Ultrabithorax
Mutations in the homeotic genes of the bithorax-complex cause transformations of cuticular patterns. Whereas abundant information exists concerning homeotic transformation of epidermis, transformations of muscles and motor neurons have been largely unexplored. An important indication of neuromuscular transformation in a segment is the expression of novel behavioral and physiological functions within that segment. In this study, some of the segmental identities of neuromuscular elements in the transformed metathorax of the bithorax-complex mutant abx bx3 pbx/Df(3R) P2 have been resolved. abx, bx and pbx are enhancers for the gene Ultrabithorax resulting in a four winged phenotype and Df(3R) refers to a deficiency in the right hand arm of chromosome 3. Thus the presence of a duplicated neural pathway for the escape-jump response was established within the metathorax. Although frequent homeotic transformation of neural elements and corresponding transformation of the tergotrochanteral ("jump") muscle are observed, corresponding transformation of flight muscles is infrequent, indicating that the presence of a motor neuron is not always sufficient to induce or determine the development of its target muscle (Schneiderman, 1993).
A developmental analysis of Drosophila Contrabithorax (Cbx) alleles offers the opportunity to examine the role of Ubx in controlling haltere morphogenesis as an alternative to, or in place of, wing morphogenesis. Several Cbx alleles are known with different spatial specificity in their wing toward haltere homeotic transformation. The molecular data on these mutations, however, does not readily explain differences among mutant phenotypes. The phenotypes
emerge from early clonality in some Cbx alleles, and from cell-cell interactions leading to recruitment of other cells to Ubx gene expression. There are mutual interactions between haltere and wing territories in pattern and dorsoventral symmetries, suggesting short distance influences or "accommodation" during cell proliferation of the anlage. These findings
are considered in an attempt to explain allele specificity in molecular and developmental terms (Gonzalez-Gaitan, 1990).
Flies doubly heterozygous for GAGA (synonym: trithorax like) and Ubx exhibit larger halteres than flies mutant for Ubx alone, and, with incomplete penetrance and variable expressivity, show homeotic transformations of the haltere and postnotum into wing. When zeste mutations are crossed into this double heterozygotic background, a similar range of phenotypes is observed. However, the fraction of animals displaying the enhanced Ubx phenotype is increased 2 to 19 fold, depending on the GAGA allele used. This increase in penetrance is observed with two different zeste alleles. Therefore, mutations in zeste increase the likelihood that limiting amouts of GAGA factor and UBX will lead to reduced expression of Ubx and to homeotic transformation of haltere into wing (Laney, 1996).
Introgression of homeotic mutations into wild-type genetic backgrounds results in a wide variety of phenotypes and implies that
major effect modifiers of extreme phenotypes are not uncommon in natural populations of Drosophila. A composite interval
mapping procedure was used to demonstrate that one major effect locus accounts for three-quarters of the variance for haltere to
wing margin transformation in Ultrabithorax flies, yet has no obvious effect on wild-type development. Several other genetic
backgrounds result in a pronounced enlargement of the haltere, significantly beyond the normal range of haploinsufficient phenotypes, suggesting
genetic variation in cofactors that mediate homeotic protein function. Introgression of Antennapedia produces lines with heritable phenotypes ranging from almost
complete suppression to perfect antennal leg formation, as well as transformations that are restricted to either the distal or proximal portion of the appendage. It is
argued that the existence of potential variance, which is genetic variation whose effects are not observable in wild-type individuals, is a prerequisite for the
uncoupling of genetic from phenotypic divergence (Gibson, 1999).
Growth and patterning during Drosophila wing development are mediated by signaling from its dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ultrabithorax (Ubx) to mediate development of a pair of tiny balancing organs, the halteres. Expression of Ubx in the haltere D/V boundary down-regulates its D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx minus clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhances the homeotic transformation of Ubx minus cells in the capitellum but also causes homeotic transformation of even Ubx plus cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles of Ubx during maintenance of cell fate and patterning in the halteres (Shashidhara, 1999).
Segment-specific differences are evident in the number of neuroblasts (NBs) that persist beyond the end of embryogenesis and proliferate during larval stages. At stage 17 of embryogenesis, all NBs have stopped dividing but can still be monitored by NB-specific expression of grainyhead. Analyses of Grh expression pattern in the CNSs of wild type embryos and of mutant embryos where cell death is suppressed, strongly suggest that a number of NBs normally die towards the end of embryogenesis. The degree of cell death shows segment-specific differences: many more NBs die in the central abdomen than in the thorax and anterior abdomen. As a consequence, when NBs resume proliferation as postembryonic NBs in the larval stages, 47 NBs are detected in each thoracic segment; about 12 are detected in the two anterior abdominal neuromeres, but only six in central abdominal segments. Furthermore, postembryonic NBs in the thorax and anterior abdomen produce hundreds of daughter cells each, whereas those in abdominal neuromeres 3-A7 give rise to only five to 15 cells. In summary there are three major factors regulating the segment-specific proliferation of NBs: (1) the period and frequency of embryonic NB proliferation; (2) the number of NBs eliminated at the end of embryogenesis, and (3) the frequency and period of postembryonic proliferation (Prokop, 1998 and references).
The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the early Drosophila embryo are identical in thoracic and abdominal segments. However, during late embryogenesis, differences in the numbers of NBs and in the extent of neuroblast proliferation arise between these regions. The homeotic genes Ultrabithorax and abdominal-A
regulate these late differences. Abdominal NBs in Ubx and abd-A mutants continue replicating DNA, and consequently the number of NBs in these mutants resembles that of thoracic neuroblasts. In embryos lacking the Antp gene, DNA synthesis in ventrolateral/lateral NBs is normal, however, additional cells are detected in ventral positions resembling the ventral patterns of the subesophageal ganglion. Therefore abd-A function is needed to repress DNA replication in some lateral NBs of abdominal neuromeres, and Antp function is required to repress DNA replication in ventral NBs of the thorax. Misexpression of either Ubx or abd-A in thoracic neuroblasts, after
segregation, is sufficient to induce abdominal behaviour in lateral neurons and subesophageal characteristics in ventral neurons. The ventral pattern appears to be due to the ability of Ubx to repress Antp expression, since the pattern of ventral neurons resembles the phenotype found in Antp mutant embryos. In wild type embryos, Abdominal-A and Ultrabithorax proteins are only detected in early neuroblasts. In stage 15 embryos no cells are found which co-express Ubx and Grh. This suggests that neither Abd-A nor Ubx are present in the NBs shortly before segment-specific differences in the numbers of cells and Grh patterns occur. Asense is expressed in NBs shortly after their segregation from the neuroectoderm and so can be used as an early marker for NBs. Ubx is detected in many NBs at stages 8-12 although there is wide variation between levels of Ubx present in different NBs and a subset of NBs contain no detectable Ubx. Similarly, Abd-A is present in many NBs at early stages. Thus both Ubx and Abd-A are present in embryonic NBs, but their expression fades before segment-specific differences become detectable (Prokop, 1998).
Transplantation experiments reveal that segment-specific behaviour is determined even prior to neuroblast segregation, that is, prior to expression of Ubx or Abd-A. When cells are heterotopically transplanted from thoracic to abdominal sites of the early gastrula neuroectoderm, 67% give rise to a large nest of postembryonic cells with postembryonic NB (pNB), consistent with the characteristics of thoracic NBs. Conversely, when cells are transplanted from abdominal to thoracic sites, all clones fail to express thoracic features and contain only embryonic cells.
It is concluded that segment-specific differences in neuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts, and executed in later programs controlling neuroblast numbers and proliferation. Two models are presented for the action of the homeotic genes. They could act as transcriptional repressors that initiate a repressed state for their target genes, which can be maintained after the proteins have disappeared, or alternatively, they may activate target genes that have the capacity for autoregulation, so that the targets maintain their own expression in the absence of homeotic proteins (Prokop, 1998).
The closely related Hox transcription factors Ultrabithorax (Ubx) and Antennapedia (Antp) respectively direct first abdominal
(A1) and second thoracic (T2) segment identities in Drosophila. It has been proposed that their functional differences derive
from their differential occupancy of DNA target sites. A hybrid version of Ubx (Ubx-VP16), which possesses a potent activation domain from the VP16 viral protein, no longer directs A1 denticle pattern in embryonic epidermal cells. Instead, it
mimics Antp in directing T2 denticle pattern, and it can rescue the cuticular loss-of-function phenotype of Antp mutants. In cells
that do not produce denticles, Ubx-VP16 appears to have largely retained its normal repressive regulatory functions. These results suggest that the modulation of Hox activation and repression functions can account for segment-specific morphological differences that are controlled by different members of the Hox family. These
results also are consistent with the idea that activity regulation underlies the phenotypic suppression phenomenon in which a more posterior Hox protein suppresses
the function of a more anterior member of the Hox cluster. The acquisition of novel activation and repression potentials in Hox proteins may be an important
mechanism underlying the generation of subtle morphological differences during evolution (Li, 1999).
Interestingly, although Ubx-VP16 acquires an Antp-like ability in denticle patterning, it preserves the Ubx ability to repress Keilin's organ development in thoracic segments. Therefore, Ubx-VP16 displays a mix
of Antp-like and Ubx-like functions, dependent on tissue types and cell positions. Since development of Keilin's organs requires the appendage-promoting
gene Distalless (Dll), the regulation
of Dll by Ubx-VP16 was examined. The expression of Dll in
thoracic appendage primordia cells is repressed by Ubx by means of the
Dll304 element, presumably by eliciting the Ubx
repression function on the element. In ectopic Ubx-VP16 embryos, both
Dll expression and
the activity of the Dll304 element are partially repressed. However, unlike ectopic Ubx,
Ubx-VP16 is capable of activating Dll304 in other cells
outside the appendage primordia.
Thus, the Ubx-like function of Ubx-VP16 in repressing Keilin's organ
development stems from retaining the Ubx repressive function upon
Dll transcription. Since this repression appears specific for
appendage primordia cells, the repression function of Ubx-VP16 is not
constitutive but rather generated in a regulated manner. Taken
together, the above results suggest that Ubx-VP16 functions are due to
normal Ubx repressive effects on some targets (e.g., Dll),
despite the attached VP16 activation domain, as well as a novel
activation function on other targets (e.g., Antp) caused by
the VP16 domain. The mix of functions that Ubx-VP16 exhibits is also often observed for natural Hox proteins (Li, 1999).
In these experiments the strength of activation function in Ubx is artificially varied. However,
the partial change in segmental identity conferred by the Ubx-VP16 protein suggests that regulating the activity state of Ubx may modulate its functional specificity in
denticle patterning. The fact that the Ubx-VP16 denticle patterning function is Antp-like suggests that the functional difference between the Ubx and Antp proteins in
diversifying denticle patterns may reside in differences in activation and repression strengths on similar target genes rather than in differences in target occupancy. This
suggestion is consistent with results indicating that Ubx and Antp recognize identical DNA sequences in vitro and regulate several common target genes
in embryos. This evidence indicates that the segment identity functions of Ubx and Ubx-VP16 are distinct, but it does not eliminate the possibility
that the VP16 domain increases activation function by altering the binding selectivity of the hybrid protein in developing embryos. This is thought to be unlikely because
the specific Ubx targets such as dpp, Antp, and Dll are all regulated, and thus presumably occupied at similar Hox sites, by both Ubx and Ubx-VP16 (Li, 1999 and references therein).
