Gene name - abdominal-A
Cytological map position - 89E2-3
Function - transcription factor
Keywords - homeotic selector - bithorax complex
Symbol - abd-A
Genetic map position - 3-58.8
Classification - homeodomain - Antp class
Cellular location - nuclear
|Recent literature||Baeza, M., Viala, S., Heim, M., Dard, A., Hudry, B., Duffraisse, M., Rogulja-Ortmann, A., Brun, C. and Merabet, S. (2015). Inhibitory activities of short linear motifs underlie Hox interactome specificity in vivo. Elife 4. PubMed ID: 25869471
Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. This study shows that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrain the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. For example AbdA is the Hox protein establishing the highest number of interactions, which is consistent with the fact that it served as a bait protein in the starting competition screen. However, the observation that Hox proteins do not interact systematically with the same set of cofactors shows their specificity. Interestingly, this specificity is not only occurring at the DNA-binding level since the loss of AbdA DNA-binding activity did not affect all interactions (18 interactions of 31 were affected). Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, these observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development.
|Beh, C. Y., El-Sharnouby, S., Chatzipli, A., Russell, S., Choo, S. W. and White, R. (2016). Roles of cofactors and chromatin accessibility in Hox protein target specificity. Epigenetics Chromatin 9: 1. PubMed ID: 26753000
The regulation of specific target genes by transcription factors is central to understanding of gene network control in developmental and physiological processes, yet how target specificity is achieved is still poorly understood. This is well illustrated by the Hox family of transcription factors as their limited in vitro DNA-binding specificity contrasts with their clear in vivo functional specificity. This study generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, following transient expression in Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. In the absence of the TALE class homeodomain cofactors Exd and Hth, Ubx and Abd-A bind at a very similar set of target sites in accessible chromatin, whereas Abd-B binds at an additional specific set of targets. Provision of Hox cofactors Exd and Hth considerably modifies the Ubx genome-wide binding profile enabling Ubx to bind at an additional novel set of targets. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in chromatin that is relatively DNase1 inaccessible prior to the expression of Hox proteins/Hox cofactors. It is concluded that there is a strong role for chromatin accessibility in Hox protein binding, and the results suggest that Hox protein competition with nucleosomes has a major role in Hox protein target specificity in vivo.
|Trujillo, G. V., Nodal, D. H., Lovato, C. V., Hendren, J. D., Helander, L. A., Lovato, T. L., Bodmer, R. and Cripps, R. M. (2016). The canonical Wingless signaling pathway is required but not sufficient for inflow tract formation in the Drosophila melanogaster heart. Dev Biol [Epub ahead of print]. PubMed ID: 26983369
The inflow tracts of the embryonic Drosophila cardiac tube, termed ostia, arise in its posterior three segments from cardiac cells that co-express the homeotic transcription factor Abdominal-A (abdA), the orphan nuclear receptor Seven-up (Svp), and the signaling molecule Wingless (Wg). To define the roles of these factors in inflow tract development, this study assessed their function in inflow tract formation. Using several criteria, it was demonstrated that abdA, svp, and wg are each critical for normal inflow tract formation. Wg acts in an autocrine manner to impact ostia fate, and it mediates this effect at least partially through the canonical Wg signaling pathway. By contrast, neither wg expression nor Wg signaling are sufficient for inflow tract formation when expressed in anterior Svp cells that do not normally form inflow tracts in the embryo. Instead, ectopic abd-A expression throughout the cardiac tube is required for the formation of ectopic inflow tracts, indicating that autocrine Wg signaling must be supplemented by additional Hox-dependent factors to effect inflow tract formation. Taken together, these studies define important cellular and molecular events that contribute to cardiac inflow tract development in Drosophila. Given the broad conservation of the cardiac regulatory network through evolution, these studies provide insight into mechanisms of cardiac development in higher animals.
