BIOLOGICAL OVERVIEW

Pair-rule and segment polarity genes are responsible for determining the uniformity of different segments, in contrast to homeodomain proteins that are responsible for establishing the diversity between segments. Abdominal-B acts in three germ cell layers to fulfill this latter function.

Abdominal-B is the last in linkage order and the most posterior acting of the linked homeodomain proteins of the bithorax. Abdominal-B is unique among the homeotics in that it is transcribed in two forms; a regulatory (r) protein and a morphogenic (m) protein. Regulatory transcripts of Abdominal-B act as repressors, suppressing embryonic ventral epidermal structures in the 8th and 9th segments of the abdomen. Thus ABD-B r and m proteins are critically involved in establishing cell fate in the tail segments of the fly.

Expression is driven by two promoters. The m form is transcribed in parasegments 10-13, corresponding to adult abdominal segments 5-8, while the regulatory protein is transcribed in parasegment 14, corresponding to adult abdominal segment 9. This division of labor does not appear until stage 13, relatively late in development. Earlier, in stage 10, both forms are transcribed in epidermis. In stage 11, both forms are found in epidermis and mesoderm. By stage 12 central nervous system (ventral cord) expression is evident for both forms. In stage 13 m expression becomes restricted to segments 11-13 for all tissues, while r expression becomes restricted to segment 14 for all tissues (De Lorenzi, 1990b). The r protein's designation as regulatory stems from its unique role in segment 14. There it suppresses myogenesis. It is believed that it also represses transcription of the m form.

The distinction between r and m functions was based on the discovery of three classes of regulatory mutations affecting Abdominal-B (Casanova, 1986). One class affects expression in five parasegments (10-14), a second affects expression in only four (parasegments 10-13) and a third class affects expression in just parasegment 14. The regulatory transcript of ABD-B is thought to suppress the proximal morphogenetic (m) function (Casanova, 1986). The smaller r protein differs from its m counterpart in its lack of an M repeat region. This is a particular amino acid segment that lies upstream of the homeobox. The M repeat is rich in glutamines, a classical transcription activation motif (De Lorenzi, 1988).

Genetic evidence shows that lines, a Drosophila segment polarity gene that has yet to be cloned, is required for the function of the Abdominal-B protein. In lines mutant embryos Abdominal-B protein expression is normal but is incapable of promoting its normal function: formation of the posterior spiracles and specification of an eighth abdominal denticle belt. The tail and A8 segment of lin embryos are highly abnormal. The A8 denticle belt is replaced by naked cuticle that occasionally forms a few denticles less pigmented than the normal ventral denticles. This abnormal A8 cuticle does not resemble the cuticle of any region of the wild-type or of the lin mutant embryo. The absence of anal pads and the abnormal hindgut suggests abnormal development of abdominal segment 11, however, other aspects of the tail development are normal, such as the formation of an anal tuft. In lin embryos the sensory organs are formed at roughly correct positions but have an abnormal shape (Castelli-Gair, 1998).

The Abd-B gene directs the formation of the posterior spiracles by controlling downstream target genes. The defects associated with lines mutation arise because in lines mutant embryos the Abdominal-B protein cannot activate its direct target empty spiracles (ems) or other downstream genes, such as cut(ct) and spalt(sal), while it can still function as a repressor of Ultrabithorax and abdominal-A. empty spiracles is one gene required for the formation of posterior spiracles. ems expression in the posterior spiracles is regulated by Abd-B. In lin embryos the transcription of ems is not activated in the posterior spiracles, showing that lin is required for Abd-B to activate its direct downstream target. The other putative Abd-B downstream targets cut and spalt are also required for the normal development of the posterior spiracles. The activation of ct and sal in the anlage of the posterior spiracles requires Abd-B function but their activation remains independent of one another and of ems, suggesting that all three genes are independently controlled by Abd-B. In lin mutants neither ct nor sal are activated in the anlage of the posterior spiracles. These results show that in lin mutant embryos, Abd-B is incapable of activating some of its targets. The requirement of lines for Abd-B function is not a specific property of the A8 segment. In wild-type embryos, ectopic Abd-B expression using the GAL4 targeting system results in the formation of ectopic posterior spiracles in segments anterior to A8. In contrast, ectopic Abd-B expression in lin mutants does not form ectopic posterior spiracles showing that no matter where the Abd-B protein is expressed in the embryo it requires lines to be fully functional (Castelli-Gair, 1998).

The effect of lin on Abd-B can be explained at the molecular level if lin is required for protein posttranscriptional modification or as a transcriptional cofactor of Abd-B. There is some evidence that the Abd-B protein is posttranslationally modified. If Lin were mediating this process, it would imply that such posttranscriptional modification is functional in vivo. Alternatively if Lines is a transcriptional cofactor of Abd-B, Lines would be interacting with Abd-B in a similar way to that proposed for Extradenticle with Ubx and Abd-A, or Ftz-F1 with Ftz. It is interesting that Exd does not have any effect on Abd-B protein binding or function, and that lin is specific for Abd-B but not for the other Hox genes tested. This suggests that different HOX proteins use different cofactors that contribute to the DNA binding specificity of the HOX proteins (Castelli-Gair, 1998).