Regulation of activation and repression functions may also be the mechanism that underlies
the phenomenon of phenotypic suppression, in which one Hox protein can dominantly suppress the function of other coexpressed Hox proteins. It has been
proposed that competition of Hox proteins for DNA binding sites is responsible for this phenomenon.
A well studied example of phenotypic suppression is the parasegment-specific transcription of the decapentaplegic gene in the visceral mesoderm (VM). dpp
is directly activated by Ubx protein in PS7 but is repressed by Abdominal-A (Abd-A) protein in PS8-12 of the VM, even when Ubx protein is ectopically expressed
in PS8-12. The repression conferred by Abd-A and the activation conferred by Ubx involves separate clusters
of Hox binding sites within the dpp674 element (34). This suggests that Abd-A does not compete with Ubx for binding to the same DNA sites to antagonize Ubx
activation on dpp. Instead, Abd-A and Ubx proteins can occupy many sites on the dpp674 element in PS8-12, but only Abd-A is capable of conferring
repression from one of the clusters of Hox sites. The Abd-A repression function can then override the Ubx activation function that is produced from another cluster
of Hox binding sites on dpp674 (Li, 1999 and references therein).
The role of activity regulation in Hox segmental specificity may provide new insight into understanding how the
Hox patterning system evolved. At an early point in metazoan evolution, prototypes of Hox genes such as Ubx and Antp were generated by the duplication
of a common ancestral gene. After the duplication event, one or both of the two copies accumulated mutations and evolved distinct functions. One
evolutionary event that altered function was the change in regulatory sequence that altered expression patterns, when compared with the ancestral copy. From the study of extant Hox genes, it is known that changes in the coding sequence during evolution have generated functional distinctions between adjacent
Hox genes. It is proposed that coding region changes that resulted in different functional specificities did so by altering activation/repression
strengths on a largely common set of downstream genes. One reason for this proposal is that mutations in coding sequences that altered binding specificity would
presumably influence target occupancy on all or most downstream genes. Therefore, the newly evolved Hox protein T would no longer regulate many of the genes
under the control of Hox protein S, and protein T would also immediately acquire a novel battery of downstream genes. It is imagined that these events would result in
striking morphological changes in the body plan that would have a low chance of surviving and being selected. However, differences in coding sequences controlling activation/repression strengths could subtly or drastically vary the amount of gene expression from one or a few
members of a common set of downstream genes (x and y for example). The difference that evolves in Hox T could be depicted as an adoption
of a novel repression ability on gene y. This mode of Hox protein evolution would be more likely to result in subtle changes in metameric morphology compatible with
survival. Occasionally some of these subtle morphological changes would result in slight advantages in natural selection for certain niches. This model also requires
changes (either preexisting or acquired) in downstream gene regulatory sequences that are near Hox binding sites, so that factors that regulate the repression strength
of Hox T could switch it into a repressive mode. This model of evolving diverse Hox functions by subtle changes in activation/repression strengths is not meant to
discount the importance of evolutionary variation in downstream genes in morphological variation (Li, 1999 and references therein).
How can it be examined whether this process has occurred in evolution? In the embryo of the crustacean Artemia, the Antp, Ubx, and abd-A homologs are
coexpressed in a trunk region that is composed wholly of appendage-bearing segments. In contrast to Drosophila, the Artemia Ubx and Abd-A homologs do
not repress Dll transcription and do not repress appendage development. There are a variety of reasons why the Artemia Ubx and Abd-A proteins might be
incapable of repressing appendages, but one possibility is that sequence motifs within the proteins that would allow them to repress the appendage enhancer of Dll are missing. This possibility may be testable by placing the Artemia versions of Ubx and Abd-A proteins in the context of Drosophila early embryonic cells and assaying their effects on appendage development (Li, 1999 and references therein).
Hox genes encode evolutionarily conserved transcription factors that control the morphological diversification along the anteroposterior
(A/P) body axis. Expressed in precise locations in the ectoderm, mesoderm, and endoderm, Hox proteins have distinct regulatory activities in
different tissues. 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).
The roles of homeotic selector genes in Drosophila was investigated in the migration and fusion of myoblasts, and in the differentiation of adult muscle fibers. Altering intrinsic homeotic identities of myoblasts does not affect their segment-specific migration patterns. By transplanting meso- and meta-thoracic myoblasts into the abdomen, it was demonstrated that the fusion abilities of myoblasts are independent of their segmental identities. However, transplanted thoracic myoblast nuclei are "entrained" by those of the host's
abdominal muscles to which they fuse and are unable to "switch on" a thoracic muscle-specific reporter gene. This process is likely to be mediated by homeotic repression, due to mis-expression of an abdominal muscle-specific homeotic gene, Ultrabithorax, in the thoracic indirect flight muscles. This results in the repression of the thoracic muscle-specific reporter gene. Removal of Ultrabithorax function, specifically from muscle cells of the first abdominal segment, results in the expression of thoracic muscle
properties. Many homeotic gene functions in muscle patterning in Drosophila could be conserved during myogenesis in other organisms (Roy, 1997).
Mutations in Ultrabithorax result in the transformation of the third thoracic
(T3) segment into the second thoracic (T2) segment. Although
it has been well established that these mutations
have striking effects on adult epidermal structures in T3,
the effect of these mutations on the adult musculature
has been controversial. In this study, a series of Ubx regulatory
mutations, anterobithorax, bithorax, postbithorax,
and bithoraxoid, as well as combinations of these alleles
were used to reevaluate the role of Ubx in the patterning
of the T3 musculature. Homeotic indirect and direct
flight muscles (IFMs and DFMs) were identified in
the transformed T3 segment of all alleles and allelic
combinations with the exception of postbithorax. The pattern and amount of these muscles were critically evaluated
and it was found that while the amount and/or quality of
homeotic IFMs increased, the amount of homeotic
DFMs did not vary significantly as the severity of the ectodermal
transformation increased (Rivlin, 2001).
The majority of adult myoblasts that contribute to the
formation of the adult flight muscles are associated with
the wing imaginal disc. Although T2 myoblasts associated
with the wing disc do not express any known Drosophila
Hox gene, T3 myoblasts associated with the haltere discs
express Antp. Despite the intrinsic differences between these two sets of myoblasts, the results suggest that T3 myoblasts are able to organize a
T2 muscle pattern in the transformed T3 segment of Ubx
mutants, implying that the ectoderm is capable of instructing
T3 myoblasts to form T2 muscles (Rivlin, 2001).
However, it is also possible that some T2 myoblasts
migrate into HT3 and contribute to the formation of
homeotic muscles. Transplantation experiments have
demonstrated that adult myoblasts from both T2 and T3
are capable of migrating throughout the body cavity and
fusing indiscriminately with developing muscles. Because there is no evidence that migrating myoblasts
can actually 'seed' the formation of an adult muscle,
and, instead, appear to only fuse with pre-existing
muscle anlagen, it is argued that the muscle anlagen that
arise in HT3 are founded by HT3 myoblasts. In addition,
HT3 myoblasts have been shown to migrate to sites in
HT3 where the IFMs and DFMs will later appear (Rivlin, 2001).
At this point the issue of whether a muscle founder is necessary for the
maintenance of adult thoracic muscles cannot be addressed. However, subsets of adult myoblasts that appear to be analogous to the muscle pioneers of insect embryos have been identified. This special class of myoblasts,
which have been called imaginal pioneers (IPs), prefigures
the development of at least three adult muscles in T2: DVM-I, -II, and the TDT. A preliminary investigation of myogenesis in the four-winged fly suggests
that the IPs for the DVMs are duplicated in the transformed
T3 segment. The presence of homologs of the T2 IPs in HT3 suggests
that, unlike the embryonic founders, the patterning of the
IPs is independent of their HOX gene identity, and, instead,
is regulated by inductive cues from the ectoderm (Rivlin, 2001).
The central role of inductive cues is illustrated in the following
model. Patterning of the adult musculature begins in the embryo with the segregation of the progenitors for the adult myoblasts. The resulting pattern of adult muscle progenitors depends autonomously on the HOX genes expressed in the
mesoderm. While the number and arrangement of these progenitors differs most notably
between the thoracic and abdominal segments, no
differences are observed between T2 and T3. During the larval
period, the progenitors divide to produce highly divergent
patterns of adult myoblasts in T2 and T3. By the
start of the third larval instar, the population of adult
myoblasts undergoes diversification with the appearance
of the IPs that prefigure the patterning of several of the
larger muscles found in the adult thorax. While the bulk of the adult myoblasts continue to proliferate, the IPs exit the cell cycle. Analysis of Ubx
mutants suggests that myoblast proliferation and diversification
are influenced by the segmental identity of the
ectoderm. In the embryo, the patterning and differentiation of
larval muscles is independent of innervation. Indeed, the genesis of the larval muscles is completed prior to the arrival of the axons. During
adult development, innervation appears to play little or
no role in establishing the thoracic muscle pattern. However,
innervation plays an important role in the proper
differentiation and maintenance of the adult muscles that
arise during metamorphosis. The central role of innervation
in adult muscle development has largely been demonstrated
by studies in lepidopterans. In the moth, innervation regulates
the proliferation and survival of nuclei in larval abdominal
muscles that serve as a scaffold for the adult-specific
muscles. In the fly, while denervation does not affect the
patterning of the IFMs, it does result in a lack of muscle
growth. Similarly, denervation
results in the reduced growth of another adult
muscle, the male-specific muscle in A5. However, unlike that observed for the IFMs, the biochemical differentiation and establishment of attachment
points, as well as the size of the male-specific muscle,
are dramatically altered in the absence of innervation.
It is likely that the maintenance of homeotic muscle
in the HT3 of the Ubx mutants is, in part, determined by
cues acquired from the transformed central nervous
system (CNS). In fact the appearance of tubular IFMs in HT3 may represent the status of IFMs that lack proper innervation during development.
In conclusion, it appears that inductive cues play a
key role in the development of adult muscle in all insects.
However, at this point little is known about the nature
of the ectodermally derived cues that influence adult
myogenesis. By clearly defining the central role of inductive
cues in the patterning of Drosophila's adult thoracic
muscles the results outlined above should have a
positive influence on future efforts to identify these cues (Rivlin, 2001).