|Wang, G., Gutzwiller, L., Li-Kroeger, D. and Gebelein, B. (2017). A Hox complex activates and potentiates the Epidermal Growth Factor signaling pathway to specify Drosophila oenocytes. PLoS Genet 13(7): e1006910. PubMed ID: 28715417
Hox transcription factors specify distinct cell types along the anterior-posterior axis of metazoans by regulating target genes that modulate signaling pathways. A well-established example is the induction of Epidermal Growth Factor (EGF) signaling by an Abdominal-A (Abd-A) Hox complex during the specification of Drosophila hepatocyte-like cells (oenocytes). Previous studies revealed that Abd-A is non-cell autonomously required to promote oenocyte fate by directly activating a gene (rhomboid) that triggers EGF secretion from sensory organ precursor (SOP) cells. Neighboring cells that receive the EGF signal initiate a largely unknown pathway to promote oenocyte fate. This study shows that Abd-A also plays a cell autonomous role in inducing oenocyte fate by activating the expression of the Pointed-P1 (PntP1) ETS transcription factor downstream of EGF signaling. Genetic studies demonstrate that both PntP1 and PntP2 are required for oenocyte specification. Moreover, PntP1 contains a conserved enhancer (PntP1OE) that is activated in oenocyte precursor cells by EGF signaling via direct regulation by the Pnt transcription factors as well as a transcription factor complex consisting of Abd-A, Extradenticle, and Homothorax. These findings demonstrate that the same Abd-A Hox complex required for sending the EGF signal from SOP cells, enhances the competency of receiving cells to select oenocyte cell fate by up-regulating PntP1. Since PntP1 is a downstream effector of EGF signaling, these findings provide insight into how a Hox factor can both trigger and potentiate the EGF signal to promote an essential cell fate along the body plan.
|Zouaz, A., Auradkar, A., Delfini, M. C., Macchi, M., Barthez, M., Ela Akoa, S., Bastianelli, L., Xie, G., Deng, W. M., Levine, S. S., Graba, Y. and Saurin, A. J. (2017). The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription. EMBO J. PubMed ID: 28871058
In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, this study uncovered a molecular mechanism that increases productive transcription. The Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.
As recently as 1986, models of the bithorax complex of genes posited not three genes, (abdominal A, Abdominal B and Ultrabithorax) as is currently assumed, but seven or eight, each independently regulated and controlled by the repressor Polycomb. This earlier model of development suggested that the cuticular phenotype of each segment depended on an array of up to eight BX-C substances elaborated in each segment. Only with the cloning of the bithorax complex genes (Regulski, 1985) was the picture more accurately understood. It then became clear that only three genes, not eight, were responsible for all the phenotypes observed.
The reason for the earlier misunderstanding is found in the existence of infra-abdominal (iab) regions; cis-acting regulatory sequences whose activities regulate the transcription of the abdominal-A and Abdominal-B genes. Ultrabithorax had already been recognized as an independent entity (Lewis, 1978). The iab regions, now termed enhancers, or locus control regions, act in cis and in many instances in trans, to regulate the structural genes. Locus control regions are able to act at a distance to enhance or diminish transcription of a structural gene. On the same chromosome, cis-acting regions are linked with their targets, while trans-acting regions are on separate chromosomes.
abdominal-A has effects that are apparent in the pattern of cuticle generated in the ectoderm and the pattern of muscle generated in mesoderm. Understanding of the effects on mesoderm predated the cloning of the homeodomain proteins. Depending on genotype, any segment can change its identity to resemble segments either more anterior or more posterior to itself. Referring to the ectodermal cuticule pattern: "Third thoracic and anterior abdominal segments can approach or achieve almost all segmental levels of development from the second thoracic to third abdominal depending on genotype" (Lewis, 1978).
The same is true for mesodermal derivatives destined to become the muscles of the fly. With ectopic mesodermal abd-A expression, one finds alterations of the mature muscle patterns of the thoracic segments and the first abdominal segment. These include a change to a more abdominal-like identity for the thoracic longitudinal muscles. In some cases, a gain of one muscle is accompanied by the loss of another (Michaelson, 1994).
Changes in regulation of the bithorax complex are found on an evolutionary scale as well. Changes in regulation by infra-abdominal regions determine how many segments, wings, arms and legs an organism develops. Without the homeodomain proteins, and especially without the regulatory changes in infra-abdominal regions, the diversity found throughout the spectrum of evolution from hydra to man would not exist.
