abdominal-A
There are reciprocal amounts of UBX and ABD-A antigens in segments of the abd-A domain. In the absence of abd-A the range of Ubx
expression expands. Thus ABD-A is a repressor of Ubx expression (Marcias, 1990).
There are 30 common binding sites for the proteins
encoded by the Ubx and abd-A genes within a negatively regulated
target, the P2 promoter of the Antennapedia (Antp) gene. By systematically mutagenizing binding
sites and observing the resulting P2 expression pattern in embryos, evidence has been found for
cell-type-specific interactions that are mediated by these sequences. In certain neuronal cells, UBX
and ABD-A proteins appear to repress by competing for common binding sites with another
homeodomain protein, which may be ANTP acting to induce P2 transcription in an
autoregulatory manner. In sets of cells that contribute to the tracheal system, UBX and ABD-A
repress by counteracting the function of a factor acting at independent sites. The latter mechanism
of repression requires only that multiple homeodomain binding sequences be present and is not
dependent on any particular binding site (Appel, 1993).
Genes that limit where the homeotic gene Sex combs reduced is transcribed can
affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is repressed by the three bithorax complex homeotic genes, thus preventing it from inducing
prothoracic structures. However, two of the
BX-C homeotic genes, Ultrabithorax and abdominal-A, have no effect on the
ability of SCR to direct the formation of salivary glands (Andrew, 1994).
In the Drosophila midgut the UBX protein activates and ABD-A represses transcription of the dpp gene. A 45bp fragment of DNA within the dpp midgut enhancer correctly responds to both UBX and ABD-A in a largely tissue-specific manner, thus representing the smallest in vivo homeotic response element identified to date (Manak, 1994).
teashirt is necessary for proper formation of anterior and central midgut
structures. ANTP activates tsh in anterior midgut mesoderm. In the central midgut mesoderm Ubx,
abd-A, dpp, and wg are required for proper tsh expression. The control of tsh by Ubx and abd-A,
and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals
as well as localized transcription regulators, the TSH transcription factor is produced in a spatial
pattern distinct from any of the homeotic genes (Mathies, 1994).
An investigation was carried out into the mechanisms by which Hox genes
compete for the control of positional identity. Functional
dominance is often observed where different Hox genes are
co-expressed, and frequently the more posteriorly
expressed Hox gene is the one that prevails, a phenomenon
known as posterior prevalence. To investigate functional dominance among Hox genes on a molecular basis, dpp674, a visceral
mesoderm-specific enhancer of decapentaplegic was used. In the visceral mesoderm, dpp is expressed in
parasegment 7 (PS7), where it is required for the formation of
the second midgut constriction. Expression of dpp is positively
regulated by Ubx in PS7, and negatively regulated by abd-A in
PS8-12. Regulation of dpp
by Ubx and abd-A takes place through a 680-bp visceral
mesoderm-specific enhancer (dpp674). This enhancer contains
Ubx/Abd-A protein binding sites defined by DNase I
protection assays. Ubx regulation of dpp674 is direct, as shown
in experiments in which expression from a mutated enhancer
is reconstituted by compensatory changes in the UBX protein
that alter its DNA-binding specificity (Capovilla, 1998 and references).
Posterior prevalence is not
adequate to describe the regulation of dpp by Hox genes.
Instead, abdominal-A dominates over
the more posterior Abdominal-B and the more
anterior Ultrabithorax. In the context of the dpp674
enhancer, abd-A functions as a repressor whereas Ubx and
Abd-B function as activators. Thus, these results suggest
that other cases of Hox competition and functional
dominance may also be understood in terms of competition
for target gene regulation, in which repression dominates
over activation (Capovilla, 1998).
The idea that posterior prevalence is the dominance of repression over
activation is supported by the observation that
abd-A functions as an activator through the 5' portion of the dpp
enhancer and as a repressor through its 3' portion. When these
portions are fused together in the full enhancer, repression by
abd-A prevails over activation. These findings suggest the possibility that other cases of
functional dominance may be explained in terms of Hox
proteins functioning as repressors and prevailing over Hox proteins
functioning as activators. For example, in accordance with
posterior prevalence, repression of Distal-less (Dll) by Ubx
prevails over Dll activation by genes of the Antennapedia
(Antp) complex. Similarly, apterous (ap)
repression by Ubx prevails over Antp activation in the central
nervous system. In contrast, and in violation of posterior prevalence,
repression of centrosomin (cnn) by Antp dominates activation
by Ubx in the visceral mesoderm. In
another case, a phosphorylation-defective Antp protein
(Antp [1,2,3,4]A ) has novel functions in addition to the wild-type
Antp functions. At least some of its novel functions are a
consequence of the ability of Antp [1,2,3,4]A to misregulate regulatory
targets of other Hox genes. Antp [1,2,3,4]A violates posterior
prevalence by repressing empty spiracles (ems) expression,
which is normally activated by Abd-B. However it is interesting
that the novel function of Antp [1,2,3,4]A as a dpp activator
cannot overcome repression by abd-A, hence respecting the phenomenon of
posterior prevalence. Thus the attractiveness
of this model is that it explains the cases of posterior prevalence
in which the posterior gene is the repressor, but it also explains
other cases of functional dominance in which posterior
prevalence is violated (Capovilla, 1998 and references).
Different Hox binding sites mediate different
transcriptional activities. Repression by Abd-A is mediated
only by certain binding sites. The ability of individual Abd-A
binding sites to mediate repression does not correlate with
their affinity for Abd-A measured in vitro. Specifically, a low-affinity site (binding site 4) is better able to
mediate repression by Abd-A than a high-affinity site (binding
site 2). These results suggest the existence of cofactors
involved in the regulation of dpp674 by Abd-A. One candidate
for such a factor is the product of extradenticle (exd); however, exd is not
required for abd-A repression of dpp or dpp674lacZ (M. Capovilla and J. Botas,
unpublished). Thus Hox specificity cannot be explained solely
by Hox/Exd cooperative binding, and unidentified cofactors
interacting differentially with Abd-A and other Hox products
probably exist. These factors may alter Abd-A binding
specificity and/or may function as corepressors or coactivators,
altering Abd-A activity as a transcription factor.