Hox genes specify the different morphologies of segments along the anteroposterior axis of animals; how these genes control complex segment
morphologies is not well understood. This study investigates how the Hox gene Ultrabithorax controls specific differences between the bristle patterns of the second and third thoracic segments (T2 and T3) of Drosophila melanogaster. Ubx blocks the development of two particular bristles on T3 at different points in sensory organ development. For the apical bristle, a precursor is singled out and undergoes a first division in both the second and third legs, but in the third leg further differentiation of the second-order precursors is blocked. For the posterior sternopleural bristle, development on T3 ceases after proneural cluster initiation. Analysis of the temporal requirement for Ubx shows that in both cases Ubx function is required shortly before bristle development is blocked. It is suggested that interactions between Ubx and the bristle patterning hierarchy have evolved independently on many occasions, affecting different molecular steps. The effects of Ubx on bristle development are highly dependent on the context of other patterning information. Suppression of bristle development or changes in bristle morphology in response to endogenous and ectopic Ubx expression is limited to bristles at specific locations (Rozowski, 2002).
In wild-type flies, apical and sternopleural bristles appear only on
the second thoracic segment, but in Ubx mutant flies, they
also develop on the third thoracic segment. Thus, directly or
indirectly, Ubx normally blocks the development of these
bristles on the third leg. To determine when the block of development
occurs for the apical and the posterior sternopleural macrochaete, early stages of sensory organ development were monitored (Rozowski, 2002).
The precursors for different types of sensory organs can be
distinguished by the combination of markers they express. A reporter construct in the neuralized gene, neu-lacZ, is
expressed in all sensory organ precursors. The homeodomain protein Cut
is expressed only in the precursors for external sensory organs, and
thus allows for distinguishing between external and internal sensory
organs (e.g., the chordotonal organs). Among the external sensory
organs, chemosensory bristle precursors can be distinguished from
mechanosensory precursors by their expression of the paired box protein
Pox-neuro. In the folded disc epithelium, the individual
mechanosensory precursors can be identified by their location in
relation to markers of joint territory and other landmarks in the
discs. When the legs elongate, the identity of the sensory organ
precursors can be assessed by comparing the position of labeled cells
with that of sensory organs in the adult leg segments. In the leg disc, the first bristle precursors develop during the third larval instar. These early segregating precursors include precursors for the tarsal
chemosensory bristles, and for a few of the largest mechanosensory bristles in the proximal leg, including the precursor for the posterior
sternopleural macrochaete. Precursors for the apical and preapical
bristles appear at about the time of puparium formation, and can be
detected reliably in the white prepupa. At this time chemosensory precursors also appear in the tibia. Precursors for the smaller mechanosensory bristles are not formed until 8-12 h after puparium formation (Rozowski, 2002).
Using sensory organ markers, it was found that sensory organ development is
blocked at different points for the apical and for the posterior
sternopleural bristles. In white prepupae, no precursor cell appears in
the third leg at the position corresponding to that of the
sternopleural precursor in the second leg. However, apical bristle precursors
are present on both the second and third legs. It is concluded that Ubx blocks some event leading to the
specification of the posterior sternopleural bristle
precursor, but that Ubx does not inhibit formation of the apical bristle precursor on T3 (Rozowski, 2002).
In the two cases studied, Ubx acts at different points in the hierarchy
of sensory organ development. Development of the posterior sternopleural bristle is intercepted in midthird larval instar, shortly
after initiation of a sternopleural proneural cluster. Ubx
function is required at and restricted to this phase. Development of
the tibial apical bristle is intercepted at 4.5-5 h APF, at the
second-order precursor stage. However, the requirement for Ubx
is not limited to this phase of sensory organ development. To suppress
the apical bristle, Ubx function is required both in the
second-order precursor cells, and prior to mother cell specification,
possibly in the proneural cluster. Ubx accomplishes apical
bristle suppression on T3 by additive mechanisms which are temporally separated (Rozowski, 2002).
It has been suggested that the placement of segment-specific
sensory organs would likely be executed by the interaction of Hox genes
with the discrete regulatory elements of the AS-C complex that
integrate prepattern factors to locate individual proneural clusters. The results presented here suggest that no exclusive
preference has been given to this mechanism. For both apical and
sternopleural bristles, a proneural cluster is initially specified but
later developmental steps are blocked (Rozowski, 2002).
It is suggested that interactions between homeotic genes and sensory organ
development have evolved independently on many occasions, affecting
different molecular steps. No evidence has been found for a potential
constraint in respect to mechanisms of bristle suppression. Evolution
appears to have fixed a specific interaction between Ubx and
the developmental pathway of individual bristles, not affecting other
morphological features (Rozowski, 2002).
Ubx blocks the development of some bristles on
the third thoracic segment (sternopleural bristles, edge
bristle, apical bristle). It alters the morphology of others
(preapical bristle, ventral basitarsal row). Yet other bristles are
indistinguishable on the second and third thoracic segments, which
suggests that they are unaffected by Ubx. It is not likely
that the proneural genes or later acting genes controlling bristle
development provide this specificity, for all of these mechanosensory
bristles express the same subset of known neural markers (Rozowski, 2002).
The differences in Ubx action might in principle result from
the differential expression of Ubx in different bristle lineages within
the T3 segment. Spatiotemporal regulation of this type allows
Ubx to define the specific segment morphologies of both third
thoracic and first abdominal segments during larval development, and instructs the distribution of trichomes during pupal leg development (Rozowski, 2002).
However, the data suggest that the regulation of Ubx levels within the
third leg disc does not play a role in eliciting the differential
response of sternopleural, apical, and preapical bristles. Ubx levels
are similar in the first- and second-order precursors of the apical and
preapical bristles. Moreover, the ectopic expression of Ubx
does not eliminate their specific responses even though it imposes
essentially the same profile of Ubx expression on a number of domains
from which different bristles will arise. This has been seen in the
regime of ectopic Ubx expression directed by the dpp-Gal4
driver, which results in the suppression of the apical but not the
preapical bristle. Similarly ubiquitous ectopic expression of Ubx in T2 during the last 24 h of the third larval instar consistently suppresses only the sternopleural bristle precursor, and not any of the seven other bristle precursors segregating at this time in the periphery of the leg disc (Rozowski, 2002).
Other factors that are differentially distributed in the disc
provide bristles with individual identities, and so restrict Ubx action. Leg patterning is already well advanced at the
late larval and early pupal stages when Ubx intercepts bristle
development. Numerous molecular markers, many of them transcription
factors, demonstrate the subdivision of the proximodistal axis into a
number of domains. Prospective joint territories are marked by complex gene expression patterns. Decapentaplegic (Dpp) and Wingless (Wg)
signaling pathways are active in dorsal and ventral leg territories, respectively. Any one of these factors may act combinatorially with Ubx to control the targets that modify bristle development (Rozowski, 2002).
It has been shown that wg null clones induced on the
ventral side of the leg eliminate the apical bristle and result in the
development of a second preapical bristle. Conversely, dpp
null clones on the dorsal side of the leg eliminate the preapical bristle and cause the formation of a second apical bristle. Clones that
constitutively activate the Wg pathway (shaggy null clones) can result in ectopic apical bristle formation on the dorsal side of
the leg next to the wild-type preapical bristle. This demonstrates an intimate link between dorsal or ventral
cell identity and sensory organ patterning and identity. However, it
cannot be concluded from these results that wg or dpp are
directly involved in modulating Ubx effects on bristle development (Rozowski, 2002 and references therein).
The idea that individual macrochaetes have distinct molecular
identities is supported by the observation that many have unique morphologies. In some cases, this is further documented by the branching pattern of their associated neurons in the leg neuromere. For
example, whereas most mechanosensory bristles show either anterior or
posterior branching in the neuromere, the apical bristle has a C-shaped
branching pattern extending into both the anterior and posterior
neuromere. These distinct molecular identities
are constituted by or derived from the fine-patterning of the leg that
is generated during larval and pupal stages (Rozowski, 2002).
It is proposed that the specific effects of Ubx on sensory organ
development depend on these additional factors. With this in mind, one can comprehend how a single homeotic gene can specify the complex differences in
sensory organ distribution and sensory organ morphology that characterize the legs and other body surfaces. The generic sensory organ program is unresponsive to the homeotic protein on its own; only in the context of other patterning information is an instruction conveyed. This allows specific responses to the Hox gene to be
elicited locally, affecting one or a small group of sensory organs, and
not all organs of the same class throughout the segment (Rozowski, 2002).
Genetic analysis shows that Engrailed has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. VP16En, a form of En that has its repressor domain replaced by the activation domain of VP16, has been used to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. The intermediate repressor activity has been identified as being encoded by sloppy paired 1 and 2 and bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, genetic evidence is provided that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. It is suggested that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so (Alexandre, 2003).
The most unexpected aspect of these results is that, in abdominal segments,
the Hox proteins Ubx and Abd-A are involved in repression by En. In formal
genetic assays, Ubx and Abd-A can substitute for Exd in helping En act on
negative targets. In the absence of Ubx, Abd-A and Exd, En can no longer
repress target genes. By contrast, two other Hox proteins (Antp and Abd-B)
appear not to be involved in En function. Antp does
not help En repress targets in vivo even though its homeodomain differs from
that of Abd-A at only five positions. Likewise, Abd-B, a more distantly
related Hox protein, is also unlikely to participate in En function. It is concluded that the role of Ubx and Abd-A in repression by En is
specific (Alexandre, 2003).
How could ectopic Ubx or Abd-A allow En to repress targets in the absence
of Exd? It could be that this is mediated by wholesale transformation of
segmental identity [although such transformation would have to be
exd/hth-independent. Alternatively, Ubx and Abd-A could have a more
immediate involvement in En function. One can envisage that they could
regulate an as yet unidentified corepressor of En (although such regulation
would not require Exd). Alternatively, and more speculatively, Ubx and Abd-A
could serve as cofactors themselves in regions of the embryo where Exd levels
are low. Again, molecular analysis of negative targets will be needed to
discriminate these possibilities (Alexandre, 2003).
Homeotic genes have not been previously implicated in En function despite
many years of genetic analysis of the Bithorax complex. It is suggested that the
role of Ubx and Abd-A in En function has been overlooked previously because,
in the absence of these two genes, Exd is upregulated in the presumptive
abdomen and thus takes over as a repression cofactor. However, the present
results establish that homeotic genes do participate in the segmentation
cascade and link two regulatory networks previously thought to be
independent (Alexandre, 2003).
The mechanisms underlying the evolution of morphology are poorly understood. Distantly related taxa
sometimes exhibit correlations between morphological differences and patterns of gene expression, but
such comparisons cannot establish how mechanisms evolve to generate diverse morphologies.
