Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated either by a single motorneuron or a select few. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. Connectin staining is first seen in one to three myoblasts on the lateral side of the body wall at late stage 11 to early stage 12. At late stage 12, small numbers of myoblasts (7-10 cells) in the ventral-lateral region of the body wall express Connectin. Some of these cells reach the size of doublet or triplet myoblasts, suggesting that cell fusion has already begun. By early stage 13, Connectin is expressed in two groups of fused cells (one in the ventral and the other in the lateral region of the body wall) that can be identified as the pioneers of individual muscle fibers. The ventral group is comprised of two muscle pioneers, which form the external oblique muscles 27 and 29. The lateral group is composed of the six muscle pioneers, which arise together and form the pleural external muscles 21-24; muscle 18, which migrates from a more dorsal position, and muscle 5 (Nose, 1992).

Connectin is expressed over the entire surface of these muscle pioneers and on their numerous filopodial extensions. No Connectin expression is seen in the majority of unfused myoblasts, however some of the myoblasts that immediately surround and are about to fuse with Connectin-positive pioneers do themselves begin to express Connectin prior to fusion. At stage 16, Connectin is expressed over the entire surface of those differentiated muscle fibers whose pioneers were Connectin positive (Nose, 1992).

At late stage 12 to early stage 13 Connectin is expressed on axons and growth cones of a subset of motoneurons exiting the CNS via both the intersegmental nerve (ISN), which innervates dorsal muscles and the segmental nerve (SN), which innervates lateral and ventral muscles. Connectin is also expressed on two identified peripheral glial cells: PG1 and PG3, and another glial-like cell, PG4. These glia may serve as guidepost cells for the motoneuron growth cones that express Connectin. At this stage PG1 sits lateral to the CNS and anterior to ventral muscle pioneers 27 and 29; this glial cell later elongates dorsoventrally to enwrap the SN. PG3 sits at the dorsal region of the body wall anterior to muscle precursor 18 and sends processes toward the CNS along the ISN; later in development, it enwraps the axons in the ISN. PG4 sits between the two ventral muscle pioneers 27 and 29, later elongates rostrocaudally, and appears at the light level to serve as a substrate for the segmental nerve c (SNc) (Nose, 1992).

During late stage 13 to stage 15 Connectin expression is observed (1) on a specific subset of motoneuron axons and growth cones in the segmental nerve a (SNa) that grow dorsally along PG1 and contacts the ventral tip of lateral muscles 21-24 that express Connectin; (2) on a subset of motoneuron axons and growth cones in the SNc that contacts Connectin-expressing glia, and (3) in several of the ventral unpaired median neurons (VUMs) in the ISN. Other specific nerves and muscles express Connectin at stage 16. By stage 17, overall expression of Connectin declines (Nose, 1992).

Connectin is transiently expressed at high levels on many longitudinal glia and on some midline glia. At stage 16, when the entire axon scaffold is formed, Connectin is expressed on many axons, many of which bundle together in specific longitudinal axon fascicles. Expression of Connectin is also observed on subsets of visceral mesoderm and on at least one nerve that innervates the gut. There is expression in the presumptive foregut and hindgut mesoderm and in a segmentally repeated subset of the presumptive midgut visceral mesoderm. It is also expressed on two to three cells in the dorsal cluster of the peripheral nervous system. There is strong labelling in the anterior central nervous system, including brain lobes and the thoracic neuromeres. Labelling is also seen associated with the gnathal sense organs from stage 11 onwards (Nose, 1992 and Gould, 1992).

During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, Connectin is a novel cell adhesion molecule whose expression suggests a role in target recognition (Nose, 1992).

In the central nervous system, Connectin is initially expressed on longitudinal glia and on a few identified neurons. These cells extend processes and connect up to form a continuous scaffold of connectin-expressing cells, presaging the development of axonal pathways. Beginning at about stage 6 connectin is expressed in a small mumber of cells in the CNS. These connectin-positive cells subsequently organize to form a continuous scaffold of connectin expression, involving both glia and neurons, in a pattern that prefigures the axonal ladder. Labelled in each segment are 4-5 longitudinal glia (LG) cells and two sets of identified neurons, a pair of VUM cells and a pair of neurons in the position of RP cells. By 8 hours the connectin-positive cells have connected together to form a continuous ladder-like array. This process involves the elongated LG cells making contacts with RP neurons in the same segment and also in the next most posterior segment. The rings of the ladder are formed by the VUM cells and their contacts with both RP neurons and the LG cells. Later, connectin is expressed on specific axons as they track along the Connectin scaffold. Glial expression then declines and Connectin appears on axons that fasciculate with pre-existing Connectin-positive bundles. Thus scaffold formation, axon pathfinding and fasciculation involve specific contacts between Connectin-positive cells. The timing and pattern of connectin expression suggest that connectin may play an important role in mediating specific interactions through homotypic cell adhesion (Meadows, 1994).