Hox genes have large expression domains, yet these genes control the formation of fine pattern elements at specific locations. The mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm has been examined. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factor and the posterior one the Odd-paired (Opa) zinc finger protein. pnt expression in the midgut visceral mesoderm (VM) commences at stage 13, in a single patch in the central midgut. This patch is just posterior to the large basophilic cells of the endoderm, which mark the future site of the central constriction. PNT mRNA is also found at the boundaries of the midgut with the foregut and hindgut, and in the visceral branches of the trachea. The spindle shape of VM cells and their separation into four patches located dorso- and ventro-laterally around the midgut clearly distinguish pnt expression in the VM from pnt expression in the trachea. At stage 16, pnt is expressed throughout the VM of the third midgut chamber. There is a slight but detectable gradient of pnt expression: the cells that border the central constriction display higher levels of pnt than the cells that border the posterior constriction. Odd-paired (opa) is produced throughout the VM of the first and fourth midgut chambers. Examination of pnt and opa reveals that the boundaries of expression of these two genes abut, and that no cells express both pnt and opa (Bilder, 1998a).
Transcription of both pnt and opa is activated by abdA but the adjacent non-overlapping patterns are not due to mutual opa-pnt regulation. Near the anterior limit of the abdA domain, two signals, Decapentaplegic (a TGFbeta) and Wingless, are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells. The two signals are known to regulate local mesodermal cell fates and to signal to the endoderm. In addition, they precisely subdivide the abdA domain: Wg acts upon anterior abdA domain cells to activate pnt transcription, while Dpp is essential in the same region to prevent abdA from activating opa transcription. pnt activation is required to determine the appropriate numbers of mesodermal cells in the third midgut chamber (Bilder, 1998a).
Early pnt mutant embryos have wild-type anterior-posterior information along the midgut axis, so the small third chamberof pnt mutants must arise from a spatially restricted reduction in VM cell number. Such a defect would change the apparent location of the posterior constriction, while not changing gene expression outside the affected domain. At stage 16, VM nuclei are aligned in four single files located at dorso- and ventro-lateral locations on each side of the midgut. The sizes of midgut chambers were measured by counting VM nuclei along a single file between the constrictions. In pnt mutant embryos, the third chamber is dramatically reduced, containing only 21 VM nuclei, as compared to 36 in wild-type embryos. The first, second and fourth midgut chambers of pnt embryos contain normal numbers of cells. The reduced size of the third midgut chamber is thus due primarily to a reduction of VM cell number in the third chamber. Neighboring chambers do not appear to expand at the expense of the third. Inappropriate cell death is not the cause of reduced VM cell numbers in pnt mutants. It is also concluded that pnt is not required for cell division in the VM. Because defects in cell death or cell proliferation do not occur in the VM of pnt mutants, the loss of third chamber VM cells must be due to a transformation of cell fate to a cell type that does not contribute to the anteroposterior size of the third chamber (Bilder, 1998a).
The following model for morphogenesis of the posterior midgut is proposed. AbdA activates three targets, in distinct subsets of its broad domain of expression: wingless at the anterior boundary of Connectin (Con) patch 7; pnt from anterior Con patch 7 to anterior Con patch 8, and opa, from anterior Con patch 8 through Con patch 11. Decapentaplegic signaling plays a central role in setting these distinct expression domains. The initial activation of wg by AbdA requires dpp. opa is activated in all abdA-expressing cells that do not receive a Dpp signal, defining the site of the posterior constriction. wg, in collaboration with abdA, activates pnt to generate the appropriate number of cells in the third midgut chamber, positioning the posterior constriction at the proper distance from the central constriction and partitioning the posterior midgut appropriately. Fine patterning of the posterior midgut is achieved by the activity of diffusible signals emanating from the central midgut, a remarkably long-range organizing effect (Bilder, 1998a).
How Hox proteins achieve tissue-specific functions and why cells lying at equivalent A/P positions but in different germ layers have distinctive responses to the same Hox protein remains to be determined. This question was examined by identifying parts of Hox proteins necessary for Hox function in different tissues. Available genetic markers allow the regulatory effects of two Hox proteins, Abdominal-A (AbdA) and Ultrabithorax (Ubx), to be distinguished in the Drosophila embryonic epidermis and visceral mesoderm (VM). Chimeric Ubx/AbdA proteins were tested in both tissues and used to identify protein sequences that endow AbdA with a different target gene specificity from Ubx. Distinct protein sequences define AbdA, as opposed to Ubx, and they function in the epidermis vs. the VM. These sequences lie mostly outside the homeodomain (HD), emphasizing the importance of non-HD residues for specific Hox activities. Hox tissue specificity is therefore achieved by sensing distinct Hox protein structures in different tissues (Chauvet, 2000).