Answers to these questions require resolution of the nature of developmental evolution within and
between closely related species. The detailed regulation of the Hox gene
Ultrabithorax patterns trichomes on the posterior femur of the second leg in Drosophila melanogaster. Evolution of Ultrabithorax has contributed to divergence of this feature among closely related
species. The requirement of Ubx in patterning trichomes on the posterior second femur was tested by generating clones of cells that lack the ability to produce Ubx protein. When these clones are produced in naked cuticle, they differentiate trichomes; thus, Ubx is needed to repress trichomes in the region. Flies carrying three copies of Ubx have significantly more naked cuticle than sibling flies carrying only one copy. Expression of high levels of Ubx during pupal development represses trichomes on most of the posterior second femur. Ubx is expressed in a proximal-distal gradient, with high levels proximally. In all of the three species studied (D. melanogaster, D. simulans, and D. virilis) most of the posterior third femur lacks trichomes and Ubx is expressed at high levels. D. virilis, in contrast to the other two species, has no naked cuticle and expresses Ubx at levels far below those seen in the posterior third leg. Inter-species crosses reveal that the cis-regulatory regions of Ultrabithorax, and not the protein itself, appear to have evolved to express Ubx at different levels (Stern, 1998).
This study provides experimental evidence that cis-regulatory evolution is one way in which conserved
proteins have promoted morphological diversity (Stern, 1998).
The developmental mechanisms that regulate the relative size and shape of organs have remained obscure despite almost a century of interest in the problem and the fact that changes in relative size represent the dominant mode of evolutionary change. This study investigates how the Hox gene Ultrabithorax instructs the legs on the third thoracic segment of Drosophila to develop with a different size and shape from the legs on the second thoracic segment. Through loss-of-function and gain-of-function experiments, it has been demonstrated that different segments of the leg, the femur and the first tarsal segment, and even different regions of the femur, regulate their size in response to Ubx expression through qualitatively different mechanisms. In some regions, Ubx acts autonomously to specify shape and size, whereas in other regions, Ubx influences size through nonautonomous mechanisms. Loss of Ubx autonomously reduces cell size in the T3 femur, but this reduction seems to be partially compensated by an increase in cell numbers, so that it is unclear what effect cell size and number directly have on femur size. Loss of Ubx has both autonomous and nonautonomous effects on cell number in different regions of the basitarsus, but again there is not a strong correlation between cell size or number and organ size. Total organ size appears to be regulated through mechanisms that operate at the level of the entire leg segment (femur or basitarsus) relatively independently of the behavior of individual subpopulations of cells within the segment (Stern, 2003).
Ubx appears to regulate the final size and shape of the third pair of legs via different mechanisms in different regions of the leg. The most obvious difference is between regulation of femur and basitarsal length, which appears mainly to involve nonautonomous regulation between all the cells of the segment, and the growth of the most proximal femur, which appears to involve autonomous influence of Ubx. In addition, the timing of these controls appears to be different. Ubx influences proximal femur shape after pupation, since the proximal femur shape could be mimicked by overexpressing Ubx in the second leg during pupal development. In contrast, Ubx is required between 24 h AEL and pupation to influence leg length, since clones were induced between 24 and 72 h AEL and overexpression of Ubx in the pupal period does not influence the length of the second leg (Stern, 2003).
The nonautonomous effect of loss of Ubx function on leg length appears to reflect a mechanism of size control whereby cells in the adjacent anterior and posterior compartments are able to communicate information about the length of the leg segment. This communication may involve signaling molecules or it may be entirely mechanical, for example, involving the detection of tension across an epithelium. Whatever this mechanism is, the truly confounding aspect of this nonautonomy is that all of the cells in the leg segment, including all of those that still express Ubx, respond to the minority of cells that have lost Ubx expression, and together they reduce leg length to the size of a leg in which none of the cells express Ubx. In other words, the absence of Ubx from some cells of the leg segment appears dominant to the expression of Ubx in the remaining cells for leg length. This does not appear to be the case for leg width (Stern, 2003).
Loss of Ubx in clones has only a small effect on femur width when the clones are found in the posterior compartment. This result is interpreted to mean either that cells respond autonomously to the expression of Ubx in determining leg width, and therefore only a small reduction could be detected in width caused by a minority of cells that had lost Ubx expression, or that the expression of Ubx in the majority of cells is largely dominant to its absence from a minority of cells, particularly if the minority is in the anterior compartment (Stern, 2003).
The loss of Ubx in cells of the ventral basitarsus causes the most surprising effect: the production of ectopic bristles, a dramatic increase in the width of the basitarsus, and a nonautonomous decrease in bristle number in the adjoining bristle row of the posterior compartment. This is the only case in which the resulting phenotypes cannot be construed as a homeotic transformation from a T3 to a T2 leg. The location of these clones, in the most ventral cells of the anterior compartment, suggests that Ubx may influence wingless (wg) signaling during the development of the T3 basitarsus, because wg is expressed in this domain and is required for driving leg growth and patterning. This view is supported, but not proven, by the observation that loss of wg function during larval development causes a similar widening of the basitarsal segments of all three pairs of legs. Tarsal widening is observed in flies carrying a temperature-sensitive allele of wg; flies were shifted to the restrictive temperature between 88 and 110 h after egg laying. This phenomenon can be seen in a first leg basitarsus. However, no differences were detected in expression of wg protein between the T2 and T3 legs and no obvious change in wg expression was detected in Ubx null clones in developing T3 leg discs (Stern, 2003).
One model consistent with the current observations is that Ubx is required early to upregulate a signaling pathway located in the ventral row of tarsal cells, but that it is required later to repress the same pathway. This two-step model is favored because uniform removal of Ubx from the entire basitarsus from the earliest stages of development (for example, in flies carrying the allelic combination abx1bx3pbx1/Df Ubx) does not cause basitarsal widening or the production of ectopic bristles, but instead transforms the T3 basitarsus to a T2 basitarsus (Stern, 2003).
An alternative interpretation is that Ubx is required to upregulate wg throughout T3 tarsal development. Late removal of Ubx may then cause a drop in wg leading to tarsal widening and the development of ectopic bristles. wg function in the basitarsus is known to be concentration dependent, with more ventral bristle fates requiring higher levels of wg activity. It is therefore worth noting that the ectopic bristles have a thick shape similar to row 8 bristles. This ectopic bristle row may therefore be interpreted as a lateral transformation caused by a slight reduction in wg level in the most ventral cells (Stern, 2003).
Whatever the true model of this function of wg may be, the nonautonomous effects observed are similar to those reported for Ubx control of wg expression in the haltere and doublesex control of wg and decapentaplegic (dpp) functions in the genital imaginal disc. However, whereas control of wg and dpp in the haltere and genital discs causes a dramatic alteration in growth patterns, the effect on the basitarsus is of much smaller magnitude. Thus, Ubx may have only a small effect on this signaling process in the basitarsus. This suggests the intriguing possibility that the major determinants of organ growth, wg and dpp, are regulated by a panoply of patterning genes that subtly control the function of these signaling molecules leading to slight alterations in organ shape in Drosophila. wg has been shown to be required to establish distal elements of the leg before 84 h AEL, but that after this time, wg is apparently required only for patterning ventral elements and also to determine the correct shape of the leg segments. It is therefore possible that other genes, such as Ubx, influence wg action in different ways if they act at different times during leg development. The wg and dpp signaling pathways might therefore commonly play a dual role of controlling proliferation and patterning of the major proximal-distal elements early during development and then contribute to more subtle effects on organ size and shape later during development (Stern, 2003).
One important caveat about the effects of clones on leg shape is that clones in the legs always run in a proximal-distal direction along the length of the leg. Therefore, while clones tend to run along the long axis of the leg and cross leg-segment boundaries, they never straddle the circumference of the leg. It may be worth considering the effects of Ubx clones on leg shape in this light. Ubx clones have a strong and nonautonomous effect on leg length and a weak and apparently autonomous effect on leg width. One possibility is that leg length is regulated either by cells at the boundaries of leg segments or by a continuous length of epithelium within a segment and that width is regulated by a contiguous circle of cells around the leg. The observed effects on leg length still require some kind of nonautonomous communication between cells throughout the leg. However, a similar mechanism may act on leg width but may have gone undetected because of the inability to eliminate Ubx from a contiguous ring of cells around the leg (Stern, 2003).
Cell number and cell size do not appear to be related in any obvious way to total leg size. These observations are consistent with a growing body of evidence that organ sizes are determined without regard to the precise regulation of cell number or cell size. These observations suggest that organ size is not directly determined by mechanisms that control cell number or size, although Ubx clearly does affect cell size and number both in the legs, as shown, and in the haltere. It is more likely that cell size and number are sufficiently plastic to allow regulation of sizes and numbers to satisfy the true, and unknown, regulator of size (Stern, 2003).
Ubx appears to modify leg shape through a variety of mechanisms and influences different parts of a single leg through different growth mechanisms. From an evolutionary perspective, this observation suggests that the control of leg shape and size by Ubx has evolved by the independent co-option of Ubx transcriptional regulation by different mechanisms of growth control in different parts of a single leg. This observation is also consistent with recent studies of the genetic architecture of variation in organ shape, which have invariably found that variation in organ shape is influenced by a large number of loci each of small effect. Patterns of allometry within and between species provide correlative evidence and have led to speculations that developmental mechanisms constrain or limit the patterns of natural variation. However, this study combined with the results of selection experiments suggests that the subcomponents of individual organs are regulated by developmental mechanisms that possess at least some independence and are therefore amenable to change by natural selection. The striking patterns of allometry observed in the natural world are therefore more likely to represent the consequence of natural selection for these particular shapes rather than mechanistic limitations on what is possible (Stern, 2003).
The proteins that regulate developmental processes in animals have
generally been well conserved during evolution. A few cases are known where
protein activities have functionally evolved. These rare examples raise the
issue of how highly conserved regulatory proteins with many roles evolve new
functions while maintaining old functions. This was investigated by
analyzing the function of the 'QA' peptide motif of the Hox protein
Ultrabithorax (Ubx), a motif that has been conserved throughout insect
evolution since its establishment early in the lineage. The QA motif was precisely deleted at the endogenous locus via allelic replacement in Drosophila
melanogaster. Although the QA motif was originally characterized as
involved in the repression of limb formation, it was found to be highly
pleiotropic. Curiously, deleting the QA motif had strong effects in some
tissues while barely affecting others, suggesting that QA function is
preferentially required for a subset of Ubx target genes. QA deletion
homozygotes had a normal complement of limbs, but, at reduced doses of
Ubx and the abdominal-A (abd-A) Hox gene, ectopic
limb primordia and adult abdominal limbs formed when the QA motif was absent.
These results show that redundancy and the additive contributions of
activity-regulating peptide motifs play important roles in moderating the
phenotypic consequences of Hox protein evolution, and that pleiotropic peptide
motifs that contribute quantitatively to several functions are subject to
intense purifying selection (Hittinger, 2005).
One of the most provocative cases of selector protein evolution correlates the acquisition of limb repression capacity by the central class Hox selector protein Ultrabithorax (Ubx) with the reduction of abdominal limb number in insects. Whereas insect Ubx possesses strong limb repression capacity when ectopically expressed in Drosophila melanogaster, crustacean (Artemia francisana) and onychophoran (Acanthokara kaputensis) Ubx do not. Sequences in the C terminus of Ubx are responsible for much of this functional divergence. A. franciscana Ubx possesses putative casein kinase II sites that modulate activity, whereas all insect Ubx orthologs contain the highly conserved C-terminal 'QA' motif required for full Ubx repression activity. This QA motif is capable of conferring limb repression activity when grafted onto onychophoran Ubx (Hittinger, 2005).