The above hypothesis on Hox functional dominance implies
that in many cases posterior Hox genes function as repressors
whereas anterior Hox genes function as activators of specific
target genes. Posterior Hox genes would generally determine
posterior body patterns by repressing target genes activated by
more anterior Hox genes. However, it is of course unlikely that
posterior Hox genes function exclusively as repressors. They
probably also function as activators of some targets; the gene ems is a good candidate for direct activation by Abd-B. In these cases the cross-regulation
between Hox genes (posterior Hox genes repress the
expression of more anterior Hox genes) would ensure the
dominance of posterior Hox genes. From the viewpoint of
evolution, the easier way to create additional posterior patterns
might be to generate ënewí Hox genes that repress existing
targets rather than activate new targets or combinations (Capovilla, 1998).
odd paired is positively regulated by Antennapedia and Abdominal-A at the location of the first and third midgut constrictions respectively. Between these domains opa is negatively regulated by Ultrabithorax and Decapentaplegic (Cimbara, 1995).
unplugged expression occurs in portions of the tracheal system that penetrate the CNS, including the cerebral branch
specific to T1. To test the possibility that genes in
the BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and in
Ubx, abd-A, Abd-B triple mutants. In Ubx mutant
embryos additional unpg expression is observed in cells surrounding
the tracheal pits of T2 and T3, indicative of a role for
Ubx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants of
posterior T2 and T3 toward a T1 identity. In abd-A mutants
embryos extra patches of unpg-expressing cells around the
tracheal pits extend posteriorly to A7, indicating a role for Abd-A
in the repression of unpg expression in the abdominal
segments. The homeotic gene abd-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triple
mutants (C. Chiang, 1995b).
There are independent enhancers for Distal-less, arrayed over 40 kb of DNA. Head and leg synthesis is independently regulated. One distal 5' enhancer sequence, an early enhancer regulating Dll expression in germ band extended embryos, is subject to repression by Ultrabithorax and abdominal-A (Vachon, 1992).
Segmental modulation
of 18 wheeler expression later in the tracheal system is dependent upon the function of the homeotic genes of
the bithorax complex. In Ultrabithorax mutant embryos, a larger, more intense patch of 18w extends to cells surrounding the tracheal pits in T2 and T3, indicative of a role for Ultrabithorax protein in repression of 18w in T2 and T3 tracheal pits and consistent with a homeotic transformation in Ubx mutants of posterior T2 and T3 towards a T1 identity. Expanded 18w similarly extends posteriorly to A6 in flies lacking both Ubx and abd-A functions and to A7 in a triple mutant also deficient in Abd-B, indicating a role for abd-A and Abd-B in the repression of 18w in the posterior abdominal segment. In the triple mutant, loss of intense posterior spiracle staining suggests that Abd-B may also be required in A8 for positive regulation of 18w. It is not known whether BX-C regulation of 18w is direct or indirect (C. Chiang 1995a).
Scabrous is a
target for UBX. Parts of the last
intron and exon of the scabrous gene contain five ATTA sequences, the core sequence shared by
most homeodomain binding sites.
Mutation of Ubx results in the ectopic transcription of sca in the first abdominal segment. Transcript localization in several combinations of deficiencies in the bithorax complex
indicates that sca is downregulated by Abdominal-A and Abdominal-B,
and suggests that it is a common target of the three genes of BX-C (Graba, 1992).
Muscle diversification in the Drosophila embryo is manifest in a stereotyped array of myofibers that
exhibit distinct segment-specific patterns. The homeotic genes of the bithorax
complex control the identities of abdominal somatic muscles and their precursors by functioning
directly in cells of the mesoderm. Whereas Ultrabithorax and abdominal-A have
equivalent functions in promoting the formation of particular muscle precursors in the anterior
abdominal segments, Abdominal-B suppresses the development of these same myogenic
cells in the posterior region of the abdomen. When expressed in the same mesodermal cells,
however, either Ubx or abd-A can override the inhibitory influence of Abd-B, suggesting that
these factors may compete in the regulation of common downstream genes. Furthermore, targeted
ectopic expression of Ubx or abd-A indicates that these homeotic genes influence muscle cell fates
by autonomous action in mesodermal cells. Muscle identity also appears to be sensitive to the level
of Ubx expression in myogenic precursors. Homeotic cues specific to
both the mesoderm and the ectoderm cooperate to specify the pattern of muscle attachment sites (Michelson, 1994).
Homeotic genes are expressed in the mesoderm,
and are regulated in a segment-specific pattern analogous to, but different from, that seen in the
ectoderm. Moreover, the effects of homeotic mutations on the muscles do not always mirror
transformations seen in the epidermis. abdominal-A may be expressed ectopically in the mesoderm without altering its expression in the overlying
ectoderm. The pattern of adult muscle precursor cells characteristic of the thorax can
be converted to that seen in the abdomen by expressing abdominal-A
specifically in the mesoderm (Greig, 1993).
Connectin is a cell-surface molecule containing leucine-rich repeats.
Connectin can mediate cell-cell adhesion, suggesting a direct link between homeotic gene
function and processes of cell-cell recognition. A 4 kb restriction enzyme fragment of Connectin, encompassing the 100 base pair clone of the Connectin promoter immunopurified with anti Ultrabithorax antibody, gives a consistent pattern of expression corresponding to a subset of the total Connectin expression pattern. High levels of expression are detected in a small group of cells in the gnathal and thoracic segments and in a posterior segment. This fragment does not produce CNS expression. The reduced levels of expression of the 4 kb fragment suggest a down regulation by Ubx and the abdominal homeotic genes. In Ubx mutants, expression is derepressed in the abdominal segments A1 and A2. Derepression is more dramatic in a Ubx/abd-A double mutant indicating that both genes repress the 4 kb construct. Antp is required for the high levels of expression found in T2 and T3 of wild type embryos (Gould, 1992).
clift/eyes absent is expressed within somatic gonad precursor cells (SGP) as these cells
first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in
gonadogenesis. In abdominal-A mutants, clift fails to be expressed, and in abd-A overexpressors, clift is likewise expressed ectopically. Despite the early expression of clift, SGP's
are specified in the absence of clift function. However, they fail to maintain
their fate; as a consequence, germ cells do not coalesce into a gonad. In
addition, using clift as a marker, it is shown that the anteroposterior and
dorsoventral position of the somatic gonadal precursor cells within a
parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with
a gene regulatory hierarchy involving abd-A within the mesoderm (Boyle, 1997).