As a guide to what protein sequences may be most important for AbdA specificity, AbdA sequences from evolutionarily close species within the insect phylum were compared. Sequence conservation extends beyond the HD, including twelve amino acids C-terminal and adjacent to the HD. This region is also well conserved among Ubx proteins. Within this region, the ten first amino acids are mostly conserved between AbdA and Ubx. This sequence will be referred to as CterC, for C-terminal conserved. Only a few residues distinguish AbdA from Ubx in the conserved HD and in the CterC, although the proteins look mostly different in other regions. The distinguishing residues are the HD amino acids 1, 12, 23, and 35 and amino acids 61, 62, 64, and 67 in the CterC (Chauvet, 2000).
Examination of AbdA protein sequences from the evolutionarily distant insects Drosophila melanogaster and Tribolium castaneum shows that the 40 amino acids preceding the HD have been significantly conserved in AbdA. This region, referred to as NterC for N terminal conserved, contains the hexapeptide, a motif required for appropriate Hox-Exd/Pbx interaction. D. melanogaster and T. castaneum AbdA proteins have the same NterC amino acids at 63% of the positions; that number increases to 78% with conservative changes taken into consideration. This score is significantly higher than the identity/similarity score (34%/47%) found in the remaining N-terminal sequences (N-ter). Apart from the hexapeptide, the NterC region of Ubx and AbdA have largely diverged (Chauvet, 2000).
It was reasoned that the few residues that differ between Ubx and AbdA in otherwise evolutionarily conserved sequences would be good candidates for specificity control. To identify sequence elements critical for AbdA character, conserved parts of the AbdA sequence were introduced into the Ubx protein and analyzed for their ability to confer AbdA-like activity. Chimeras A-F contain combinations of point mutations introduced into Ubx to test the functional importance of the HD and CterC residues that are found specifically in AbdA. Chimeras G and H are protein sequence switches addressing the function of the NterC and of the region following the AbdA CterC sequence (C-ter). All of the protein-coding constructs were fused to a heat-inducible promoter and introduced into the fly by using P-mediated germ-line transformation. Eggs from these transgenic lines were immunostained with an antibody directed against the N-terminal sequences of Ubx, allowing detection of chimeras A-H. After heat induction, all chimeric proteins accumulate at similar levels in the nucleus (Chauvet, 2000).
Eight amino acids distinguish Ubx and AbdA within the HD and CterC region. Chimera A consists of a Ubx protein where all eight amino acids have been changed into the corresponding AbdA residues. The effects on cuticle development of ubiquitous expression of chimera A were compared with those of ubiquitous expression of Ubx and abdA. The number, identity, and spatial organization of denticles readily distinguish these two segments. Uniform Ubx expression transforms anterior segments into extra A1 segments, whereas abdA turns them into A2. Chimera A has the same effect as ubiquitous abdA expression. The three thoracic segments (T1-T3) and the first abdominal segment A1 have been transformed toward an A2 identity. In the epidermis, therefore, chimera A has acquired AbdA-like character (Chauvet, 2000).
Ubx and abdA differentially activate the target genes wg and dpp during midgut morphogenesis. In the midgut mesoderm, dpp is expressed in parasegment (PS) 3-4 and PS7, with Ubx activating dpp in PS7. When Ubx is expressed ubiquitously, dpp is ectopically activated in PS anterior to PS7. Ubiquitous abdA expression represses dpp in all of the VM. Ectopic Ubx does not affect wg transcription that continues to occur only in its normal place, PS8. Ubiquitous abdA expression does induce ectopic wg transcription in anterior regions. Chimera A behaves like Ubx, because it still activates dpp while leaving wg expression unaffected. It was noted, however, that the activation of dpp by chimera A does not occur in all VM cells anterior to PS7. This suggests that the chimera is transcriptionally less potent and/or that it is more sensitive to phenotypic suppression by the more anteriorily expressed Scr and Antp genes. In any case, these results unambiguously show that in the VM, chimera A retains Ubx specificity and has not acquired, as in the epidermis, an AbdA-like character (Chauvet, 2000).