The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). The VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. Mesodemal cells located just under the engrailed-expressing cells in the posterior ectodermal compartment have been called the mesodermal "P domain." The dorsal-most cells of the mesodermal P domain in each PS express the homeobox gene bagpipe (bap); they detach from the mesodermal fold and move inward toward the center of the embryo. These bap-expressing cells form the VM progenitor groups. The VM cells initiate expression of Fasciclin III (FasIII) as they migrate to join each other and form a continuous band of VM running along each side of the embryo. Thus all the VM derive from the posterior parts of the initial mesoderm metameres. As VM progenitors merge to form a continuous band running anterior to posterior along the embryo, expression of connectin (con) occurs in 11 metameric patches within the VM, revealing VM subdivisions analogous to ectodermal compartments (Bilder, 1998).

The VM subdivisions, and the metameric expression of con, form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog (hh) and wingless (wg) and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Con in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific enhancers (Bilder, 1998).

Since Con expression marks the imprint of ectodermal PS boundaries on the VM, Con patches can be used to precisely map the domains of Hox gene transcription in relation to Con patches. teashirt is expressed in two domains. The anterior midgut domain extends from visceral mesoderm segment (VS) 4 to mid-VS 6, where it shares a posterior boundary with Antennapedia; the central midgut domain extends several cells to either side of the VS 8 boundary. dpp is also expressed in two domains: at the gastric caeca, it is found in the A domain of VS2 and the P domain of VS 3, while in the central midgut it extends from the A domain of VS 6 to terminate just anterior to the VS 8 boundary. wg is expressed just anterior to the VS 8 boundary, with some cells after stage 12 lying in VS 8. pnt is expressed throughout VS 8, although expression is not seen until early stage 13. At stage 13, the two domains of odd paired (opa) expression extend from the P domain of VS 4 to the VS 6 boundary and from VS 9 through VS 11 (Bilder, 1998).

Several Hox targets appear to respect the PS subdivision organization of the VM. The initial VM expression of opa is seen only adjacent to Con patches, in A domains of VS 3-5 and 8-11. Similarly, wg is limited to a subset of abdA-expressing cells: those at the border of VS 8. wg is activated by abdA and dpp. Ectopic expression of abdA leads to induction of wg in a single posterior patch. Strikingly, the sites of ectopic wg induction in both genotypes align with the VS boundaries: in cells just anterior to VS 3, 5, and 6 in ectopic AbdA embryos and anterior to VS 9 in ectopic Dpp embryos. these results suggest that metameric subdivisions in the VM limit Hox gene activation of VM targets (such as wg) to restricted areas. It is suggested that divergent Hox expression in the VM has its basis in tissue-specific regulation of Hox expression in the VM and this expression is governed by unknown regulators that control VM-specific Hox enhancers (Bilder, 1998).

connectin mutants do not show dramatic neuromuscular defects, and ectopic expression studies so far have not supported an adhesion role. connectin mutants do, however, have a readily identifiable phenotype: the normally connectin-positive pleural muscles fail to adhere closely together. Other connectin positive-muscles, 27 and 29, do not show any alteration in morphology, but these muscles, though relatively close, do not actually contact each other in the wild type. An in vivo adhesion role is supported by misexpression studies, which result in excessive adhesion of normally connectin-negative muscles. Misexpression also causes defects in axon pathfinding. While a previous study interpreted similar defects as indicating a repulsion role for connectin, it is argued that the phenotypes are consistent with connectin's adhesion role (Raghavan, 1997).

beaten path (beat) is a gene required for the selective defasciculation of motor axons at axonal pathfinding choice points. In beat mutant embryos, motor axons fail to defasciculate and bypass their targets. This phenotype is suppressed by mutations in FasII and Con, two genes encoding cell adhesion molecules expressed on motor axons, suggesting that beat provides an antiadhesive function. beat encodes a novel secreted protein that is expressed by motoneurons during outgrowth (Fambrough, 1996).

Connectin is ectopically expressed on ventral muscles normally innervated by SNb motoneurons. The SNb growth cones change both their morphology and their trajectory when they encounter ectopic Connectin-positive ventral muscles, displaying "bypass," "detour," and "stall" phenotypes. Moreover, SNb synapse formation is prevented by Connectin expression on ventral muscles. These results reveal a repulsive function for Connectin during motoneuron growth cone guidance and synapse formation (Nose, 1994).

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. By manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, further evidence is provided that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons (Yu, 2000).

The failure of axonal defasciculation observed in Sema1a mutant embryos is likely due to the lack of Sema-1a-mediated repulsion among motor axons along efferent trajectories. Reducing adhesion by removal of attractive cues, the CAMs Fas II and Conn, rescues characteristic hyperfasciculation defects (both ISNb and SNa phenotypes) of Sema1a mutants. In contrast, increasing adhesion by overexpression of the CAM Fas II enhances the hyperfasciculation defects in the ISNb and SNa pathways in Sema1a mutant embryos. In addition, reduction in the level of Fas II will also suppress CNS fasciculation defects in Sema1a mutant embryos. These experiments complement those previous studies showing that FasII loss of function can suppress defasciculation defects observed in PlexA mutants. Further, they show that mutations in genes encoding different classes of CAMs, including both Ig superfamily members and LRR-containing proteins, genetically interact with Sema1a mutants, suggesting that CAM-specific signaling events are not involved in this interaction. Taken together, these results demonstrate that Sema-1a regulates axonal fasciculation at specific choice points by countering the attractive functions of at least two CAMs, Fas II and Conn (Yu, 2000).

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date revised:  21 November 2016 

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