The sufficiency of the QA motif to confer limb repression capacity suggests that its acquisition during early insect evolution could have played an important role in the evolution of insects lacking adult abdominal limbs. However, little is known about the role of the QA motif in normal development. For example, is the QA motif required for abdominal limb repression in insects? Is this motif dedicated to limb repression, or is it pleiotropic? What would be the phenotypic consequence of removing such a conserved part of an integral patterning gene (Hittinger, 2005)?
To characterize the genetic and phenotypic role of the QA peptide motif of Ubx, this motif was precisely deleted at the endogenous Ubx locus via allelic replacement in D. melanogaster. The effects of deleting the QA motif were strong in some tissues but barely detectable in others. This finding of differential pleiotropy suggests that peptide motifs in selector proteins can conditionally modulate selector activity and need not be uniformly pleiotropic across all tissues. The requirement for the QA motif for limb repression was found to be dose dependent and partially redundant with the Abdominal-A (Abd-A) Hox protein, suggesting that redundancy and the additive contributions of peptide motifs play important roles in modulating the phenotypic consequences of selector protein evolution (Hittinger, 2005).
The genetic deletion of the QA motif of Ubx produced a surprisingly subtle
but highly pleiotropic homozygous phenotype. The QA motif
is partially redundant with Abd-A in A1 for limb repression, is one of several
motifs within Ubx that quantitatively affect Ubx activity, and that
reducing Ubx or Abd-A levels uncovers a requirement for the QA motif in limb
repression. The QA motif is preferentially required for a subset of
Ubx-regulated developmental processes, a characteristic that is termed here differential pleiotropy. The conservation of the QA motif throughout the insect lineage suggests some of its many functions are crucial for the proper patterning and fitness of insects. These findings offer a conceptual framework for
understanding how pleiotropy, redundancy and selection interact to guide the
evolution of selector proteins and the morphology they govern (Hittinger, 2005).
Selector genes encode proteins that regulate many downstream target genes,
often in several different tissues. Therefore, coding sequence mutations are expected to be highly pleiotropic and generally deleterious, especially when the selector is expressed in several different tissues. It is clear that regions of selector proteins such as the
DNA-binding domain are likely to affect protein activity uniformly wherever
the protein is expressed. However, it is uncertain to what extent peptide
motifs are preferentially used in the regulation of a subset of selector
targets. The UbxDeltaQA allele
allowed a genetic test of whether the QA motif is uniformly pleiotropic
or differentially pleiotropic. The reversal of the genotypic series for
UbxDeltaQA/UbxDeltaQA and Ubx–/Ubx+ demonstrates a differential
requirement for QA function between these tissues (Hittinger, 2005).
Hox selector proteins, such as Ubx, may accomplish their diverse genetic
and regulatory functions by using distinct peptide motifs for the regulation
of subsets of target genes. Ubx is expressed throughout development in many
tissue types, suggesting that distinct activation and repression motifs
exist. In an accompanying study, Tour (2005) describes at least three motifs
that quantitatively and differentially affect the expression of specific
target genes when ectopically expressed, suggesting that Ubx contains several
differentially pleiotropic peptide motifs that influence the expression of Ubx
target genes. The YPWM motif interacts with Exd and is
differentially pleiotropic at least in part because nuclear Exd is not present
in all regions where Ubx is active and required.
Detailed studies on the derived Hox protein Fushi Tarazu (Ftz) have also
demonstrated that beetle (Tribolium castaneum) Ftz has distinct
homeotic and segmentation functions that are differentially mediated by a YPWM
motif and a nuclear receptor box or 'LXXLL' motif, respectively
(Lohr, 2001; Lohr, 2005). By
contrast, use of different peptide motifs on different targets may not be a
necessary feature of selector proteins dedicated to one cell type, such as the
mouse photoreceptor selector Crx, or dedicated to either activation or repression,
such as the posterior compartment selector Engrailed (Hittinger, 2005 and references therein).
The QA motif is not strictly necessary for limb repression in A1 at any
stage of development because of the additive roles played by other peptide
motifs in Ubx and because it is partially redundant with the Hox protein Abd-A. Extensive limb
derepression was oberved in A1 in embryos and adults when both the QA motif was absent and
when the Ubx and abd-A doses were reduced but not when
either was manipulated singly. The partial redundancy of the Ubx and Abd-A in
limb repression is mechanistically explained by their direct repression of the
Dll limb primordia enhancer through the same binding site. The
absence of ectopic limb primordia or limbs on the more posterior abdominal
segments of UbxDeltaQA/Ubx–
abd-A– flies suggests that the higher level and broader
expression of Abd-A are sufficient to repress limb formation in more posterior
segments (A2-A7) (Hittinger, 2005).
Compared with the relatively rapid turnover of cis-regulatory
elements, the evolution of selector protein function appears to be a rare
occurrence, owing, at least in part, to the pleiotropic consequences of
mutations in protein coding regions. By
contrast, many cis-regulatory elements have a modular architecture
and mutations in these elements can more easily adjust the expression of a
single gene in a single tissue. Analogously, the differential pleiotropy
observed for the QA motif may provide a degree of modularity to some selector
proteins. If natural selection can quantitatively alter a specific trait by
modifying selector protein sequence and accrue minimal pleiotropic fitness
trade-offs in other tissues, this route might be taken if the fitness gains
are great compared with any offsets, if genetic suppressors arise, or if it is
the most readily available path (Hittinger, 2005).
Redundancy may further limit the number of functions subject to intense
purifying selection. For example, if a selector protein performs n
functions but n-1 are redundant with the function of other
selectors, natural selection may be free to modulate the nth function
through coding changes with limited effects on the other traits. The two most
extreme cases of the evolution of Hox protein function have involved Hox genes
that were co-opted for other regulatory functions. The
ancestral Hox3 and Ftz expression domains both overlapped with multiple Hox
proteins, suggesting they were at least partially redundant with
neighboring Hox genes during their co-option. It is proposed that the rare
instances of the evolution of selector protein function tend to be facilitated
when a combination of redundancy and the differential pleiotropy of peptide
motifs alleviates the constraints on selector protein evolution (Hittinger, 2005).
There is an intuitive but misleading contradiction between the
UbxDeltaQA/UbxDeltaQA phenotype and the macroevolutionary time-span over which the motif has been
conserved. The QA motif has been conserved in all insects, but the phenotype
observed in
UbxDeltaQA/UbxDeltaQA
D. melanogaster affected traits that vary between insects, not between
insects and other arthropods. This suggests that, as Ubx has acquired
different genetic targets in different insect lineages, so has the QA motif. Some of the phenotypic effects of deleting the QA motif are mitigated by the contributions of other motifs, redundancy with Abd-A, and differential pleiotropy. Yet, it has also been argued that these same forces could facilitate the evolution of selector function under the right combination of circumstances. Why, then, is the QA motif still present in all insect orders studied (Hittinger, 2005)?
Sudden variation in all of the traits governed by the pleiotropic QA motif would probably not be tolerated in a natural, competitive environment. Even
though UbxDeltaQA/UbxDeltaQA flies are viable and fertile and have a modest phenotype from a developmental
perspective, natural selection acts on genetic variation that has a selection
coefficient as small as the inverse of twice the effective population size. For insects, which are likely to have effective population sizes of 105 to
106, the difference between the production of an average of one
fewer offspring out of a million literally makes the difference between
variation that is tolerated and that which is selected against. Therefore,
despite a turnover of targets and traits governed, pleiotropic peptide motifs
that subtly modulate selector protein function can experience consistent purifying selection that preserves them across vast periods of time (Hittinger, 2005).
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).
Different proliferation of neuroblast 6-4 (NB6-4) in the thorax and abdomen produces segmental specific expression pattern of several neuroblast marker genes. NB6-4 is divided to form four medial-most cell body glia (MM-CBG) per segment in thorax and two MM-CBG per segment in abdomen. Since homeotic genes determine the identities of embryonic segments along the A/P axis, whether temporal and specific expression of homeotic genes affects MM-CBG patterns in thorax and abdomen was ivestigated. A Ubx loss-of-function mutation was found to hardly affect MM-CBG formation, whereas abd-A and Abd-B caused the transformation of abdominal MM-CBG to their thoracic counterparts. In contrast, gain-of-function mutants of Ubx, abd-A and Abd-B genes reduced the number of thoracic MM-CBG, indicating that thoracic MM-CBG resembled abdominal MM-CBG. However, mutations in Polycomb group (PcG) genes, which are negative transregulators of homeotic genes, did not cause the thoracic to abdominal MM-CBG pattern transformation although the number of MM-CBG in a few per-cent of embryos were partially reduced or abnormally patterned. These results indicate that temporal and spatial expression of the homeotic genes is important to determine segmental-specificity of NB6-4 daughter cells along the anterior-posterior (A/P) axis (Kang, 2006).
In the Drosophila embryonic central nervous system (CNS),
about 30 glia are produced in a stereotyped pattern in each
hemisegment, and certain of these glia are
arranged in different patterns between segments along the
A/P axis. Thus, it is important to understand how the regional specificity of
certain glia is determined and maintained during nervous
system development. repo is essentially required for the
differentiation and maintenance of glia. Moreover, some of these repo expressing cells, MM-CBG, show different patterns along the A/P
axis. In the present study, MM-CBG pattern abnormalities were examined in BX-C and its negative transregulator, PcG mutant embryos (Kang, 2006).
The data showed that Ubx loss-of-function mutation did
not cause the homeotic transformation of the abdominal
MM-CBG pattern to the thoracic one. However, a loss-of-function
mutation in the abd-A gene caused the transformation
of abdominal MM-CBG into a thoracic pattern. Abd-B
mutant embryos also showed transformation of MM-CBG
in its functional domain. These results indicate that unlike
Ubx, abd-A and Abd-B genes are involved in the segment-specific
MM-CBG pattern formation. The role of BX-C on
MM-CBG formation was confirmed using gain-of-function
BX-C mutation. Ectopic expression of BX-C with sca-GAL4/UAS system caused thoracic MM-CBG to follow the abdominal pattern of MM-CBG. Unlike the result
shown in Ubx loss-of-function mutant embryos, four thoracic
MM-CBG were frequently reduced to two or three
MM-CBG in Ubx gain-of-function mutant embryos, suggesting
that Ubx might be involved in MM-CBG pattern
formation. The Abd-A and Abd-B proteins driven by sca-GAL4 driver changed the thoracic MM-CBG pattern to the abdominal one. It was suggested that Abd-A and Abd-B
proteins repress the proliferation of MM-CBG through inhibition
of CycE in the abdomen, which makes two MMCBG
per abdominal segment and four MM-CBG per thoracic segment (Kang, 2006).