The activities of chimeras B-F in the epidermis were studied to assess the individual contributions of the eight amino acids that distinguish AbdA from Ubx in the HD and CterC region. Changing only one residue in the N-terminal arm of the HD, either amino acid 1 (chimera E) or 12 (chimera F), is not sufficient to give the protein AbdA character in epidermal patterning. Both chimeras induce, like Ubx, ectopic A1 metameres. However, simultaneously changing both amino acids 1 and 12 (chimera C) directs the formation of A2-like segments in place of thoracic and A1 segments. The transformation toward an A2 identity is less complete than with chimera A. Such weak A2-like transformations are also observed when the changes in the protein concern the four residues in the CterC region (chimera D). Among chimeras B-F, the only protein that has an efficient AbdA effect is chimera B. This indicates that amino acids 1 and 12 of the HD, as well as amino acids 61, 62, 64, and 67 of the CterC region, are collectively required for full-potency AbdA effects. HD residues 23 and 35 appear to be unnecessary for AbdA-like activity (Chauvet, 2000).
Because chimera A does not have AbdA-like effects in the VM, the possible contributions of two additional protein sequences were investigated. The importance of the AbdA C-ter region, which is significantly longer than the Ubx C-ter region, was tested by using chimera G. In chimera G, the Ubx HD and downstream sequences are replaced by AbdA sequences. Chimera G behaves like a Ubx protein in the VM: it retains the ability to activate dpp while leaving wg expression unaffected. Sequences downstream of the AbdA HD are therefore not sufficient to convey AbdA function in the VM (Chauvet, 2000).
The function of the NterC sequence was analyzed by using chimera H, which has Ubx sequences through amino acid 234 and AbdA sequences thereafter, including the AbdA NterC, HD, and C-tail. Chimera H therefore tests the contribution of the NterC region to AbdA activity in the VM, particularly in comparison to chimera G. The results show that chimera H has AbdA effects but not Ubx effects: it represses dpp expression and induces anterior ectopic expression of wg. In the VM, therefore, the hexapeptide-containing NterC sequence confers AbdA activity, in contrast to the HD and CterC sequences important for AbdA character in the epidermis (Chauvet, 2000).
Is the NterC region on its own sufficient to confer to an otherwise Ubx protein an AbdA activity in the VM? The UbxC1 mutation is a chromosome rearrangement that leads to an AbdA/Ubx fusion product. The resulting protein consists of AbdA N-ter and NterC sequences joined to the Ubx HD, CterC, and C-ter. The hybrid gene is expressed in the posterior VM in the abdA expression domain. The mutation can therefore be used to determine whether the AbdA NterC region is sufficient to provide AbdA activity in the VM. In homozygous UbxC1 embryos, wg is not activated in VM PS8 by the C1 fusion product. In the same mutant context, expression of dpp in the anterior VM (PS3-4) is never affected, whereas expression in the central part of the midgut is severely reduced or abolished. A minority of mutant embryos display posterior ectopic expression within the expression domain of the fusion protein C1. These observations indicate that the C1 fusion protein has retained some transcriptional activity in the VM and that the level of Dpp signaling is very low in most mutant embryos (Chauvet, 2000).
wg activation by abdA critically depends on Dpp signaling in the central midgut. The absence of wg expression in UbxC1 homozygous embryos might thus be the result of reduced Dpp signaling rather than the inability of the C1 fusion protein to activate wg. To discriminate between these possibilities, wg expression was analyzed in homozygous UbxC1 embryos, where a high level of Dpp activity was provided by a heat-inducible transgene (HS.dpp). Even in such a context, no wg expression is observed. Taken together, these experiments indicate that the C1 fusion protein is not capable of providing AbdA-like function in the VM. The AbdA NterC region, although necessary to impart AbdA-like function, is not sufficient on its own. Additional AbdA sequences lying in the HD and/or C-terminal sequences are required as well (Chauvet, 2000).
The effects of ectopically expressed Hox or Hox chimeric proteins depend in some instances on ectopic activation of endogenous Hox gene activity. It was tested whether the switches to AbdA-like function obtained with chimera A in the epidermis and chimera H in the VM require the endogenous abdA gene. Embryos uniformly expressing chimera A or chimera H were stained with a probe corresponding to the N-ter sequences of AbdA, allowing the detection of the endogenous abdA gene exclusively. In both cases, no ectopic expression of abdA was observed, either in the epidermis or in the VM. The AbdA-like activities of chimeras A and H therefore do not rely on activation of the endogenous abdA gene. Instead, both chimeras have acquired an AbdA activity (Chauvet, 2000).