During gastrulation, the Drosophila mesoderm invaginates and forms a single cell layer in close
juxtaposition to the overlying ectoderm. Subsequently, particular cell types within the mesoderm are
specified along the anteroposterior and dorsoventral axes. The exact developmental pathways that
guide the specification of different cell types within the mesoderm are not well understood. The developmental relationship between two mesodermal tissues in the Drosophila embryo,
the gonadal mesoderm and the fat body, has been analyzed. Both tissues arise from lateral mesoderm within the even-skipped
domain. Whereas in the eve domain of parasegments 10-12 gonadal mesoderm develops from
dorsolateral mesoderm and fat body from ventrolateral mesoderm, in parasegments 4-9 only fat body is
specified. The cell fate decision between gonadal mesoderm and fat body
identity within dorsolateral mesoderm along the anteroposterior axis is determined by the combined
actions of genes including abdA, AbdB and srp; while srp promotes fat body development, abdA allows
gonadal mesoderm to develop by repressing srp function. Genetic analysis suggests that before stage 10 of embryogenesis, gonadal mesoderm and the fat body
have not yet been specified as different cell types, but exist as a common pool of precursor cells
requiring the functions of the tin, zfh-1 and cli genes for their development (Moore, 1998).
Abd-A regulates the segmental identity of neural elements in the peripheral nervous system. Anti-POXN stains cells in the PNS that give rise to poly-innervated sensory organs. Some of these stain-accepting cells produce structures that are homologous to one another but are still different from one another, depending on their location ( thorax or abdomen). A dorsal row of POXN-positive cells become kölbchen in the thorax (dorsal pits), but become small sensory hairs in the abdomen. These sense organs differ in both their position and in their differentiation. In thoracic segments T2 and T3, the dorsal POXN-positive cells migrate to a more ventral position than do those in the abdomen. Both differential migration and the terminal differentiation of these precursors are determined by abdominal-A (Castelli-Gair, 1994).
homothorax expression is regulated by genes of the bithorax
complex. Starting at germband extension, and throughout the rest of
embryonic development, hth expression is modulated in a
segment-specific fashion. Most notable is the repression of hth
expression in the ectoderm and VNC of abdominal segments
during late stages of embryonic development. The genes of the homeotic complexes are the major regulators of segmental identity. hth expression has therefore been analyzed in embryos deficient for the abd-A gene, or the abd-A and Ubx genes together. In the absence of abd-A activity, hth expression is derepressed in the abdominal ectoderm in cells
along the segment boundaries. In the absence of both abd-A and Ubx, the derepression is more prominent; hth is expressed in ectodermal cells throughout the segment. In addition, a uniform level of the Hth protein is observed in all the thoracic and abdominal neuromers of
Df(3R)Ubx 109 homozygous embryos (Kurant, 1998).
An immunopurification method has been used to clone target genes of the
Antennapedia protein (ANTP). centrosomin (cnn) is expressed in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS). In
the VM, Antp and abdominal-A negatively regulate cnn, while Ultrabithorax shows
positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and
abd-A. Evidence suggests that the expression of the cnn gene in the VM
correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second
midgut construction (Heuer, 1995).
Signaling by the Epidermal growth factor receptor (EGFR) plays a critical role in the
segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a
dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval
abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of argos. These homeotic genes account for the main difference in shape between
abdominal and thoracic denticle belts (Szuts, 1997).
Diversification of Drosophila segmental morphologies requires the function of Hox transcription factors. However, little information is available that describes pathways through which Hox activities effect the discrete cellular changes that diversify
segmental architecture. Serrate is a Hox
gene target. Serrate acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for
morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox
genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal
segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of
rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head,
Deformed is required to activate Serrate transcription in the maxillary segment, a region where Serrate is required for normal mouth
hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the
Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function (Wiellette, 1999).
Ser transcripts in the trunk are first detected at the extended
germband stage in ventral patches in the middle of abdominal
segments A2-A8 and in offset lateral patches. The ventral regions
of thoracic segments do not exhibit Ser expression at this stage.
As the germband retracts, the abdominal stripes intensify and
develop sharp anterior borders. The first abdominal
segment (A1) is unique: Ser expression begins later than in the
other abdominal segments and forms a narrower stripe after
germband retraction. After germband retraction,
Ser transcripts can also be detected in the ventral regions of
thoracic segments in broad, faint patches.
Embryos mutant in all genes of the Bithorax Complex (BX-C),
Ubx, abd-A and Abdominal-B (Abd-B), develop thoracic-type
denticles throughout the trunk region. Consistent with this
transformation, stage 11 and 12 BX-C mutant embryos have
no Ser expression in ventral regions. Ventral Ser
expression does begin in BX-C mutants after germband
retraction, but the location and level of expression matches that
of the thoracic segments. As expected, Ubx mutant
embryos show a transformation of abdominal- to thoracic-type
Ser expression only in A1; abd-A mutants show Ser transcript
stripes in A2-A8 that are similar to the wild-type A1 pattern,
and Abd-B mutants display no change in A1-A8 ventral Ser
transcription. Thus Ubx function is sufficient
to activate some Ser expression in the center of each segment,
but abd-A function is required for the earlier, broader pattern
of Ser transcription in A2-A8, a transcript pattern that
correlates with complete diversification of denticle belts.
Embryos lacking all trunk Hox functions express Ser at the
margins of the anterior part of each trunk segment and at lower
levels in the center of this region, a pattern almost the inverse of that seen in wild type. Transcription of Ser in the posterior-most region of each segment, probably corresponding to the posterior compartment, is completely suppressed. The delimitation of Ser expression to reiterated
subsegmental stripes in the embryonic metameres suggests
that segment polarity genes also regulate the Ser transcript
pattern. ptc mutants lack ventral abdominal Ser transcripts, correlating with the loss of
denticle diversity and number in ptc denticle belts. Ser transcription in wingless (wg) mutants appears in broad stripes, while hedgehog (hh) and engrailed (en) mutant embryos exhibit Ser transcription throughout almost the entire ventral epidermis of the abdominal segments. Broadened patterns of Ser transcription in these segment polarity mutants correspond to expanded fields of denticles that lack significant diversity of denticle type (Wiellette, 1999).
A model is presented for the roles of Ser, rho and Hox genes in the generation of denticle belt patterns in the thorax and abdomen. Three Hox genes (Antp, Ubx, and abdA) serve to establish the segmentally specific levels of Ser expression in the third abdominal segment and in the first two thoracic segments respectively. Ser is activated
at stage 11 in abdominal parasegments by Ubx and abd-A functions
but not in thoracic parasegments where Antp is the principal
Hox function. Ubx function is required for the A1-type abdominal expression pattern of Ser, which is narrower and
fainter than the pattern in other abdominal segments. This pattern
correlates with a narrower, less complex denticle pattern in A1 than
in more posterior segments. abd-A function is required for the
wider, more abundant Ser stripes in A2-A8.