Abdominal-B is required to specify the posterior abdomen and the genitalia. Homologs of Abdominal-B in other species are also needed to determine the posterior part of the body. The function of Abdominal-B in the formation of Drosophila genitalia has been studied, and the absence of Abdominal-B in the genital disc of Drosophila has been shown to transform male and female genitalia into leg or, less frequently, into antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less expression depends on the antagonistic activities of the Abdominal-B gene (as a repressor), and of the decapentaplegic and wingless genes (as activators). Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. These results suggest that Abdominal-B forms genitalia by modifying an underlying positional information and repressing appendage development. It is proposed that the genital primordia should be subdivided into two regions, one of them competent to be transformed into an appendage in the absence of Abdominal-B (Estrada, 2001).

Abd-B clones were induced, and they transform posterior abdominal segments into more anterior ones but are normal in the analia. Rare clones transform to distal antennae (second and/or third antennal segment and arista). Transformations to legs or antennae are cell autonomous. The formation of legs requires the activity of genes such as homothorax (hth), dac and Dll, which specify the proximal, medial and distal parts of the leg, respectively. Dll expression in wild-type discs is regulated by the combined activities of wingless and dpp in the genital primordia, and is confined to two groups of cells in male and female discs, the female domains being smaller and expressing lower levels of Dll protein. Since Abd-B is transcribed in the entire genital primordia of the two sexes, some cells co-express Abd-B and Dll. In the male disc, hth is not expressed in the Dll-expressing cells and is also excluded from a large group of cells surrounding them. Levels of antibody signal vary within the disc, and are higher in the female repressed primordium. In females, the hth domain of expression occupies the whole primordium. Lower levels of Hth are detected in a region encompassing the Dll-expressing cells, whereas higher levels are observed in the male repressed primordium. In both sexes, hth expression is absent from the anal primordium. dac is expressed differently in male and female genital primordia: in male discs, Dac protein is detected in two broad lateral bands, while in female discs it is found in the central region, almost coincident with the wg-expressing region. Therefore, the expression patterns of hth, dac and Dll differ substantially from those observed in legs (Estrada, 2001).

It is known that expression of Dll is not required to make male genitalia and that it has only a minor role in the formation of the female one. To ascertain the role of hth in the genitalia, hth minus clones were induced during the third larval period and they were examined in the adult structures. In the female genitalia, hth minus clones cause extra growths with additional vaginal teeth. In males, these clones show occasionally some abnormalities in the clasper teeth. hth clones in the analia are wild type. Possible interactions between Dll and hth in the genital disc were sought. In these experiments, unless stated, the results apply both to male and female genital primordia. Dll minus clones in the Dll domain of the male disc have no hth expression. Similarly, in hth minus clones Dll is not ectopically expressed. Dll was also expressed ectopically and the effect on hth expression was examined. Dll-expressing cells close to the wild-type Dll domain repress hth expression, although not all the cells do so. By contrast, clones far from the Dll domain do not affect hth expression (Estrada, 2001).

To characterize the transformation of genitalia into leg or antennal tissues, Abd-B minus clones were examined. Abd-B minus clones in the genital primordia tend to segregate from the rest of the tissue, round up and have smooth borders, suggesting they have acquired different affinities. By contrast, clones in the analia have indented borders and do not segregate. Abd-B minus clones in the genital primordium close to the normal Dll domain show ectopic, cell-autonomous Dll expression, whereas those far apart do not show such expression. dac is also activated cell autonomously in many Abd-B minus clones. As expected, Dll target genes, such as Bar, also become activated in these clones (Estrada, 2001).

Abd-B minus clones exhibit differential effects on hth, depending on their position: those close to the Dll domain show no hth expression, whereas those located away from the Dll domain show a slight increase in hth signal. Clones in intermediate positions do not significantly change hth levels. This distribution, however, is clearer in females, since in males there is a wide region with no hth expression. The repression of hth observed in some Abd-B minus clones may be mediated by the ectopic Dll (Estrada, 2001).

In the genital disc, the transcription of Dll depends, as in the leg disc, on dpp and wg signals. Abd-B represses Dll expression. Moreover, increasing Abd-B levels in the Dll domain suppresses Dll transcription. The antagonistic activities of dpp/wg and Abd-B in determining the Dll distribution was analyzed. Mutations in PKA ectopically activate wg and dpp expression. PKA minus clones in the genital primordia activate Dll, although only in some places. This activation is not mediated by changes in Abd-B levels. Similarly, although Dll is derepressed in many late Abd-B minus clones, derepression of either dpp or wg was not observed. It is concluded that there is an antagonism between the activation of Dll by dpp/wg signaling and its repression by Abd-B. This is not mediated by changes in the expression of either dpp, wg or Abd-B (Estrada, 2001).

