hartenstein, 1996 and references).
The optic lobes of the adult brain are derived from neuroblasts organized during the third instar larvae into two columnar epithelia: the inner proliferation center (ipc) and the outer proliferation center (opc).
First instar: A total of 30 to 40 precursor cells of the optic anlagen are found superficially in the lateral cell body layer of each brain hemisphere at the time of hatching (the transition from embryonic development to the first larval stage). These cells differ from the remaining cells of the hemisphere due to their somewhat smaller size. Within the early first instar, labelled nuclei appear in these smaller cells. The cells become larger, ellipsoid and epithelially arranged. From the second half of the first instar onwards two different epithelia can be distinguished; these develop into the opc and ipc.
Second instar: No obvious pattern of labeling is detectable until the end of the second instar, when the production of postmitotic neurons begins. At the end of the second instar, there are about 700 neuroblasts in the opc and about 400 neuroblasts in the ipc. Subsequently, a proliferation zone is formed at the medial edge of the opc. Mitotic figures and some small ganglion mother cells can be distinguished adjacent to the neuroblasts. The number of cells produced by this proliferation zone amounts to approximately 40,000, consisting of the medulla, the outermost cell layer of the optic lobe (Hofbauer, 1991).
Third instar: During the middle of the third instar, a second proliferation zone (still part of the opc) develops at the lateral rim of the opc. This zone consists of neuroblasts that have become separated from the main anlage by a deep furrow. Ganglion mother cells and ganglion cells are produced at the inner edge of this crescent. However, this zone yields many fewer cells than found in the medulla anlagen (about 6000 at 25 hours after puparium formation). These cells form the lamina (Hofbauer, 1991). Since mitotically active lamina precursor cells, which normally produce lamina neurons, are absent in mutants that lack retinal innervation, it is concluded that the arrival of photoreceptor axons in the larval brain initiates cell division to produce lamina neurons (Selleck, 1991).
Two different populations of ganglion cells originate from the lateral proliferation zone of the ipc. Most of the cells differentiate to become the cell body layer of the lobula plate. The other cells participate in the formation of the inner medulla neuropil. About 15000 cells are generated from the lateral proliferation zone. An additional small dorsal proliferation zone develops from the ipc and these cells become part of the lobula cell body layer (Hofbauer, 1991).
Ganglion cells begin to grow axons shortly after their final mitosis: corresponding to the gradient of cell proliferation, there is also a gradient of differentiation in each developing neuropil. The axons of the lamina cells grow centrally, forming a fine fiber sheet at the inner margin of the lamina cell body area at right angles to the medulla neuropil. When differentiation of the lamina starts during the middle of the third instar, the youngest part of the lamina neuropil is in contact with the youngest, most lateral part of the medulla neuropil. The cells of the lobula complex send their axons centrally and form a neuropil opposite and almost parallel to the medulla neuropil. Lobula and lobula plate will develop from this fiber mass. The first fibers connecting the different visual neuropils appear very early, at a time when only the minor part of the ganglion cells are postmitotic. Photoreceptor cell axons start growing into the brain during the middle of the third instar, beginning at the posterior edge of the developing eye and progressing towards the anterior. At the same time, the proliferation of lamina ganglion cell progenitors begins and the newly generated lamina cells become connected with newly ingrowing photoreceptor cells (Hofbauer, 1991).
The lamina is a ganglion layer of the visual center of the brain that processes information received from R1-R6 photoreceptor neurons located in the ommatidia of compound eyes. The R1-R6 photoreceptors send their axons to the lamina, where they are distributed in a "neurocrystalline" array of postsynaptic "cartridge" units. Each cartidge unit contains a set of R1-R6 axons and five lamina neurons and is associated with glia of four defined types.
In eyeless mutants, the lamina is completely absent, and the other visual ganglia are reduced in size. The dependence of lamina development on innervation from the compound eye is due to direct coupling of neurogenesis to the arrival of retinal axons from the eye. As photoreceptor cell differentiation and ommatidial assembly sweep across the eye disc epithelium, the arrival of axons from each new ommatidial photoreceptor cell cluster triggers a corresponding group of lamina precursor cells (LPC) to undergo a final cell division and commence differentiation. In animals lacking retinal innervation, LPCs fail to undergo their terminal division and remain in G1 arrest until eliminated by cell death. Direct contact with retinal axons triggers the transition of G1-phase LPCs into S Phase.