Expression of Ser in the embryonic epidermis results in
context-dependent responses, including rho expression,
denticle belt patterning and normal development of the mouth
hooks. These embryonic roles of Ser are apparently different
from its roles in wing margin determination and wing
outgrowth. One similarity is the short range over which Ser
function is exerted, either at the anterior border of its ventral
A2-A8 expression pattern, or at the dorsal/ventral margin of its
expression boundary in the wing pouch.
The spitz-group gene rho can potentiate Egfr activation via
the Spitz (Spi) ligand. Egfr activation is required from late stage
11 to early stage 13 for patterning of the denticle belts, and rho, unlike spi and Egfr, has a
spatially and temporally regulated expression pattern. Abdomen-specific rho expression is required for
patterning of abdominal denticle rows 1 through 4, probably
by allowing secretion of Spitz protein from denticle row 2 and
3 cells, which activates Egfr in neighboring cells. Ser
function is required for activation of the abdomen-specific
posterior row of rho transcription, expression of which is also
dependent on Ubx/abd-A. The evidence
presented in this paper suggests that Ser provides a critical
intermediate that translates broad Hox and segment polarity
domains into narrow stripes of rho expression, which then specify
diversification at the single cell level (Wiellette, 1999 and references).
The Hox/homeotic genes encode transcription factors that generate segmental diversity during Drosophila
development. At the level of the whole animal, they are believed to carry out this role by regulating a large number of
downstream genes. This study addresses the unresolved issue of how many Hox target genes are sufficient to define the
identity of a single cell. Focus was placed on the larval oenocyte, which is restricted to the abdomen and induced in response
to a non-cell autonomous, transient and highly selective input from abdominal A (abdA). Hox mutant rescue
assays were used to demonstrate that this function of abdA can be reconstituted by providing Rhomboid (Rho), a processing factor for the EGF receptor ligand, secreted Spitz. Thus, in order to make an oenocyte, abdA regulates just one principal target, rho, that acts at the top of a complex hierarchy of cell-differentiation genes. These studies strongly suggest that, in at least some contexts, Hox genes directly control only a few functional targets
within each nucleus. This raises the possibility that much of the overall Hox downstream complexity results from cascades of indirect regulation and
cell-to-cell heterogeneity (Brodu, 2002).
Oenocytes are present in clusters of approximately six cells in each of the abdominal segments A1-A7. In the thorax, there is no Egfr induction around the chordotonal organ precursor called C1 and no specific serial homolog of the oenocyte. In order to score unambiguously the presence of oenocytes in a range of different genetic backgrounds, a panel of seven immediate-early, early and late markers were identified. To determine why oenocyte formation is restricted to the abdomen, embryos lacking various Hox genes or extradenticle (exd), which encodes a Hox co-factor, were examined. These experiments indicate that oenocyte formation requires exd and abdA but not two other Hox genes that are also expressed in the abdomen: Antp and Ubx. To assess whether oenocytes form in the absence of all Hox functions, the T1 segment was examined in embryos lacking Sex combs reduced (Scr) and Antp activities. No oenocytes are produced in this context, and therefore these cells are not part of the ground state. However, the ground state does contain both the signaling and responding cell types involved in oenocyte induction: C1 and the Sal-positive dorsal ectoderm (Brodu, 2002). signal
Two types of GAL4/UAS assays were used to test the involvement of genes in the formation of oenocytes. The ectopic assay tested whether gene products can trigger oenocyte formation in the T1-T3 thoracic segments while the rescue assay tested the potential of genes to overcome the oenocyte deficit in abdA mutants. en-GAL4 was used to express AbdA or Ubx in ectodermal stripes that include the oenocyte precursors. In this ectopic assay, only AbdA was able to produce oenocytes in T1-T3. Together with the preceding results, this indicates that abdA provides a highly selective patterning input and is both necessary and sufficient for the oenocyte fate (Brodu, 2002).
At the time of oenocyte induction during stage 11, a transient burst of AbdA expression is found in both C1 and oenocyte precursors. To ascertain where abdA function is required, two drivers were used that, unlike en-GAL4, have complementary expression in the oenocyte precursors (sal-GAL4, or in the C1 lineage (ato-GAL4). Driving AbdA with ato-GAL4 is sufficient to induce a late oenocyte marker in thoracic segments and also to rescue oenocyte formation in abdA mutants. In each assay, using sal-GAL4 to drive AbdA in the dorsal ectoderm fails to produce oenocytes and using both drivers together does not augment the numbers of oenocytes formed with ato-GAL4 alone. These experiments demonstrate clearly that abdA is required in the C1 lineage but not in the presumptive oenocyte itself, despite being transiently expressed there. It therefore follows that although abdA switches on an extensive hierarchy of early-to-late differentiation genes within the oenocyte, all this regulation must be indirect (Brodu, 2002).
Potential abdA targets were sought from among the genes known to play a role in the specification or function of C1. This particular sensory organ precursor produces a type of stretch receptor, the chordotonal organ, that is defined by the proneural gene atonal (ato). ato is also required for oenocyte formation but it is similarly expressed in thoracic and abdominal C1, is not regulated by abdA and is downregulated prior to oenocyte induction. rho, a gene downstream of ato and rate-limiting for the production of sSpi by cleavage from an inactive membrane-bound precursor (mSpi) in the Golgi apparatus, was examined. Like ato, rho is also required for oenocyte formation. Rho protein is first expressed in C1 at stage 10, after it has delaminated from the dorsal ectoderm. As with Ato at this stage, early Rho is present at similar levels in thoracic and abdominal C1 precursors and is not under abdA control. During stage 11, however, thoracic Rho becomes extinguished while abdominal Rho persists at a similar level in the C1 lineage. Unlike the early expression, this late phase correlates with the time of oenocyte induction and is missing in abdA mutants. Furthermore, driving AbdA in the C1 lineage during stage 11, either in the thorax of a wild-type embryo, or in the abdomen of an abdA mutant, is sufficient to prolong Rho expression. Together, these results indicate that the maintenance but not the establishment of Rho expression is under abdA control. Analysis of a rho-lacZ line, expressed at stage 11 but not stage 10, suggests that this late regulation is at the transcriptional level and is mediated by a different enhancer than that controlling the early phase of expression (Brodu, 2002).
Next, it was asked whether the rather simple Rho timing difference between the thorax and the abdomen is responsible for deciding whether a segment is going to form oenocytes. en-GAL4 or ato-GAL4 were used to extend the time-window of Rho expression in the thorax. Remarkably, using either driver results in the formation of bona fide oenocytes, albeit that they are frequently unclustered and dorsally misplaced. The sufficiency of rho in the absence of AbdA can be clearly demonstrated since ato-GAL4 driven expression of Rho rescues oenocyte formation in abdA mutants. Hence, prolonging the expression of Rho in the C1 lineage is all that is needed to reconstitute the oenocyte identity function of abdA. Next, ato-GAL4 was used to drive AbdA or Rho expression in Scr;Antp double homozygotes. Since ectopic oenocytes are formed in both cases in the mutant T1 segment, representing the ground state, any redundant requirement in the responding ectoderm arising from functional equivalence of Hox proteins can be ruled out (Brodu, 2002).
