Connectin

Promoter Structure

A 4 kb restriction enzyme fragment of Connectin, encompassing the 100 base pair clone of the Connectin promoter immunopurified with anti Ultrabithorax antibody, gives a consistent pattern of expression corresponding to a subset of the total Connectin expression pattern. High levels of expression are detected in a small group of cells in the gnathal and thoracic segments and in a posterior segment. This fragment does not produce CNS expression. The reduced levels of expression of the 4 kb fragment suggest a down regulation by Ubx and the abdominal homeotic genes. In Ubx mutants, expression is derepressed in the abdominal segments A1 and A2. Derepression is more dramatic in a Ubx/abd-A double mutant indicating that both genes repress the 4 kb construct. Antp is required for the high levels of expression found in T2 and T3 of wild type embryos. There is an additional pattern of expression in the visceral mesoderm of the midgut, with a smaller 1 kb construct that is not observed with the 4 kb construct. The visceral mesoderm expression corresponds to the developing second midgut constriction and corresponds to a subset of parasegment 7. This is particularly interesting as Ubx is expressed in PS7 in the visceral mesoderm and Ubx function is required for the formation of the second midgut constriction (Gould, 1992).

Transcriptional Regulation

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two effector genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. It is argued that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity. In wild-type embryos, all neurons that express engrailed also express invected. The converse is not true, however. Neurons, which lie anterior to the predominant Engrailed/Invected stripe in the CNS, express invected but not engrailed. This is the best example to date of differing expression of engrailed and invected in an identified cell type. Connectin is also expressed in SNa and SNc motor neurons, which are Engrailed negative. When Engrailed is expressed in all neurons, Connectin is not downregulated but slightly upregulated in these motor neurons, in contrast to the effect on interneurons. Since Engrailed can act either as a repressor or as an activator, it is possible that ectopic Engrailed directly activates Connectin in the motor neurons (Siegler, 1999).

The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis. In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).

In Drosophila, trunk visceral mesoderm (VM), a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. These findings have been extended by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided in the A/P axis into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts a feedback effect on trachea and ectodermal derivatives (Hosono, 2003).

VM is presently considered to develop in two steps under the control of ectodermal Hh and Wg signals. First, by stage 10 (when four mesodermal primordia have become specified), VM competent or bap expression regions are promoted by hh but repressed by wg, via a direct targetor, slp. The second surge of hh and wg activity at stages 10-11 is responsible for subdividing VM-PSs into two regions: con positive and negative. These results indicate that the expression of four other VM-metameric genes, hh, tin, bnl and bap, is also regulated by the second surge of hh and wg activity at stages 10-11 (Hosono, 2003).

To examine the regulation of VM-metameric genes with changing the activity of hh and/or wg, it may be necessary to evaluate the effects of possible change in cell number on VM-PS subdivision or VM-PS cell specification. In temperature-sensitive mutants of hh and wg shifted-up from stage 10, the number of VM cells positive to FAS3 at mid stage 11 on is essentially identical to that of wild type, indicating that VM-cell number change is negligible under the conditions used, while the expression of some VM metameric genes appear compromised. In hh^ts mutants, VM-hh and con are not expressed, though tin, bnl and bap are expressed. In wg^ts mutants, VM-hh and tin are not expressed, but con is expressed. All these observations are totally in agreement with those found in simple loss-of-function and overexpression experiments; under these conditions, the formation of a VM competent region should be hindered. Thus, these results may indicate that ectodermal Hh and Wg regulate directly, but in different ways, the expression of metameric genes in VM; VM-hh expression requires both Hh and Wg. tin, bnl and bap are positively regulated by Wg alone, and con is activated by Hh and repressed by Wg (Hosono, 2003).

In view of morphological changes in a VM competent region and consideration of these findings on VM gene regulation, the following model for VM-PS cell specification is proposed. At stage 10 to early stage 11, anterior terminal cells of VM-PSs are presumed to be situated near an ectodermal AP border, where they are capable of continuously receiving Wg and Hh signals, and Wg confers competence on these cells to express tin/bnl/bap. Wg and Hh are responsible for inducing VM-hh, and Hh, for con expression. In the anterior-most cells, con expression is reduced, which would be expected in view of repression by high Wg signal. The different thresholds of hh for con and VM-hh expression may explain why the con area expands more posteriorly compared with that of VM-hh. Posterior terminal VM cells, when formed, are situated far from Wg expressed on the ectodermal PS border. But as they migrate posteriorly and close to the posteriorly neighboring AP border by early stage 11, they become capable of receiving Wg and acquire competence to express tin/bnl/bap. Thus, the tin/bnl/bap domain would appear regulated by spatially and temporally distinct Wg signals. The two-step induction of tin/bnl/bap expression is supported by experiments using the wg^ts mutant, where, either posterior or anterior expression within one patch can be differentially turned off. Indeed, a stepwise activation of tin/bnl expression is seen in VM-PSs around stage 11. tin and bnl metameric expression became apparent almost simultaneously at mid-stage 11, and preliminary experiments have shown that neither tin nor bnl misexpression can induce the ectopic expression of any other metameric genes examined here. Thus, tin and bnl expression might be initiated in a mutually independent manner (Hosono, 2003).

This VM-PS subdivision model should be modified when applied to thoracic segments, where hh may not be the sole determinant of con expression (Hosono, 2003).

This study strongly suggests that metameric VM-hh is required for the maintenance of its own as well as metameric con expression, although the latter becomes independent of VM-hh at late stages. That Ptc, a direct target of hh, is upregulated in each VM-hh expression domain at stage 12, at that time VM is far away from the epidermis or ectodermal Hh sources, is additional evidence supporting the notion that hh signaling caused by metameric VM-hh is operative in VM (Hosono, 2003).

Protein Interactions

Connectin can function as a homophilic cell adhesion molecule. Cultured cells transduced with Connectin cDNA form large aggregates consisting of hundreds of thousands of cells. When mixed with non-expressing cells, only the expressing cells aggregate (Nose, 1992)

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