To characterize this antagonism further, Abd-B minus clones that were made were also unable to transduce the dpp signal. This signal requires the presence of the type II receptor encoded by the gene punt. In put;Abd-B double mutant clones, Dll is not activated, indicating that, in the absence of Abd-B, Dpp (and possibly Wg) are still required to activate Dll. Abd-B minus clones far from the wild-type Dll domain fail to activate Dll ectopically, suggesting that activation of Dll in the absence of Abd-B depends on the range of diffusion of Dpp and Wg, as in the leg disc and in the anal primordium (Estrada, 2001).

Dll is required for the development of legs and antennae, and induces these structures when expressed ectopically in the wing or eye-antennal discs. However, although Dll is also expressed in the genital primordia this expression does not lead to the formation of any of these appendages. To test if Abd-B prevents Dll function Abd-B was eliminated in Dll-expressing cells; these cells formed leg tissue. However, it is possible that the high levels of Dll observed in these mutant cells account for the leg transformation. To test this, use was made of the GAL4/UAS system to increase Dll expression in the genital disc (dpp-GAL4/ UAS-Dll flies). Male and female genitalia of this genotype are abnormal, but not transformed into leg. To extend these observations, the ability of Dll to promote Bar transcription, a gene expressed in the leg disc and activated by Dll, was examined. Bar is not expressed in the female genital primordium and only in a few cells within the Dll domain in the male genital primordium; however, Abd-B minus clones show Bar expression in both sexes. When Dll is ectopically expressed in the genital disc, Bar expression is activated in some of the cells that express Dll. These results suggest that, in females, Dll levels are insufficient to activate Bar when Abd-B is present, but that increasing Dll expression or removing Abd-B activates Bar transcription. Abd-B, therefore, prevents some Dll activity in females. In males, although there is Bar transcription, leg tissue is not formed, probably because Abd-B modifies or prevents the activation of other Dll target genes. A similar case has been reported in the wing disc: ectopic Dll activates bric a brac, a gene downstream of Dll, both in the wing pouch and the body wall region of the wing disc; however, legs appear in the wing, but not in the notum (Estrada, 2001).

The Hox gene Antennapedia is involved in leg development. Therefore, an examination was performed to see whether Antp is derepressed in Abd-B minus clones. Antp is not transcribed in the wild-type genital disc, but some Abd-B clones show Antp signal. The presence of the Antp product, however, is not required to transform the genitalia into a leg, since Antp:Abd-B double mutant clones still form ectopic legs. This result is consistent with the view that the role of Antp in leg specification is simply to repress hth expression. It seems that Dll alone is able to direct leg development, provided that Hox and hth genes are not transcribed. Under these conditions, leg tissue can be formed in several appendages: leg, wing, antennal and genital primordia (Estrada, 2001).

Ubx and abdominal A expression were examined in Abd-B minus clones. Ubx was not derepressed in these clones, whereas some clones presented weak ectopic abd-A expression, but only in some cells (Estrada, 2001).

GENE STRUCTURE

Four classes of overlapping transcripts are generated from the Abd-B gene The transcription initiation sites for the class A (4.6 kb) and class B (3.4kb) transcripts show that they are generated from separate promoters. Both of these transcripts are present throughout the period during which the ABD-B subfunctions are required. A mutation that inactivates the morphogenetic function is associated with a 411bp deletion of the initiation site for the 4.6 kb RNA. Regulatory function mutations disrupt the transcription unit for the 3.4 kb RNA, but not the 4.6 kb RNA. A morphogenetic (m) function is assigned to the 4.6 kb RNA and a regulatory (r) function to the 3.4 kb RNA.

A 7.8 kb RNA expressed during embryogenesis may also contribute to the regulatory function. Sequence analysis of cDNAs indicates that the 4.6 kb RNA encodes the 55-kD morphogenetic protein, whereas the 3.4 kb RNA encodes a 30-kD regulatory protein. The m and r proteins share a carboxy-terminal sequence that includes the homeodomain, but the r protein lacks a glutamine-rich amino-terminal domain found in the m protein (Celniker,1989 and Zavortink, 1989).

Exons - four for class A transcript and five for class B transcript

Bases in 3' UTR - 1604

PROTEIN STRUCTURE

There are two variants to the ABD-B protein. One, a morphogenetic function can be assigned to the 55 KD protein and a second, regulatory function can be assigned to a 30 KD protein (Boulet, 1991).

Amino Acids

The class r 4.6 kb transcript encodes for a protein of 493 amino acids. The class m 3.4 kb transcript encodes for a protein of 270 amino acids (Zavortink, 1989).

See Four paralogous Hox clusters of mammals for homologies of Abd-B with mammalian Hox cluster genes.

date revised: 25 APR 97  

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

Please e-mail comments/corrections to brodyt@codon.nih.gov