The above experiments do not reveal whether C1 also produces some other oenocyte signal that is normally present in both thorax and abdomen. Addressing this issue, en-GAL4 was used to express Rho in a genetic background lacking ato, and therefore missing a functional C1 cell. In this mutant context, oenocytes can still be induced, indicating that the only role that C1 plays during oenocyte specification is to express Rho and thus provide a source of sSpi signal (Brodu, 2002).
Maintenance of Rho expression by abdA is predicted to extend the period of Spi secretion, so that the abdominal C1 lineage signals for longer than its thoracic homolog. To test directly whether prolonging active ligand production could induce oenocytes, ato-GAL4 was used to drive a constitutively active form of sSpi, in the C1 lineage after stage 10. This resulted in ectopic oenocytes in the thorax and, more importantly, rescued oenocyte formation in abdA mutants. Providing sSpi prematurely, from stage 9 onwards using en-GAL4, also produces thoracic and rescued abdominal oenocytes but the onset of induction remains restricted to the normal time window during stage 11. Together, these results demonstrate that the oenocyte specification function of abdA can be rescued by adding back either Rho or sSpi in C1 during the period of ectodermal competence. Given that the oenocyte role of abdA is synonymous with prolonging Rho and thus sSpi synthesis in C1, then activating the Egfr in the dorsal ectoderm at the appropriate time would be expected to have the same effect. Consistent with this prediction, expressing constitutively active Egfr (EgfrACT) under the control of sal-GAL4 is sufficient to trigger oenocyte formation in abdA mutants, completely rescuing their number, position and clustering (Brodu, 2002).
Oenocyte formation is under the positive control of AbdA and its co-factor Exd. The temporally restricted pulse of AbdA expression in C1 reflects a transient function in prolonging the oenocyte-inducing signal during stage 11. This type of hit-and-run Hox function appears to be widespread and has previously been observed for other ectodermal derivatives. The misexpression experiments clearly indicate that the oenocyte-promoting role of AbdA is highly selective and can not be substituted for by Ubx. This is explained in molecular terms, since only AbdA is capable of maintaining the transcription of rho in the C1 lineage. Such selectivity contrasts with the equivalent biological activities of Ubx and AbdA proteins in promoting haltere formation. In this regard, it is noted that exd is required to make an oenocyte but not a haltere and therefore may allow these two Hox proteins to discriminate between different targets (Brodu, 2002).
In the absence of any Hox input, oenocytes are completely missing and therefore are not an overt part of the ground state. At first sight, it might seem that for cell types that have no morphological representation in the ground state, such as oenocytes, Hox genes must necessarily play a classic instructive role in defining the appropriate pathway of differentiation. However, as will now be argued, this is not the only way that Hox genes can direct the formation of segment-specific cell types. Two lines of evidence suggest that the sSpi signal from C1 is permissive in the sense that it does not itself contain any oenocyte specificity information: (1) providing ectopic sSpi signal outside of a restricted dorsal zone around C1 fails to induce oenocytes; (2) the degree of sSpi signaling influences the number of induced cells rather than their identity. In contrast, it has been demonstrated that all of the cell-type specificity information is encoded in the dorsal ectoderm as an oenocyte prepattern. One crucial component of this prepattern is encoded by sal. The Sal zinc-finger transcription factor acts to prime the Egfr response in favour of the oenocyte fate. In its absence, there is a fate switch and sSpi signaling now induces secondary chordotonal organs. Thus, it has been shown that oenocyte specificity is provided by the sal-dependent prepattern and not by the sSpi-inducing signal (Brodu, 2002).
The segmental restriction of oenocyte induction has been analysed and evidence is provided supporting a model where there is no Hox input into the prepattern but the timing of the sSpi-inducing signal is controlled by abdA. Together with the previous finding that sSpi signaling is permissive, it is now concluded that abdA does not directly specify the oenocyte identity, rather it determines which segments will form oenocytes. This involves modifying the signaling properties of C1, a serially reiterated cell type that is part of the ground state. In turn, this provides a permissive trigger that uncovers a cryptic oenocyte identity also present in the ground state. Hence ato and sal, two of the genes that contribute to the ground state, are essential for specifying the C1 cell type and the complete oenocyte prepattern, respectively. Another important feature of this model is that the dorsal ectoderm is not competent for oenocyte induction until stage 11. This implies that if competence were to be acquired earlier, when C1 expresses Rho in both the thorax and abdomen, then oenocytes would be produced in all trunk segments independent of Hox genes (Brodu, 2002).
The 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes
Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides an assay to analyze the sequence
requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent Exd 5'-TGAT core site
are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding
specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed
to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A-mut, which on the
consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. It has been observed, however, that the bx1:A-mut site
is regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of
action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions (White, 2000).
A homeotic response element that mediates
repression by the Hox genes Ubx and abd-A has been built. The element is based on two short sequence modules; the 21 bp binding site Grainyhead binding site element
(Gbe) for the transcription factor GRH and the 26 bp UBX/
ABD-A bx1 footprint site from the Dll regulatory
sequences. On its own the Gbe mediates uniform epidermal
activation. However, the combination of the Gbe and the
bx1 element produces a homeotically modulated response.
Specifically in the domain of expression of the Hox genes
Ubx and abd-A the epidermal expression is repressed. This
homeotic element thus successfully recapitulates the
features of endogenous regulatory elements from homeotic
target genes giving tissue-specific regulation by multiple
homeotic genes (e.g. connectin). However, while endogenous target
gene regulatory elements tend to be large (typically covering several kb) and are correspondingly difficult to analyze,
this constructed element is appealingly simple and provides
a manageable system for analyzing the specificity of the
homeotic response (White, 2000).
The bx1 element contains the notable sequences 5' -
TAAT (the core Hox binding site) and 5' -TGAT (the core
Exd binding site). These two sequences are contained
within the 5' -TGATTTAATT which is similar to the
proposed PBX/HOX consensus site 5' -TGATNNAT[g/t][g/a]. Mutation of the 5' -TAAT
site within bx1 abolishes the abdominal repression
conferred by this element, presumably by reducing the affinity of the site for Ubx and Abd-A proteins. Thus, both Ubx and Abd-A appear to bind in vivo to the same site in the
bx1 element. Mutation of the 5' -TGAT putative Exd binding site also abolishes the abdominal repression, suggesting
that both Ubx and Abd-A require the binding of the cofactor Exd in order to function as repressors on this construct (White, 2000).
Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. This study describes a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. Fifteen deficiencies were found that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways; this suggests an extensive use of signaling in the control of Hox gene function (Marabet, 2002).
This study surveyed 60% of the genome and 11 genomic regions were found acting as recessive activators of ems HRE; 4 were found acting as recessive repressors of ems HRE, and 18 were found acting as recessive repressors of wg HRE. So far, the only known gene in addition to AbdB required for ems activation is lines. Df(2R)H3E1, which uncovers lines, has been recovered from the screen for AbdB modifiers. A search for discrete mutations that reproduce the deficiency phenotypes allowed identification of four ems HRE modifier genes: dally, ds, scw, and ttk. Although ttk and scw have already been linked to filzkörper development, none of the four genes had previously been involved in the control of ems expression in posterior spiracles. The screen for AbdA modifiers was restricted to genomic regions leading to ectopic activation of the wg HRE; these response elements relate to functions that repress the enhancer. Accordingly, genomic regions or genes already known to play a role in wg activation, such as abdA, exd, hth, or genes coding for components of the Dpp signaling pathway, were not recovered. Five mutations at specific loci reproduce the phenotypes caused by original deficiencies. Four of these mutations identify tsl, ttk, and genes encoding a putative MPK and a putative CBP as candidate modifiers of wg HRE. None of these genes has so far been involved in the regulation of wg in the visceral mesoderm (Marabet, 2002).
Three of the four candidate genes identified from the ems screen encode molecules acting in or acting in connection with signal transduction pathways. The Scw protein is a secreted factor of the TGF-ß family. The loss of ems expression induced by Brk, a potent repressor of the Dpp/TGF-ß target gene, strongly supports this hypothesis. The involvement of additional signaling pathways in the regulation of ems is more indirectly suggested by the identification of ds and dally that act in connection with several signaling pathways. ds codes for a calcium-dependent cell adhesion molecule of the cadherin superfamily and genetically interacts with shotgun and rhomboid, two genes involved in epidermal growth factor (EGF) signaling, as well as with armadillo (arm), which produces a nuclear effector of the Wg transduction pathway. dally encodes a heparin sulfate proteoglycan involved in the reception of Wg. Although additional experiments are required to firmly establish the involvement of the Wg and EGF pathways, the integration of multiple signals seems to be required for accurate ems regulation by AbdB (Marabet, 2002).
Two modifier genes obtained from the wg screen are presumably involved in the signal transduction cascade. The first, tsl, encodes a ligand for the RTK Torso receptor and the second encodes a putative MKP. Signaling by Ras/MAPK could thus be part of the genetic network that controls wg expression in the midgut, which has been confirmed by showing that wg transcription is impaired by a constitutive active form of Ras. Interestingly, the Ras/MAPK pathway has been implicated in regulation of the Ubx and lab enhancer in the central midgut, and the ETS-domain-containing transcription factor Pointed, which acts as a nuclear effector of the Ras/MAPK pathway, is expressed in the third midgut chamber (Marabet, 2002).
Several modifiers of wg and ems HRE activities identified in this study encode molecules acting in signal transduction cascades. This indicates that signaling processes play important roles in the control of Hox gene function and extends previous observations from a screen for modifiers of a dominant Pb phenotype. Understanding how cell signaling and transcriptional control by Hox protein are mechanistically integrated requires further study (Marabet, 2002).
Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).
AbdA is expressed and is active in the third and fourth compartments of the midgut (PS8-PS12), and yet it activates the wg target gene only in PS8. Dpp secreted from PS7 is shown to provide the spatial information required for PS8-localized wg activation and, acting through a newly identified 546 bp enhancer, AbdA and Mad, a transcriptional effector of the Dpp pathway, directly control wg transcription. The convergence of Hox function and Dpp signaling therefore occurs at the levels of DNA and transcription, and endows AbdA with PS8-specific regulatory properties (Grienenberger, 2003).
To identify the enhancer responsible for wg expression in the VM,
subfragments of a 9kb genomic region known to drive wg embryonic
expression were
analyzed in transgenic lines transformed with lacZ reporter
constructs. The
smallest fragment that drives accurate expression in the VM is a 546 bp
XhoI/ClaI (XC) restriction fragment. Its activity is first
detected during germ-band retraction, when wg transcripts are visualized in the VM by in
situ hybridization,
and only in PS8 VM cells. During subsequent development, XC enhancer activity
still mimics wg expression, and is associated with the site of central midgut
constriction formation. Thus, from early on to the end of embryogenesis, the XC
enhancer exclusively and accurately recapitulates wg spatiotemporal
expression in the VM (Grienenberger, 2003).
To address whether AbdA and Dpp signaling could directly regulate wg, the sequence of the XC enhancer was examined for the presence of putative binding sites for AbdA and for Mad/Medea (referred to as DRS, for Dpp response sequence), the canonical transcriptional effectors of the Dpp signaling pathway known to recognize identical target sequences. Since genetic and molecular data led to the proposal that, in Drosophila, the CRE sequences to which Creb proteins bind are required to respond to Dpp in addition to DRSs, potential Creb binding sites were sought. Six TAAT core sequences and four sequences resembling the consensual Hox/Pbx binding sites (TGATNNATG/TG/A) were identified as potentially mediating AbdA function. The Hox/Pbx 3 and 2 sequences strongly match the consensus, with seven or six of the eight consensus nucleotides conserved, respectively. Hox/Pbx sequences 1 and 4 only have five of the eight consensus nucleotides conserved. The XC fragment contains three sequences matching DRSs and two potential CRE sites (Grienenberger, 2003).
To assess the evolutionary conservation of the XC enhancer, an homologous fragment from Drosophila virilis was isolated and analyzed for its in vivo activity by transgenesis in Drosophila melanogaster. The D. virilis fragment drives expression in a pattern very similar to that of the XC enhancer, suggesting that sequences conserved between these two enhancers may be important for wg regulation in the midgut. Sequence comparison, including sequences from D. pseudoobscura, revealed that a majority of the TAAT core motifs, the DRSs and the putative Creb-binding sequences are evolutionarily conserved, whereas sequences that match heterodimeric Hox/Pbx consensus binding sites are not. The existence of two large conserved sequences, Box 1 and 2, is noted. Since Box1 lies in a fragment that does not drive reporter gene expression in transgenic flies, particular attention was paid to Box2 (Grienenberger, 2003).