Most of the Hox protein specificity information has been found to lie within the HD. Indeed, in all but two of the chimeras already analyzed, the HD must be switched to change a Hox protein's function into that of another Hox protein. Residues in the N-terminal arm of the HD have been shown necessary and sometimes sufficient to define functional specificity. In agreement with these findings, the results presented here indicate that amino acids 1 and 12, in the N-terminal arm, significantly contribute to defining AbdA character (Chauvet, 2000).
For several reasons, the idea that the HD contains most of the specificity information appears overemphasized. (1) The switch of specificity is often only partial. For example, when the Deformed (Dfd) HD is replaced by that of Ubx, the chimeric protein activates the Antp gene, a target of Ubx, whereas failing to carry out a Dfd function, autoactivation of Dfd transcription. The chimeric protein has therefore acquired the target specificity of Ubx. However, the chimera did not have all of the regulatory specificity of Ubx because Ubx normally represses Antp (Chauvet, 2000).
(2) The switch of the HD alone sometimes does not confer an identity switch: replacing the HD of Ubx by that of Antp results in a chimera that behaves like Ubx, promoting A1 identity in the epidermis. Similarly, the effect of a Hox-A4/Hox-C8 chimera on vertebral patterning does not follow the identity of the HD. The Ubx/AbdA chimera data show that, in vivo, switching the HD is not necessary for changing Hox protein specificity. Chimera A activates dpp in the VM and has no effects on wg expression, indicating that it has Ubx character, despite having the AbdA HD (Chauvet, 2000).
(3) Sequences outside the HD have been shown to provide important functions for Hox gene activity. This includes the C-tail for Ubx, Antp, Scr, and Dfd along with an acidic N-region preceding the HD in the Dfd protein. The results presented here demonstrate that two sets of non-HD sequences contribute to Hox specificity. On the C-terminal side, four residues adjacent to the HD are necessary to impart AbdA-like character in the epidermis. This region is evolutionarily conserved; it has been proposed to be part of a coiled-coil structure, and might constitute an interface for interacting proteins. On the N-terminal side, the NterC region is required to define mesodermal specificity (Chauvet, 2000).
The role of the hexapeptide YPWM in the formation of Hox-Exd/Pbx heterodimers in vitro has been extensively studied, yet its in vivo function has not been established. An interesting feature of the NterC region is that it contains the hexapeptide motif. The requirement of Exd for target gene regulation in the VM suggests that the motif is involved in AbdA mesodermal function. Several lines of evidence, however, do not favor the hypothesis that the YPWM motif itself provides the key for AbdA VM specificity. (1) Ubx and AbdA share the YPWM motif, so sequences other than the motif itself must contribute to distinguishing AbdA from Ubx. (2) Exd that contacts the motif is required for Hox protein activity in several tissues. (3) The conservation within NterC regions of AbdA proteins evolutionarily as distant as D. melanogaster and T. castaneum is not restricted to the hexapeptide motif. One would thus expect these evolutionarily conserved sequences in AbdA, that have diverged in Ubx, to provide the VM-specificity information. The NterC domain therefore likely coordinates the contribution of Exd and putative tissue-specific cofactors for accurate AbdA mesodermal activity (Chauvet, 2000).
In the Drosophila larval cardiac tube, aorta and heart differentiation are controlled by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA), respectively. There is evidence that the cardiac tube undergoes extensive morphological and functional changes during metamorphosis to form the adult organ, but both the origin of adult cardiac tube myocytes and the underlying genetic control have not been established. Using in vivo time-lapse analysis, this study shows that the adult fruit fly cardiac tube is formed during metamorphosis by the reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation. The genetic control of the process, which is cell autonomously ensured by the modulation of Ubx expression and AbdA activity, has been characterized. Larval aorta myocytes are remodelled to differentiate into the functional adult heart, in a process that requires the regulation of Ubx expression. Conversely, the shape, polarity, function and molecular characteristics of the surviving larval contractile heart myocytes are profoundly transformed as these cells are reprogrammed to form the adult terminal chamber. This process is mediated by the regulation of AbdA protein function, which is successively required within these persisting myocytes for the acquisition of both larval and adult differentiated states. Importantly, AbdA specificity is switched at metamorphosis to induce a novel genetic program that leads to differentiation of the terminal chamber. Finally, the steroid hormone ecdysone controls cardiac tube remodelling by impinging on both the regulation of Ubx expression and the modification of AbdA function. These results shed light on the genetic control of one in vivo occurring remodelling process, which involves a steroid-dependent modification of Hox expression and function (Monier, 2005).