PcG mutation causes the ectopic expressions of abd-A
and Abd-B genes in the anterior of their functional domains. It is presumed
that the ectopic thoracic expressions of abd-A and
Abd-B genes would transform thoracic MM-CBG to an
abdominal one as shown in the gain-of-function BX-C mutation,
because the thoracic pattern of the epidermis and
central nervous system are transformed to the abdominal
segments in these two mutants. However, PcG mutant embryos
showed little evidence of an abnormal MM-CBG pattern in the thorax because most PcG mutant embryos showed wild type thoracic MM-CBG pattern. This was
confirmed using a gcm enhancer trap line. A gmc driven reporter was expressed only in the MM-CBG of the abd-A domain. Although
Pc zygotic, esc and pho maternal effect mutations
caused the ectopic expressions of abd-A and Abd-B in the
CNS from head to tail, the anterior boundary of gcm-lacZ expression did not move to more anterior segments. In addition, thorax-specific eg expression pattern was unchanged in PcG mutant embryos (Kang, 2006).
These observations indicate that temporal and spatial homeotic
gene expression is important in MM-CBG pattern
formation. The homeotic gene products driven by sca-
GAL4 driver are present in the neuroectoderm from embryonic
stage 8, which clearly changes the thoracic MM-CBG
pattern. However, derepressed BX-C gene products caused
by PcG mutations do not affect MM-CBG pattern. Ubx, abd-A and Abd-B genes begin to be weakly misexpressed from stage 11 and shows strong ectopic expression at stage 13 in Pc and esc mutant embryos. In wild type embryos MM-CBG appears to proliferate once between stage 11 and 12, and
become four cells per segment in the thorax, while there is
no cell division of MM-CBG in the abdomen because Abd-
A and Abd-B proteins repress CycE expression. So PcG mutants seems to
cause the ectopic expression of the BX-C genes after MMCBG are already determined to be prolifered in the thorax. Early segment-specific commitment of NB6-4 progeny cells also supports this conclusion. When BX-C genes are overexpressed from stage 10 using eg-GAL4, thoracic MM-CBG pattern was not changed. Taken together, temporal and spatial expression of the homeotic genes is important to determine segmental-specificity of MM-CBG along the anterior-posterior (A/P) axis (Kang, 2006).
Hox genes control regional identity along the anterior-posterior axis in various animals. Each region contains morphological characteristics specific to that region as well as some that are shared by several different regions. The mechanism by which one Hox gene regulates region-specific characteristics has been extensively analyzed. However, little attention has been paid to the mechanism by which different Hox genes regulate the same characteristics in different regions. This study shows that two Hox genes in Drosophila, Sex combs reduced and Ultrabithorax, employ different mechanisms to achieve the same out-put, the absence of sternopleural bristles, in the prothorax and metathorax, respectively. Sternopleural bristles are characteristics of the mesothorax, and it was found that spineless is involved in their development. Analysis of the regulatory relationship between Hox genes and spineless indicated that ss expression is repressed by Sex combs reduced in the prothorax. Since sole misexpression of ss could induce ectopic sternopleural bristle formation in the prothorax irrespective of the expression of Sex combs reduced, spineless repression appears to be critical for inhibition of sternopleural bristles by Sex combs reduced. In contrast, spineless is expressed in the metathorax independently of Ultrabithorax activity, indicating that Ultrabithorax blocks sternopleural bristle formation through mechanisms other than spineless repression. This finding indicates that the same characteristics can be achieved in different segments by different Hox genes acting in different ways (Tsubota, 2008).
This study found that three genes, Antp, ss and al, are involved in sternopleural bristle formation. In the al mutant, no appreciable Ac expression in the T2 leg disc is detected and sternopleural bristles are not formed, indicating that the requirement of al is absolute. In contrast, Ac expression is detectable in the ss mutant T2 leg disc and in the Antp mutant clones, indicating that the requirement of both ss and Antp for ac expression is not absolute. However, sternopleural bristles were never found in the ss mutant, despite the fact that Antp expression was unaffected in the ss mutant clone in the T2 leg disc. In contrast, Antp mutant cells, in which ss is expressed normally, formed sternopleural bristles. In addition, sole misexpression of ss in the T1 segment produces sternopleural bristles ectopically, while that of Antp did not. Therefore, ss appears to be necessary and sufficient for sternopleural bristle formation, while Antp appears to be insufficient and not necessarily required. Moreover, Ac expression is ectopically induced in the T1 leg disc by misexpression of ss but not of Antp and in the ss mutant T2 leg disc is very weak, highly restricted, and only transient. This indicates that ss but not Antp appears to be one of the major activators of ac expression. Taken together, ss appears to be much more fundamental for sternopleural bristle formation than Antp (Tsubota, 2008).
The initiation of ac expression coincides with the initiation of ss expression. Since al and Antp are already expressed before ac induction in the early third instar stage, the timing of ac induction may be determined by the regulation of ss expression. Interestingly, the residual Ac expression seen in the ss mutant leg disc is first observed in the mid third instar as in the wild-type leg disc. This implies that at least one additional gene (referred to as X hereafter), whose expression or function is activated at the same stage as the initiation of ss expression, may be involved in ac induction. One possibility may be a gene functioning in hormonal regulation. Nonetheless, the ability of the sole misexpression of ss to induce ectopic ac expression and sternopleural bristle formation strongly indicates that ss is much more fundamental than X (Tsubota, 2008).
The restriction of ac expression to the overlap between the ss and al expression domains indicates the importance of determining the distal limit of ss expression and the proximal limit of al expression. Analysis of clones lacking ss activity or misexpressing ss indicates that ss has a repressive activity on al expression. How can al be expressed in the overlap domain? In the overlap domain, ss represses al expression when misexpressed at high levels but does not when misexpressed at approximately endogenous levels. The level of ectopic Al expression in the ss mutant clone located in a region proximal to the normal al expression domain is lower than that of endogenous Al expression. Moreover, Al expression in the wild-type leg disc gradually decays at its proximal edges. Considering all of these observations, the following hypothesis is suggested: al expression is activated according to the proximodistal information and the proximal limit of the al expression domain may be determined by a balance between activation according to the proximodistal information and repression by ss. The activation force may dominate the repressive activity of ss in the overlapping region but may gradually decay towards the proximal edges of the al expression domain. In contrast, ss expression does not appear to be regulated by al. As with the case of al activation, it may be possible that ss is repressed according to the proximodistal information (Tsubota, 2008).
The morphological identities of the T1 and T3 segments, including the absence of sternopleural bristles, are determined by Scr and Ubx, respectively. Analyses of the T1 leg disc with Scr mutant clones and the T2 leg disc with ectopic Scr activity indicate that both ss and Antp are repressed by Scr in the T1 leg disc. In addition, there is a possibility that the expression or function of gene X is repressed by Scr. Weak Ac expression is transiently observed in the ss mutant T2 leg disc, indicating that ac expression can be weakly activated without ss activity in the presence of gene X and Antp activity. In addition, Scr does not appear to repress ac expression directly, since ectopic induction of ac by ss misexpression in the T1 leg disc was not associated with an alteration in Scr expression. If gene X is active in the T1 leg disc, sole misexpression of Antp is expected to activate ac expression at least weakly and transiently. However, no ectopic Ac expression was found upon sole misexpression of Antp. Therefore, the activity of gene X is likely to be repressed in the T1 leg disc. For evaluating the significance of these three genes on Scr-dependent inhibition of sternopleural bristle formation, the ability of ss misexpression to induce ectopic ac expression and sternopleural bristle formation without affecting Scr expression is of crucial importance. At present, whether ac expression and sternopleural bristle formation can be induced solely by ss or only in a combination of ss and Antp and/or gene X is unclear. However, ss misexpression induced Antp expression and, thus, at least ss and Antp were coexpressed upon sole misexpression of ss. As for gene X, if it is not activated by ss misexpression, the results indicate that ac expression and sternopleural bristle formation can be induced without gene X activity at least in the presence of both ss and Antp expression. In contrast, if ac expression and sternopleural bristle formation require gene X activity, ss misexpression must activate gene X. After all, the results indicate that sole misexpression of ss can fulfill at least a minimum requirement for ac expression and sternopleural bristle formation. In other words, if Scr could not repress ss expression, ac expression would be activated and sternopleural bristles would be formed irrespective of the expression and function of Antp and gene X. Therefore, Scr must repress ss expression and this appears to be a key step to block sternopleural bristle formation in the T1 segment (Tsubota, 2008).
In contrast to the T1 leg disc, strong Ss expression was observed in the wild-type T3 leg disc and it is unaltered in Ubx mutant clones. Therefore, Ubx appears to act through a mechanism unrelated to ss expression. How does Ubx function? Simultaneous expression of both ss and Antp seemed insufficient for ac expression and sternopleural bristle formation in the T3 segment, since Antp misexpression failed to induce Ac expression in the T3 leg disc, in which ss is prominently expressed. It may be possible that Ubx represses ac expression directly. Alternatively, Ubx may compromise the function of the Ss protein directly or indirectly through regulation of its downstream gene products. Another possibility is that Ubx acts through repression of gene X activity. These possibilities are not mutually exclusive with each other (Tsubota, 2008).
The occurrence of ac expression and sternopleural bristle formation in the absence of Antp activity indicates that the absence of sternopleural bristles is not the ground state. However, the number of sternopleural bristles is variable in that condition, indicating that the complete formation of sternopleural bristles is not also the ground state. Since ss misexpression experiment suggests that sternopleural bristles can be formed as long as ss is expressed, one possible aspect of the ground state may be the expression of ss and the production of at least some kind of bristles. Antp may have acquired the ability to modify this state to produce the current-type of sternopleural bristles. On the other hand, Scr may have evolved the ability to block sternopleural bristle formation by acquiring the activity to repress ss expression and Ubx by acquiring another, yet unknown function. Taken together, the current state of sternopleural bristles in all three thoracic segments appears to be the derived state (Tsubota, 2008).
The generation of morphological diversity among segmental units of the nervous system is crucial for correct matching of neurons with their targets and for formation of functional neuromuscular networks. However, the mechanisms leading to segment diversity remain largely unknown. This paper reports that the Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate segment-specific survival of differentiated motoneurons in the ventral nerve cord of Drosophila embryos. Ubx is required to activate segment-specific apoptosis in these cells, and their survival depends on Antp. Expression of the Ubx protein is strongly upregulated in the motoneurons shortly before they undergo apoptosis, and these results indicate that this late upregulation is required to activate reaper-dependent cell death. Ubx executes this role by counteracting the function of Antp in promoting cell survival. Thus, two Hox genes contribute to segment patterning and diversity in the embryonic CNS by carrying out opposing roles in the survival of specific differentiated motoneurons (Rogulja-Ortmann, 2008).