Morphogenesis in the lamina is structured in a linear fashion, taking place sequentially as axonic innervation arrives in the brain. Morphogenesis takes place along a furrow that moves across the developing lamina, in a manner directly related to the progression of the morphogenetic furrow of the eye optic disc. Arrival of axons trigger LPCs to divide at the posterior margin of the lamina furrow, initiating the events that give rise to lamina neurons. In the lamina, the LPC progeny are organized into columns in precise register with the axons that regulate their differentiation. LPC progeny and glia of several types assume stereotyped positions in the lateral medial axis. The glia include medulla, marginal, epithelial and stellate types.
The secreted protein Hedgehog has been identified as the signal transmitted along retinal axons which serves as the inductive signal triggering neurogenesis in the lamina. patched is expressed in two classes of lamina glia and in subretinal glia cells in the optic stalk and the eye disc, cell populations that are in close proximity to retinal axons. Ectopic hh expression in the brains of eyeless flies induces lamina differentiation, but is not sufficient to induce Elav, a late marker. This suggests that HH alone is not sufficient for the later events of lamina development that include the specification of LPC progeny as lamina neurons (Huang, 1996).
The target of HH in the developing visual system is wingless, which in turn targets decapentaplegic and Distal-less. The lamina neurons and the cortical neurons that contribute axons to the medulla neuropil derive from a neuroblast population (OPC or outer proliferation center) that divides throughout most of larval development. Although cells expressing wg constitute only a small fraction of the OPC, the inactivation of wg at early times results in the later absence of nearly the entire target structure. Inactivation of wg at later times results in the formation of pregressively more complete central target structures. The expression of wg in the developing visual system is consistent with a role in organizing the dorsoventral axis. wg expression begins in a single ventral cell at approximately 10 hr of larval develpment. About 6 hr later, wg expression begins in a single dorsal cell. For the remaining stages of larval development wg is expressed in two OPC domains that define the dorsal and ventral termini of the developing target regions. These observations suggest that wg has a nonautonomous role in organizing the target region of retinal axons. The fact that early wg expression occurs prior to axon ingrowth, indicates that HH is required for the maintenance of wg expression and not for its initiation (Kaphingst, 1994).
Wingless regulates the onset and maintenance of dpp expression. Approximately 14 hr after the onset of wg expression, dpp expression begins in single cell domains immediately adjacent to the wg-expressing cells, and is maintained throughout larval development as these cell populations divide up to and including the period of retinal axon ingrowth. In dpp mutants many OPC progeny fail to down-regulate the expression of the cell adhesion molecule fasII, fail to express neuron markers, and fail to contribute axons to the medulla neuropil (Kaphingst, 1994).
Distal-less expression is found in wg-expressing cells adjacent to the dpp domains. dll expression is significantly greater in the dorsal domain. The involvement of dll in neurogenesis in Drosophila has yet to be documented (Kaphingst, 1994).
Minibrain is a serine/threonine kinase involved in proliferation of cells of the opc. When the development of optic lobes in minibrain mutants is compared with that of wild type, it is found that the opcsin mutants attain an irregular structure. The opcs are distinguishable in third instar larvae, appearing in tightly packed, ribbon-like layers of cells, demarcated from the hemisphere. The proliferating neuroblasts appear as a very regular dome-like pattern in wild-type brain. One thick and two thin ribbons of bromodeoxuridine (BrdU) labeled cells (indicating cells that have gone through the DNA synthetic S phase) can be distinguished in the distal brain hemispheres. This regular spatial distribution of BrdU labeled neuroblasts is disturbed in the opcs of mnb mutants. In strong alleles, this ribbon of opc neuroblasts is condensed, and the BrdU-labelled nuclei are not distributed evenly. The regular structure of the thin ribbons disappears, resulting in a scattered distribution of labeled nuclei in these areas. It is important to note that in most cases the altered opc structure does not apparently change the number of labeled cells in comparison with wild type. Nevertheless, abnormally large cells with dark nuclei (probably degenerating cells) are frequently seen near to neuroblast-like cells. In comparison with wild type, mutants are missing a clear demarcation between neuroblasts and gangion cell bodies. In contrast to mutant opcs, no detectable structural alteration is seen in mutant ipcs. From observations of pupal brains, it is concluded that the minibrain phenotype is primarily caused by the inability of mutants to generate a sufficient number of optic lobe and central brain neurons during postembryonic development (Tejedor, 1995).