Hox signaling integration was examined to determine whether signaling pathways contribute towards specifying how AbdA, a widely expressed Hox selector protein, controls the development of distinct pattern elements at different locations. Dpp signal secreted from PS7 provides the positional cue responsible for localized activation of wg by AbdA. Biochemical and reverse genetics experiments have established that AbdA and Mad directly regulate wg transcription through the XC enhancer, which thus serves as an integrator of Hox and Dpp input. AbdA is impotent with respect to this enhancer in the absence of the Dpp signal, though it can function perfectly well on other genes without Dpp. Therefore, functional interactions between selector proteins and signaling pathways confer specificity to signaling pathways, and reciprocally confer functional diversity to selector proteins (Grienenberger, 2003).
This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).
Further studies are required to understand how Hox selector proteins functionally interact with nuclear effectors of signaling pathways to generate specific transcriptional patterns. In the control of wg by AbdA, several scenarios can be envisioned. In one, the effect of the Dpp transcriptional effector Mad on AbdA activity would be indirect, by antagonizing the function of a repressor that would otherwise act on the XC enhancer to prevent wg expression. The absence of a binding site for this hypothetical repressor in Box2 could explain how Box2 drives AbdA-dependent transcription even without Dpp transcriptional effector binding sites. In a second scenario, Dpp transcriptional effectors would more directly control the activity of AbdA by influencing its DNA binding or transregulatory properties. A direct interaction of HoxC8 and Smad1 has been reported to induce osteoblast differentiation in mammals, suggesting that the coordinate action of AbdA and Dpp signaling might rely on direct AbdA-Mad interaction. In wg regulation, the situation may be different, as additional regulatory inputs are involved. bin and hth are essential, and Wg signaling is required for accurate levels of wg expression. The contribution of Creb might indicate that the Ras/Mapk signaling pathway is involved as well. Ras signaling has been proposed to play a permissive role by acting on CRE sequences of the Ubx and lab enhancers. These observations suggest that AbdA and Hox proteins in general attain specificity and diversity by participating in a variety of protein interactions in enhancer-binding complexes (Grienenberger, 2003).
During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).
The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior–posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior–posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior–posterior axis (Gebelein, 2004).
This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior–posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).
Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior–posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).
Exd and Hth, as well as their vertebrate counterparts, are used as Hox
cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).
To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).
One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).
To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).
Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).
Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).
On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).
To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).
On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).
Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).
Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).
The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).
Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).
According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).
Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).
Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).
The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior–posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).
These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).
These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).
Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).
This study examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. This study shows that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages (Berger, 2005).
In Drosophila, individual neuroblasts deriving from corresponding neuroectodermal positions among thoracic and abdominal segments generally acquire similar fates. However, some of these serially homologous neuroblasts produce lineages with segment-specific differences that contribute to structural and functional diversity within the CNS. The NB6-4 lineage was selected as a model to determine how this diversity evolves from a basic developmental ground state. As an experimental system, NB6-4 has an additional advantage, since Eagle (Eg) is expressed in all the cells of both thoracic and abdominal lineages and can thus be used as a lineage marker (Berger, 2005).
First the expression patterns of different homeotic genes were examined in thoracic and abdominal lineages of NB6-4. Antennapedia (Antp) is expressed in NB6-4t lineages of thoracic segments T1-T3. Abdominal A (Abd-A) is expressed in the NB6-4a lineage of abdominal segments A1-A6, whereas Abdominal B (Abd-B) is expressed in the NB6-4a lineage of segments A7-A8. Whereas loss of Antp function does not affect the NB6-4t lineage in any of the thoracic segments, loss-of-function mutations in abd-A and Abd-B cause NB6-4a-to-NB6-4t homeotic transformations in their corresponding segments. Interestingly, Ultrabithorax (Ubx), which is expressed in most of the cells of T3, is specifically absent in the NB6-4t lineage of that segment, and its loss-of-function alleles do not show any thoracic phenotypes. However, overexpression of Ubx as well as abd-A causes NB6-4t-to-NB6-4a transformations. Thus, it seems that the NB6-4t fate is the ground state and the NB6-4a state is imposed by the function of homeotic genes of the bithorax-complex (BX-C). This is consistent with previous reports that the T2 state is the ground state (for epidermis, including adult appendages) and other segmental identities are conferred by the function of homeotic genes (Berger, 2005).
The mechanism was examined by which abd-A or Abd-B specify the NB6-4a lineage compared with the NB6-4t lineage. As the mode and number of mitoses is the most obvious characteristic by which the NB6-4a lineage differs from NB6-4t, it was wondered whether factors regulating the cell cycle might be involved in controlling NB6-4 cell fate. One major factor that regulates the cell cycle is the G1 Cyclin CycE, which is needed for various aspects of the G1-to-S-phase transition (Berger, 2005).
To examine possible effects on cell fate decisions in the NB6-4t lineage, CycEAR95-mutant embryos were stained for gcm transcripts and Pros and Repo proteins. In wild-type embryos, gcm is initially distributed to both daughter cells during the first division of NB6-4t, but subsequently gets rapidly removed in the cell that functions as a neuronal precursor. Pros is transferred asymmetrically into only one cell, where it is needed to maintain and enhance the expression of gcm, thereby promoting glial cell fate. In CycEAR95 embryos, even at late stages (up to stage 14), gcm mRNA is strongly expressed in both daughter cells after the first division of NB6-4t. Even distribution of Pros was observed in both daughter cells, which could be the cause of continued expression of gcm. Furthermore, the glial marker Repo revealed that both cells differentiate as glial cells. The NB6-4a lineage is not affected in CycEAR95-mutant embryos, suggesting that the requirement for zygotic CycE is specific to NB6-4t (Berger, 2005).
Whether ectopic expression of CycE in abdominal lineages causes the opposite effect was tested. The sca-GAL4 line was used to drive UAS-CycE to achieve early expression in the neuroectoderm. An asymmetric distribution of Pros to one of the two progeny cells was observed just after the first division of NB6-4a. At later stages an increase was observed in the number of cells in the NB6-4a lineage (up to 5 cells). Some of these cells migrated medially, as NB6-4 glial cells normally do, maintaining Pros expression at a lower level. They also expressed Repo, which confirmed their glial identity. Other cells stayed in a dorso-lateral position and did not stain for Repo, suggesting neuronal identity (Berger, 2005).