Over the course of development, clear differences emerge between thoracic and abdominal segments in the form of specialized functional networks that support regional locomotion and sensory requirements. These differences are the result of
region-specific division patterns of the neural progenitors (neuroblasts) and
of the different numbers and types of neural cells that the neuroblasts
generate. Regulation of CNS cell number is achieved through control
of cell proliferation and death, and both of these processes have been shown
to be orchestrated by Hox genes, in vertebrates and invertebrates. It has
recently also been reported that mutations in Hox genes affect locomotor
behavior in larval crawling, suggesting that they provide positional information for neurons and thus regulate the formation of neuromuscular networks that control region-specific peristaltic locomotion. The
Drosophila Hox genes Ubx and Antp regulate
segment-specific survival of two differentiated motoneurons, and may thus
pattern neuromuscular networks by regulating the segment-specific presence of
individual motoneurons. This study shows that Ubx is necessary and sufficient to induce apoptosis, and that it does so by impeding the positive effect of Antp on the survival of these cells (Rogulja-Ortmann, 2008).
The pattern of Ubx expression in the GW and NB2-4t motoneurons and
their respective lineages is interesting. In early stages, the Ubx
expression pattern conforms to its role as a homeotic gene: it is expressed
from the posterior half of segment T3 to the anterior half of A7. The levels
of Ubx protein in NB7-3 and NB2-4 and their progeny are not particularly high
at these stages, but removal of both Ubx and abdA, for
example, results in an NB7-3 lineage that is typical for T1 and T2, suggesting that Ubx determines the segmental identity
of this neuroblast. At a later developmental stage, Ubx expression is
strongly upregulated in the NB7-3 and NB2-4 progeny in a segment-specific
manner. This dynamic pattern of expression implies two roles of Ubx
in these lineages: (1) an early role in establishing tagma-specific identity
of the neuroblast, such as has already been shown for NB1-1;
and (2) a late role in inducing apoptosis of the motoneurons. Heat-shock
experiments confirm that, at least in NB7-3, it is this late upregulation of
Ubx that leads to apoptosis. NB2-4t could not be tested for the
proapoptotic function of Ubx in the heat-shock experiments. However, it is believed that Ubx plays a late role in this lineage as well: since Ubx does not play an early role in specifying the T3 identity of the NB2-4t neuroblast, this suggests that the proapoptotic function of Ubx must
be executed at a later point in development. Also, in
poxN-Gal4,UAS-GFP/UAS-Ubx embryos, the NB2-4t
lineage does not appear to be transformed into its abdominal counterpart, since
two closely positioned dorsal motoneurons were observed in thoracic segments. In
these embryos, Ubx is still capable of inducing apoptosis of the
anterior motoneuron in all thoracic segments. Taken together, these data
indicate that, also in this lineage, Ubx is needed at a late
developmental stage to induce apoptosis. Dual requirements for Hox genes have
also been described in determining thoracic bristle patterns and
in cardiac tube organogenesis. It is not clear how the late expression of
Ubx is regulated. It has been suggested that this might depend on
genes that define the differences between cell types, and it will be
interesting to see whether this is also the case for the NB7-3 neurons (Rogulja-Ortmann, 2008).
Interestingly, the late Ubx expression in NB7-3 extends to the
second thoracic segment, but in a cell-specific manner: the GW motoneuron does
not activate Ubx expression, whereas the EW interneurons do. These
findings evoke the following questions: (1) why is it that the EWs in T2 to A7
do not undergo apoptosis although they also upregulate Ubx? and (2)
what represses Ubx expression in the T2 GW (Rogulja-Ortmann, 2008)?
Regarding the first question, it is likely that the differentiation program
of the EWs creates a different cellular context in which Ubx is
unable to induce apoptosis. The results of ectopic Ubx expression
experiments support this assumption: whereas GW undergoes apoptosis, the EWs
do so very rarely, although they express Ubx at equally high levels.
In addition, GW seems to acquire the competence to undergo
Ubx-dependent apoptosis rather late, as en-Gal drives
expression strongly from earlier stages but apoptosis does not occur until
stage 15. This would suggest that the susceptibility to apoptosis of at least
some motoneurons is coupled to differentiation. The context-dependent ability
of Ubx to activate apoptosis also holds true for the NB2-4t lineage:
the anterior motoneuron is susceptible to Ubx-induced apoptosis,
whereas the posterior one is not, even when Ubx is overexpressed (Rogulja-Ortmann, 2008).
Preliminary attempts to determine at least some of the factors
contributing to apoptosis-susceptibility were not successful. For the NB7-3
lineage, abdA expression was examined and it was found that, at the onset of GW apoptosis, it is weakly expressed only in the EWs from A1 to A7, but not in
GW. However, the survival of the EWs is not impaired in abdA mutants. The cofactors homothorax (hth) and extradenticle (exd), which are known to be required for some functions of homeotic genes, were tested, but compelling evidence was obtained for their involvement in the apoptosis of these cells. Mutants of other factors that are differentially expressed in GW and EWs (numb, zfh1) were also examined, and no indication was found that any of these is involved in the differential effect of Ubx on cell survival (Rogulja-Ortmann, 2008).
Regarding the question of differential Ubx expression in NB7-3
cells of T2, this is an intriguing observation that might prove to be key in
determining the developmental signal that upregulates Ubx late in
development. One candidate for repressing Ubx expression in GW is the
gap gene hb. It is known to repress Ubx in early embryonic
development, and hb overexpression can suppress Ubx in the
NB7-3 lineage. However, hb is also necessary
to activate Antp expression and thereby specify the second thoracic
segment. In hb mutants, T2 is not present and it was thus impossible to test whether this is the factor repressing Ubx expression in GW. Other obvious candidates are the Polycomb group (PcG) proteins, well-known repressors of Hox genes. It has recently been shown that, contrary to what had been
believed for a long time, target gene repression by these proteins is not
necessarily maintained throughout development, but can be reversed in certain
developmental contexts. It is conceivable that repression by PcG proteins could be lifted in some cells (e.g. EWs in T2) and not in others (e.g. GW). However,
the question would still remain as to how the difference between the GW and EW
neurons is established specifically in this segment. Alternatively,
differential Ubx regulation might be effected via micro RNAs or
non-coding RNAs (Rogulja-Ortmann, 2008).
It was also shown that Antp is required for GW survival in all
segments, and that Ubx counteracts Antp in T3 to A7 to
induce apoptosis. Although the lower percentage of dying abdominal GWs in
Antp mutants (69%) as compared with wild type (81.2%) might indicate a
proapoptotic function of Antp in the abdomen, it is believed that this is
not the case because Antp overexpression actually reduces the amount
of abdominal GW apoptosis more than twofold.
Moreover, removing Antp function in a Ubx heterozygous
background increases GW apoptosis (both in T3 and in abdomen) in a
dose-dependent manner, and removing both Ubx and Antp results in a recurrence of GW apoptosis, albeit with low penetrance, lending further support to a pro-survival function of Antp (Rogulja-Ortmann, 2008).
It is not clear at which level these two factors interact. GW apoptosis is
inhibited both in T3 and in abdominal segments of Ubx mutants.
However, Antp expression in Ubx mutants is upregulated only
in T3, and remains low in abdominal segments, suggesting that here
Ubx does not induce apoptosis through Antp repression. In
addition, the pattern and levels of Ubx expression do not change at
all in Antp mutants, indicating that in this context Antp
does not promote survival via Ubx regulation. It is therefore proposed
that in the wild type, Antp and Ubx might compete for a co-factor or for a
target enhancer, rather than cross-regulating each other. The proapoptotic
gene rpr, which is transcriptionally activated in both GW and MNa
motoneurons, is a candidate target. In fact, several binding sites
for both Ubx and Antp were found in the enhancer of the rpr gene. It will be interesting to see whether Antp can prevent activation of the
apoptotic machinery by affecting an upstream factor or through direct
repression of rpr. The presence of several Antp binding sites in the
rpr enhancer permits such speculation, and although it awaits
experimental validation, this does suggest a model for the antagonistic
effects of Antp and Ubx on cell survival. According to this model, Ubx and
Antp compete for sites in the rpr enhancer. In cells that express
Antp at high level, this would repress rpr transcription. Ubx would
overcome repression by Antp and activate rpr transcription to induce
apoptosis. Such positive Hox regulation of rpr has already been
demonstrated in shaping segment borders in Drosophila embryos, where
Deformed directly activates rpr transcription. In
addition, antagonistic transcriptional regulation of the P2 Antp
promoter in the embryonic ventral nerve cord has been demonstrated for Antp
and Ubx. In this case, Antp positively autoregulates its own P2
promoter in the thoracic segments, and Ubx competes with Antp for the same
binding sites and thus prevents high-level expression of Antp in the
more posterior segments (Rogulja-Ortmann, 2008).
A requirement for Hox genes in segment-specific cell survival has already
been shown for the MP2 and MP1 pioneer neurons, where AbdB expression is necessary for survival of these neurons in the three most-posterior abdominal segments. In the more anterior segments, the dMP2 and MP1 motoneurons undergo apoptosis at the end of embryonic development, after they have completed their role in pioneering axonal tracts. The surviving dMP2 neurons innervate the hindgut and differentiate into insulinergic neurons. The exact function and targets of the GW and the anterior NB2-4t motoneurons in the first and second thoracic segment are unclear, as is the reason for their removal in the relevant segments. The surviving GW and MNa might exert a region-specific neurosecretory function and thus modulate neuronal or muscle activity, as has been described for neurosecretory cells in the larval brain, the processes of which arborize on the wall of the anterior aorta adjacent to the ring gland. Alternatively, the elimination of certain outward-projecting neurons in the posterior thoracic and/or abdominal segments might be related to the pattern of muscle fibers, which differs considerably between the thorax and the abdomen. It is suggested that Hox-regulated segment-specific motoneuron survival is a part of the patterning process that enables formation of region-specific functional neuromuscular networks (Rogulja-Ortmann, 2008).
Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. This study found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. Double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles (LaBeau, 2009).
In Drosophila, the seven pairs of embryonic alary muscles attach to Svp pericardial cells along the dorsal vessel as it migrates dorsally towards its final location. The alary muscles persist throughout larval development, playing what are thought to be important roles in stabilizing the location of the heart in the body cavity. In addition, modified alary muscles are also found in the adult, and there is evidence from some insects that contraction of these adult muscles is concordant with heart beating. These data suggest important functions for the alary muscles throughout the life cycle (LaBeau, 2009).
Attachment of the alary muscles to the cardiac tube occurs in the vicinity of the Svp pericardial cells. The data clearly identify processes emanating from the alary muscles towards the pericardial cells. It is reasonable to propose that the Svp pericardial cells produce or present some molecule(s) to which the alary muscles attach, although the nature of this molecule has yet to be defined. This suggestion is consistent with observations that in the developing pupa the pericardial cells and alary muscles are connected by significant amounts of connective tissue. In addition, the expression of this unknown molecule must be independent of svp function, since in svp mutants the patterning of the alary muscles appears largely normal. The cardiac tube expresses several secreted molecules which are known to function in cell attraction. However an enrichment for any of these in the Svp pericardial cells has not been reported (LaBeau, 2009).