Cell proliferation within the optic lobe anlagen is dependent on ecdysteroids during metamorphosis of the moth Manduca sexta. Cultured tissues were used to show that ecdysteroids must be maintained above a sharp threshold concentration to sustain proliferation. Proliferation can be turned on and off repeatedly simply by shifting the ecdysteroid concentration to levels above or below this threshold. In subthreshold hormone, cells arrest in the G2 phase of the cell cycle. Ecdysteroid control of proliferation is distinguished from differentiative and maturational responses to ecdysteroids by requiring tonic exposure to the hormone and lower levels of 20-hydroxyecdysone, and by being sensitive to either 20-hydroxyecdysone or its precursor, ecdysone. These characteristics allow optic lobe development to be divided into two ecdysteroid-dependent phases. Initially, moderate levels of ecdysteroid stimulate proliferation. Later, high levels of 20-hydroxyecdysone trigger a wave of apoptosis within the anlage that marks completion of its proliferative phase (Champlin, 1998).
The optic lobe of Manduca sexta, like that of Drosophila, is composed of three ganglia, the lamina, medulla and lobula. The neurons of the optic ganglia are progeny of neuroblasts in the optic lobe anlage (OA). In Manduca, the neuroblasts are arranged in a double-banded bracelet that comprises the inner and outer OA. During early larval stages, expansion of the neuroblast population occurs by symmetric cell divisions. In the final larval instar, the neuroblasts switch to the asymmetric divisions that lead to neuron production. The smaller daughter of each asymmetric division, the ganglion mother cell (GMC), divides to produce neurons. GMCs of the inner OA produce the neurons of the lobula. The medial margin of the outer OA is composed of the neuroblasts and GMCs that produce the neurons of the medulla (medulla precursor cells, MPC), while the lateral margin is composed of the neuroblasts and GMCs that produce the neurons of the lamina (lamina precursor cells, LPC) (Champlin, 1998).
Proliferation of the LPC has been shown to be regulated by incoming retinal afferents. Transection of the optic nerve in Manduca also leads to disruption of LPC proliferation. Communication via retinal afferents provides a coordinating link between the developing retina and production of lamina neurons. Ecdysteroids, by contrast, control production of neurons throughout the optic lobe. The precursors for the medullar and lobular neurons both require ecdysteroids for proliferation. The optic nerve is severed in cultures making it difficult to determine if the same is true for the LPCs. Despite these conditions, , arrested LPCs still enter mitosis in response to 20E. It appears that neural precursors throughout the optic lobe have a similar ecdysone-dependent G2 checkpoint. The failure of LPCs in cultured brains to incorporate BUdR suggests that 20E allows the cells to divide but they then arrest in G1 in the absence of innervation. This interpretation is consistent with data from Drosophila that LPCs arrest in G1 in mutants lacking proper retinal innervation. Thus, the cell cycle of LPCs in Manduca may have two developmentally regulated control points, stimulation by retinal afferents being required for progression through G1 and stimulation by ecdysteroids being required for progression through G2 (Champlin, 1998).
MPCs are found to proliferate in an ecdysteroid-dependent manner, even in small pieces of the outer OA, clearly indicating that other long-range signals are not required for these cells. Cells throughout the OA, including the MPCs, express the ecdysteroid receptor; therefore, these cells could be responding directly to ecdysteroids. An important issue is whether the ecdysteroid-dependent proliferation of the MPC is a direct response to the steroid or due to short-range signals from neighboring cells. Evidence for communication between surrounding glia and neuroblasts is seen for the protein product of the anachronism locus, which is secreted by nearby glial cells and inhibits proliferation of optic lobe neuroblasts in Drosophila. The fact that the MPCs appear to respond to ecdysteroid as a unit suggests that there may be coordinating signals within the OA of Manduca (Champlin, 1998).
Champlin, D. T. and Truman, J. W. (1998). Ecdysteroid control of cell proliferation during optic lobe neurogenesis in the moth Manduca sexta. Development 125(2): 269 - 277
Hofbauer, A. and Campos-Ortega, J. A. (1990). Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster. Roux's Arch. Dev. Biol. 198: 264-274
Huang, Z. and Kunes, S. (1996). Hedgehog, transmitted along retinal axons, triggers neurogenesis in the developing visual centers of the Drosophila brain. Cell 86: 411-422
Kaphingst, K. and Kunes, S. (1994). Pattern formation in the visual centers of the Drosophila brain: wingless acts via decapentplegic to specify the dorsoventral axis. Cell 78: 437-448
Selleck, S. B. and Steller, H. (1991). The influence of retinal innervation on neurogenesis in the first optic ganglion of Drosophila. Neuron 6: 83-99
Tejedor, F., et al. (1995). minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila. Neuron 14: 287-301
Separate sections of The Interactive Fly group genes according to their involvement in glia morphogenesis and axonogenesis.
genes expressed in brain morphogenesis
Genes involved in organ development
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