To further investigate if ectopic CycE had indeed induced a neuronal sublineage in NB6-4a, and to test whether CycE can function cell-autonomously, a cell transplantation technique was employed. Single progenitor cells (stage 7) from the abdominal neuroectoderm of horseradish peroxidase (HRP)-labelled donor embryos overexpressing CycE were transplanted into the abdominal neuroectoderm of unlabelled wild-type hosts (at the same stage). The lineages produced by the transplanted cells were identified by morphological criteria. In all six cases, where cell clones were derived from NB6-4a, they were composed of both glial cells and neurons exhibiting their respective characteristic structures and positions. Because the clones are located in a wild-type abdominal environment, this experiment provides evidence that ectopic expression of CycE causes asymmetric division of NB6-4a and confers neuronal identity to one part of the lineage in a cell-autonomous manner. In these single-cell transplantation experiments, similar observations were made for NB1-1 and NB5-4, which also generate segment-specific lineages. Thus, CycE seems to have a general role in establishing segment-specific differences in neuroblast lineages (Berger, 2005).
Next whether the requirement for CycE to specify the neuronal lineage in NB6-4t is due to altered cell-cycle phases was examined. In string mutants, NB6-4t (whose proliferation is blocked before its first division) expresses gcm mRNA, as well as Pros and hunchback protein, although it does not differentiate as a glial cell. The composition of NB6-4t and NB6-4a lineages were further analysed in embryos mutant for other factors that interact with CycE in cell-cycle regulation. dacapo (dap) is the Drosophila homologue of members of the p21/p27Cip/Kip inhibitor family, which specifically block CycE-cdk complexes. Interestingly, in dap-null-mutant embryos an additional glial cell was observed in the NB6-4a lineage, but the appearance of any neuron-like cells was not observed. Consistent with these results, overexpression of Dap resulted in a reduction in the number of neurons in the NB6-4t lineage (from the normal number of 6 to 2-4), but homeotic transformation of the lineage did not occur. The number of glial cells was never affected, neither in the thorax nor in the abdomen. Ectopic expression of p21, the human homologue of the Drosophila dacapo gene, generated similar phenotypes (Berger, 2005).
The influence of the transcription factor dE2F, which mediates the activation of several genes needed for the initiation of S phase, was tested. In dE2F-mutant embryos, unlike in CycE mutants, no homeotic transformation of NB6-4t to NB6-4a was observed, although the number of neurons was reduced from 5-6 to 2-4. Ectopic expression of dE2F resulted in an increase in cell number in some abdominal hemisegments. In only a small percentage of those embryos, cells at lateral positions in abdominal segments did not show expression of gcm or Repo, suggesting their neuronal identity. Thus, although dE2F activation in the CNS depends on CycE, ectopic expression of dE2F cannot fully bypass the requirement for CycE in a NB6-4a-to-NB6-4t transformation. Similarly, ectopic expression of Rbf, a potent inhibitor of E2F target genes, did not cause any changes in the segregation of gcm mRNA, Pros or Repo in the NB6-4t lineage (Berger, 2005).
Whether interfering with another checkpoint of the cell cycle, the transition from G2 to M phase, affects NB6-4 cell fate was tested. Previous studies show that loss of CycA function prevents further mitosis after the first division of NB6-4t. However, the first division of NB6-4t follows the normal pattern; it gives rise to one glial and one neuronal cell. Similar effects were observed in CycA mutants. The cyclin-dependent kinase cdc2 heterodimerizes with CycA and CycB, and high levels of cdc2 expression have been shown to be required for maintaining the asymmetry of neuroblast divisions. In a cdc2 loss-of-function background, NB6-4t generated a normal lineage consisting of two glial cells and five to six dorso-lateral neurons. These observations show that NB6-4 cell fates do not change after manipulation of the transition from G2 to M phase (Berger, 2005).
These results suggest a critical role for CycE per se in regulating the NB6-4t lineage. Therefore whether CycE itself is differentially expressed between thoracic and abdominal NB6-4 lineages was tested. In situ hybridization with CycE RNA on wild-type embryos revealed that CycE is expressed just before the first division in NB6-4t. After the first division, CycE mRNA was detected in the neuronal precursor only and not in the glial precursor. In abdominal segments, no CycE expression was detected in NB6-4a before or after the division. Consistent with the role of CycE in specifying the NB6-4t lineage, notable levels of CycE transcripts were detected in the homeotically transformed NB6-4a lineages in abd-A-mutant embryos. Conversely, overexpression of abd-A caused down-regulation of CycE levels in thoracic segments and homeotic transformation of NB6-4t to NB6-4a. The importance of CycE in generating neuronal cells in the NB6-4t lineage was confirmed in an epistasis experiment involving abd-A and CycE mutants. As described above, loss of abd-A leads to transformation of NB6-4a to NB6-4t. Such homeotic transformation was suppressed by mutations in CycE, suggesting an absolute requirement for CycE in specifying the NB6-4t lineage. Finally, nine potential AbdA-binding sites (five of which are evolutionarily conserved in Drosophila pseudoobscura) were identified in a 5.0-kb enhancer fragment of CycE that is known to harbour cis-acting sequences for driving CycE expression in the CNS (Berger, 2005).
It is concluded that, in addition to its role in cell proliferation, CycE is necessary and sufficient for the specification of cell fate in the NB6-4 lineage. These results suggest that the function of CycE in regulating cell fate in NB6-4 lineages is independent, albeit partially, of its role in cell proliferation. The absence of any cell fate changes in the loss-of-function mutants of string, dap, cdc2 or CycA and in the Dap or Rbf gain-of-function genetic background may be attributed to the presence of CycE, which is strongly expressed in the NB6-4t, but not the NB6-4a, lineage. However, CycE may still function by controlling cell-cycle progression. NB6-4a, which does not express CycE, divides once followed by a cell-cycle arrest, presumably in G1. After the first division in the thorax, one daughter cell expresses high levels of CycE and divides roughly three times to generate neuronal cells. Therefore, this daughter cell presumably progresses through S phase. Chromatin reorganization during S phase might allow cell fate regulators to access their target genes, driving neuronal differentiation. Contrary to this interpretation, the other daughter cell of NB6-4t, which does not express CycE, divides twice but still generates glial cells. Thus, it remains to be investigated whether the role of CycE in neuronal cell fate determination is entirely independent of its role in cell proliferation. The results on the role of CycE in specifying neuronal compared with glial cell fate in the CNS are consistent with data from Xenopus on the role of cyclin-cdk complexes in specifying neuronal cell fate, inhibition of which promotes glial cell fate. In addition, this study shows that homeotic genes contribute to regional diversification of cell types in the CNS through the regulation of CycE levels (Berger, 2005).
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