The data also demonstrate that normal alary muscle patterning is under the direct control of the Hox genes Ubx and abd-A. This finding is consistent with previous research on the role of the Bithorax Complex in patterning of cardiac and skeletal muscle within the mesoderm, and the more general function of the Hox genes in controlling AP diversity. It is noted that the domains of Hox gene function in alary muscles bear a closer resemblance to their expression in developing skeletal muscles rather than Hox gene expression in the cardiac tube. This observation is consistent with the conclusion that the alary muscles are skeletal muscle derivatives based upon their multinucleate nature (LaBeau, 2009).
Previous research further illustrates the requirement of the Hox genes abd-A and Ubx in the normal development and patterning of Svp cardial and pericardial cells. Experiments manipulated both abd-A and Ubx in knockout as well as over-expression conditions, and the nature of the current results are in general consistent with previous findings for the cardiac Svp cells: over-expression of abd-A and Ubx produced three additional sets of Svp expressing cardial cells; and knockout conditions produced either no change in Svp cardial cell number (for abd-A mutants), loss of anterior Svp cardial cells (for Ubx mutants), or almost no sets of Svp cardial cells (for Ubx abd-A mutants) (LaBeau, 2009).
Orthologs of Drosophila Hox genes are detected in the developing human heart, as well as being widely expressed in the neighboring viscera such as the lungs, spleen, liver, pancreas and epidermis. In other vertebrates, Hox genes have been generally (although not specifically) implicated in cardiac development. A recent genome-scale study of skeletal muscle development also established an AP pattern of Hox gene expression in the developing embryonic skeletal myoblasts. Together, these studies support a general role for Hox gene patterning of muscle derivatives broadly across the Animal Kingdom (LaBeau, 2009).
What are the embryonic origins of the alary muscles? As indicated previously, this question is difficult to answer absent a specific marker for the alary muscles early in embryonic development, and it must be noted that these analyses are therefore by necessity end-point assays carried out at stage 16. Nevertheless given the syncytial nature of the alary muscles, they likely arise from specific founders cells specified at particular locations in the somatic mesoderm. Furthermore, since there is only one alary muscle which arises in each hemisegment, it can be proposed that the founder for this muscle arises from an asymmetrical cell division. Experiments were carried out to test this hypothesis using mutants for sanpodo and numb, which are genes in the asymmetric cell division pathway. While sanpodo mutants produced individuals with a partial loss of alary muscles, the numb mutants were so disrupted at the level of the whole organism that it was not possible to discern any sign of the forming alary muscles if present. This issue might be addressed in the future via analysis of alary muscle precursor formation in these mutants, once suitable markers become available. Alternatively, a strategy for following cell lineages in founder cells might prove useful (LaBeau, 2009).
Are there mammalian versions of the alary muscles? While in some cases it is difficult to assign directly homologous structures between insects and mammals, there are a number of ligaments known to stabilize the location of the heart in mammals. These include in particular the ligaments which attach the outer pericardial layer to the diaphragm and spinal column, as well as the sternopericardiac ligaments which connect the pericardium to the sternum. Since the requirement for structures to stabilize the heart within the body cavity appears to be conserved, the molecular mechanisms responsible for their development might also bear some resemblances to each other (LaBeau, 2009).
In the Drosophila ventral nerve cord, the three pairs of Capability neuropeptide-expressing Va neurons are exclusively found in the second, third and fourth abdominal segments (A2-A4). To address the underlying mechanisms behind such segment-specific cell specification, the developmental specification of these neurons was followed. Va neurons are initially generated in all ventral nerve cord segments and progress along a common differentiation path. However, their terminal differentiation only manifests itself in A2-A4, due to two distinct mechanisms: segment-specific programmed cell death (PCD) in posterior segments, and differentiation to an alternative identity in segments anterior to A2. Genetic analyses reveal that the Hox homeotic genes are involved in the segment-specific appearance of Va neurons. In posterior segments, the Hox gene Abdominal-B exerts a pro-apoptotic role on Va neurons, which involves the function of several RHG genes. Strikingly, this role of Abd-B is completely opposite to its role in the segment-specific apoptosis of other classes of neuropeptide neurons, the dMP2 and MP1 neurons, where Abd-B acts in an anti-apoptotic manner. In segments A2-A4 abdominal A was found to be important for the terminal differentiation of Va cell fate. In the A1 segment, Ultrabithorax acts to specify an alternate Va neuron fate. In contrast, in thoracic segments, Antennapedia suppresses the Va cell fate. Thus, Hox genes act in a multi-faceted manner to control the segment-specific appearance of the Va neuropeptide neurons in the ventral nerve cord (Suska, 2011).
Addressed here is the segment-specific appearance of one peptidergic neuronal subtype, the Capa-expressing Va neurons. One pair of Va neurons is initially generated in each segment of the VNC. At embryonic stage 14, differentiation begins and the cells commence the expression of the transcription factors Dac and Dimm. Only after this process is initiated, at stage 16, the posteriorly expressed Hox gene Abd-B triggers PCD in segments A5 to A8. This PCD involves the RHG motif genes, and mutant analysis indicates that grim, or grim and hid play the most important roles. As development progresses, the Va neurons in abdominal segments A2-A4 are further specialized under the influence of abd-A, which results in expression of the Capa neuropeptide at stage 17. The single pair of Dimm/Dac-expressing Va neurons in the first abdominal segment is present into larval stages, but does not express Capa. These alternate Va neurons depend upon Ubx for their Dimm expression, but it is unclear if they differentiate into peptidergic neurons, and if so, which neuropeptide gene they express. In thoracic segments, Antp is involved in the down-regulation of Dac and Dimm. These studies unravel a complex interplay of Hox gene input critical for the segment-specific survival and differentiation of the Va neurons and thereby highlight the involvement of Hox genes during the process of shaping the segment-specific structures of the nervous system (Suska, 2011).
Ectopic appearance of Capa expression through ectopic expression of abd-A indicates that abd-A is an important partner in the combinatorial code of transcription factors necessary for initiating the expression of Capa. The roles of Ubx and Antp are not as straightforward to assess. Ubx showed a participation in the specification of the Va neurons in more anterior segments of the VNC, mainly the thoracic area. Ectopic Ubx expression resulted in maintained Dac/Dimm expression in thoracic Va cells into late embryonic stages (18hAEL). Its endogenous role seems to be confined to segment A1, which is characterized by co-expression of Dac/Dimm and a lack of Capa. The role this pair of neurons plays is unknown, as they are not known to express any neuropeptide. The mutant analysis indicates a possible role of Antp in the down-regulation of Dac/Dimm in thoracic Va neurons. The ectopic expression of Antp however could not override specification signals provided by the other factors (Suska, 2011).
Several studies have identified roles for Hox genes in specifying neuronal subtypes. Of particular interest for the current study are previous findings that Antp acts at a late stage to specify two other neuropeptide cells; the thoracic Nplp1 and FMRFa neurons of the Apterous (Ap) cluster. In this study, Antp first acts together with the temporal gene castor to activate expression of the collier gene, an EBF family member, thus triggering specification of a transient 'generic' Ap cluster neurons identity. Subsequently, Antp acts in a feedforward manner with collier to activate late cell fate determinants, such as dimm, and ultimately the Nplp1 and FMRFa neuropeptide genes. Currently, the neuroblast origin of the Va neurons is unclear. Double-labeling with the neuroblast row 5-6 marker GooseberryNeuro indicates that Va neurons originate from a row 5 neuroblast. As the neuroblast origin of the Va neurons is established, and this lineage mapped, it will be possible to place the generation of Va neurons within a lineage tree. This will furthermore allow identification of the temporal window that generates Va neurons (Suska, 2011).
Programmed cell death plays a critical role in the generation of segmental diversity. Studies in the Drosophila embryo have revealed that this can act both at the level of progenitor and postmitotic, even differentiated cells. In progenitors, PCD acts to remove many abdominal neuroblasts after they have completed their lineages and become quiescent. This ensures that as neuroblasts re-enter proliferative states in the larvae, the abdomen has very few quiescent neuroblasts that can enter the cell cycle. Thus, in the adult CNS, the abdomen will end up containing substantially fewer neurons and glia. In postmitotic cells, PCD acts in two apparently different ways: (1) to remove certain postmitotic cells immediately after mitosis, or (2) to remove differentiated neurons. A particularly relevant case to the studies presented is the removal of the peptidergic dMP2 and MP1 neurons. These cells are generated in all VNC segments, extend axons to pioneer critical axon tracts, and subsequently undergo PCD in all segments but the A6-A8 segments. Strikingly, here Abd-B has an anti-apoptotic and promotes peptidergic identity role, while in the Va neurons it has a pro-apoptotic role. Moreover, the cell death of both MP1 and Va neurons also depends upon the RHG genes. These results suggest that Abd-B acts in an opposing manner, pro- versus anti-apoptotic, by differentially controlling the same PCD pathway in related neurons. An attractive and simple model for this dual role of Abd-B would be that MP1 and Va neurons express different regulatory genes, which can act with Abd-B to trigger either survival or death. Further studies of PCD in the dMP2, MP1 and Va neurons may help shed light on the molecular genetic mechanisms behind these dual roles of Abd-B (Suska, 2011).
Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. This study shows that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. A similar role in cell segregation was shown for the Abdominal-B Hox gene. The results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells (Curt, 2013).
The sorting out of cells with distinct Hox activity in Drosophila has been reported before and in the case of the Hox gene Deformed a possible function in cell segregation has been assigned to such activity. This study has observed some cases that show that Ubx is needed to maintain segregation of cells from different segments during pupation. It is possible that Drosophila Hox genes may have a function in cell segregation during this pupal stage, where cells from different discs and histoblast nests fuse to develop the adult cuticle. The mechanism of segregation seems to rely on the confrontation of cells with different Hox function and not on the absolute levels of Hox expression. This implies that Hox activity in neighboring cells may be checked through proteins at the cell membrane whose expression or levels must be controlled by Hox genes. In the embryo, the Hox gene Abd-B has been shown to regulate molecules like cadherins, and such proteins may mediate segregation between adjacent cells with distinct Hox input (Curt, 2013).
In vertebrates, cells from different rhombomeres are also almost completely prevented from freely mixing. As was shown in this study for Drosophila, it has been proposed that the tension provided by the activity of actomyosin molecules, controlled by Hox genes, could prevent mixing of cells in the vertebrate's rhombomeres. Hox-directed cell segregation, therefore, prevents cells with different Hox code to intermingle, and therefore the appearance of homeotic transformations. This function of Hox genes may be an old one in evolution, required in animals in which development of different body regions is not coupled to the mechanisms of segmentation. In Drosophila, this role of Hox genes may not be needed in cells that are physically separated during most of development (as in imaginal discs and histoblasts from different segments) or superseded by the activity of proteins like Engrailed and Hedgehog, but the maintenance of different affinities by Hox genes and signaling pathways through myosin accumulation may be a general mechanism to segregate cell populations in different species (Curt, 2013).
Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved (Liu, 2018).
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Ultrabithorax:
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