The morphological transformations of the cardiac tube during metamorphosis were visualised by polymerised actin (F-actin) staining with phalloidin, which reveals myofibril organisation and enables visualisation of the general morphology of the cardiac tube. Major morphological and functional changes accompany the remodelling of the cardiac tube. In order to unambiguously determine the origin of the adult cardiac myocytes, cardiac tube-specific expression of GFP was analyzed throughout metamorphosis. This approach indicated that the adult organ arises by the remodelling of the larval cardiac tube by a continuous and progressive process. Time-lapse analysis of cardiac tube remodelling, performed from 27 to 64 hours after puparium formation (APF), demonstrates that adult myocytes directly derive from larval myocytes without cell proliferation. In addition, cell tracing experiments show that the adult heart is formed by the myocytes that constituted the larval posterior aorta, and by segment A5 myocytes, which are a part of the larval heart and which constitute the posterior tip of the adult heart (the terminal chamber). Both approaches thus indicate that the adult heart is formed by persisting larval myocytes that are remodelled without proliferation or the addition of new cells. One identity for each adult cardiac myocyte can therefore be inferred from its larval origin (Monier, 2005).
Transdifferentiation is the process by which a cell committed to one phenotypic fate acquires a different fate, implying that some characteristics are lost while novel ones are acquired. This study suggests that myocytes in segment A5 undergo a transdifferentiation process during cardiac tube remodelling: their shape and polarity are changed and they undergo transcriptional reprogramming. In addition, their physiological function is modified: they lose the rhythmic contractile activity they display during larval life, but gain specific innervations, implying that they may become involved in the regulation of cardiac activity. Indeed, adult A5 cardiac myocytes are directly innervated, whereas anterior innervations are targeted to ventral imaginal muscles but not to A1-A4 cardiac myocytes. Neurons innervating the terminal chamber have been described as being positive for Cardioacceleratory peptide and might be involved in the regulation of the anterograde heart beat (Monier, 2005).
Much attention has been given to transdifferentiation in recent years; however, the majority of studies have used artificial systems and the genetic control of the process is still largely unknown. This study has investigated the genetic and molecular control of a naturally occurring transdifferentiation process: the metamorphosis of the Drosophila larval cardiac tube. This situation offers the opportunity to analyse cell plasticity in an intact system, in which cells can be individually identified (Monier, 2005).
Like myocytes in segment A5, those in segments A1-A4 also gain a number of novel properties during remodelling. For example, Tin-expressing myocytes increase their myofibrillar content, activate Ih channel (Ih) transcription and acquire contractile activity; svp-expressing cells transiently express wg and differentiate into functional ostiae. However, this may not represent transdifferentiation per se, since these cells do not appear to lose any of the differentiated characteristics they would have acquired during embryonic or larval life. Rather, it is considered that larval A1-A4 myocytes are committed to differentiate into adult heart myocytes, but do not represent, on their own, a fully differentiated state. Interestingly, their final differentiation relies on an ecdysone-dependent modulation of Ubx expression: Ubx expression has to be downregulated in Tin-expressing myocytes, while its maintenance, and probably its overexpression, is required in svp-expressing cells. Ubx function, however, appears to be unchanged in these cells. Indeed, when Ubx is overexpressed in the embryonic cardiac tube, it is able to activate wg expression in A1-A4 svp-expressing myocytes, and to repress Ih transcription in Tin-expressing myocytes. This clearly indicates that, at least at the wg and Ih transcriptional level, Ubx activity is unmodified during remodelling (Monier, 2005).
By contrast, the reprogramming of A5 myocytes depends on the modification of AbdA activity. Although abdA is required, during embryogenesis, for Ih transcription and for the acquisition of rhythmic contractile activity by the A5 myocytes that constitute part of the beating larval heart, its cellular function is modified at metamorphosis. During cardiac tube remodelling, abdA is indeed required for Ih repression, and for changes in the shape and polarity of myocytes, as well as for the acquisition of specific innervations. In these myocytes, the functional outcome of AbdA activity is thus dramatically different during embryogenesis and metamorphosis, indicating that abdA is instrumental in myocyte reprogramming (Monier, 2005).
The developmental switch triggering cardiac tube remodelling is mediated by ecdysone, which impinges on Ubx expression and on AbdA activity. Interestingly, like retinoic acid (RA) in the developing vertebrate hindbrain, the outcome of ecdysone activity during the remodelling of segments A1-A4 is a 'posterior transformation': adult A1-A4 myocytes acquire properties characteristic of larval A5-A7 posterior myocytes. This is the first reported example of a steroid function in axial patterning in arthropods. In addition, it is shown that, like RA, which modulates Hox expression in vertebrates, the ecdysone signalling pathway modulates Ubx expression. These observations strongly suggest that steroid activity on axial patterning is conserved from flies to vertebrates (Monier, 2005).
In contrast to its effects on Ubx, ecdysone does not affect abdA expression but impinges on its activity. This is the first observation of such an activity for a steroid hormone and it certainly warrants further study in other animal models. Is this also a property of steroids (such as RA) in vertebrates? To date, the only known activity of RA on Hox gene function is a modulation of their transcriptional expression. The data presented in this study recommend a re-examination of RA/Hox functional interactions in vertebrates (Monier, 2005).
Cellular identities along the rostrocaudal axis mainly depend on the Hox code, i.e., on the combinatorial expression of Hox genes. The fact that, as is shown in this study, a steroid hormone can additionally control Hox activity greatly increases the number of cellular identities that can potentially be determined by the same Hox code. In addition, the situation described in this study concerning AbdA activity indicates the importance of the developmental context upon Hox specificity. An important unanticipated result from the present study is that the change of AbdA activity can occur in the same cells, leading to a reprogramming between two differentiated states that are both controlled by the same Hox gene (Monier, 2005).
A future challenge will be to understand the molecular basis of the steroid-dependent Hox activity switch. Ecdysone signalling might change AbdA transcriptional activity, by specifying new transcriptional targets or by switching its activity from an activator to a repressor (and vice versa) on the same transcriptional targets. What could be the molecular mechanism by which EcR activation impinges on AbdA activity? Given the pivotal role played by transcriptional cofactors in Hox function, one hypothesis is that EcR activates or represses such a Hox cofactor. Exd/Hth are obvious candidates but do not seem to be involved; they are not expressed in the cardiac myocytes, neither during embryogenesis nor during the remodelling process. Conversely, there is evidence that Hox transcriptional activity can be modified by phosphorylation. An alternate possibility would therefore be that EcR activates or represses the expression of some kinases or phosophatases, which would in turn modify AbdA activity. In any case, elucidation of the molecular mechanisms will rely on the identification of direct abdA targets in the cardiac tube. wg and Ih are potential candidates, and are currently under investigation (Monier, 2005).
In conclusion, this study may shed light on Hox gene function and regulation in other cell reprogramming processes, such as pathological remodelling of the human heart, in which steroids appear to play a key role. In addition, cellular therapies for inherited myopathies are based on the cellular plasticity of the donor cells (either satellite cells or muscle-derived stem cells). Cell transplantations for the treatment of muscular dystrophies appear promising, and understanding the molecular basis of one example of myocyte reprogramming should help to unravel the underlying mechanisms (Monier, 2005).
abdominal-A is one of three homeotic genes of the bithorax complex (BX-C) and is situated between Ultrabithorax and abdominal-B, and flanked by regulatory regions infra-abdominal 2 and 3.
A comparison of the homeobox sequence of bithorax complex genes ANTP, UBX and ABD-A shows great intra-complex similarity, with only a few amino acid substitutions noted. A YPWM motif is shared by UBX, ANTP, DFD, SCR and LAB as well as an OPA repeat downstream of the homeobox sequence. Outside the homeodomain, the sequence of Abd-A has diverged from these other proteins (Karch, 1990 and Duboule, 1994).
date revised: 12 August 2000
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