even-skipped
The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation. Chip and its
mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however,
are required for segmentation, nor for expression of most genes known to be regulated by Chip. Chip also interacts
with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and Chip potentiates activity of
one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in
segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers (Torigoi, 2000).
Full-length Chip interacts with the HD proteins Bicoid (Bcd) and Ftz,
and with a fragment of the Su(Hw) insulator protein. The HD protein Otd binds almost as efficiently as does Bcd and Ftz to Chip, but the Eve HD protein binds poorly, a result possibly attributable to improper folding of the in
vitro-translated protein. The domains of Chip involved in homotypic and heterotypic interactions include the LIM interaction domain (LID) and the self-interaction domain (SID). Deletion of the LID reduces interaction with
Apterous. That deletion,
however, has no effect on interaction with Bcd, Ftz, Su(Hw)DeltaCTD, or
Chip. In contrast, two other deletion
mutants, ChipDelta404-465 and ChipDelta441-454, reduce binding to Bcd,
Ftz, Su(Hw)DeltaCTD, and Chip but have little effect on binding to
Apterous. On the basis of this and
additional deletion mutants, Chip residues 439-456 are identified as the region that interacts with
the HD proteins, Su(Hw), and with Chip itself. This region is termed
the other interaction domain (OID) (Torigoi, 2000).
Chip potentiates Bcd activity in the Drosophila embryo when the Bcd activity is low. This effect
is consistent with previous studies on the expression of segmentation
genes in embryos lacking maternal Chip activity. Embryos contain a
gradient of Bcd protein, with a high concentration at the anterior end
and a low concentration at the posterior end. Loss of maternal
Chip strongly reduces all seven blastoderm stripes of Eve protein
produced by the eve pair-rule gene. Many, if not all of
these stripes are also regulated by Bcd, even though most occur in
regions with low to intermediate Bcd concentrations. The
eve stripes are activated by several remote enhancers located ~1.5-9 kb from the promoter, and Bcd-binding sites
are critical for activation by at least the stripe 2 enhancer.
It is likely, therefore, that Chip increases eve expression
at least in part by increasing binding of Bcd to the enhancers. Accumulation of the Hb protein is not substantially affected by loss of
maternal Chip even though hb expression is dependent on
Bcd and several Bcd-binding sites just upstream of the promoter. This lack of an effect of Chip is not unexpected, however,
because hb is expressed in the anterior end where the Bcd
concentration is the highest (Torigoi, 2000 and references therein).
The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is
critical for striped patterns of pair-rule gene expression in the Drosophila embryo. To test
whether these domains function as sources of morphogenetic activity, the stripe 2 enhancer
of the pair-rule gene even-skipped was used to express kni in an ectopic position.
Manipulating the stripe 2-kni expression constructs and examining transgenic lines with
different insertion sites led to the establishment of a series of independent lines that
display consistently different levels and developmental profiles of expression. Individual
lines show specific disruptions in pair-rule patterning that are correlated with the level
and timing of ectopic expression (Kosman, 1997).
It is likely the KNI functions as a repressor to set the posterior border of eve stripe three. To test whether the early repression of eve stripe 3 is mediated through the eve stripe three enhancer, stripe 2-kni constructs were crossed with a line carrying lacZ under the control of this enhancer. Ectopic kni specifically represses the stripe 3 enhancer in a dose-dependent manner. Stripe 2-kni causes disruption of runt stripes 2 and 3, but has no effect on stripe 1. The repression of stripe 3 increases in proportion to the level of ectopic kni, a response similar to that seen for eve stripe 3. Different levels of ectopic kni cause disruptions of fushi tarazu stripes 2 and 3, but have no effect on the expression of ftz stripe 1. It is possible that these effects are indirect and may be mediated through other segmentation genes but this possibility is made unlikely by the fact that hairy expression is virtually unaffected in stripe 2-kni embryos. These results suggest that the ectopic domain of kni acts as a
source for morphogenetic activity that specifies regions in the embryo where pair-rule genes
can be activated or repressed. Evidence is presented that the level and timing of
expression, as well as protein diffusion, are important for determining the specific responses
of target genes (Kosman, 1997).
Transient over-expression of runt under the control of a Drosophila heat-shock
promoter causes stripe-specific defects in the expression patterns of pair-rule genes hairy and
even-skipped (Tsai, 1994).
Although many of the genes that pattern the segmented
body plan of the Drosophila embryo are known, there
remains much to learn in terms of how these genes and
their products interact with one another. Like many of
these gene products, the protein encoded by the pair-rule
gene odd-skipped (Odd) is a DNA-binding transcription
factor. Genetic experiments have suggested several
candidate target genes for Odd, all of which appear to be
negatively regulated. Pulses of ectopic Odd
expression have been used to test the response of these and other
segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype
and a pair-rule phenotype restricted to the dorsal half of the
embryo.
The head defects only phenotype prevails when Odd is
induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat
shocks are administered between
2:50 and 3:10 AEL. The results are complex, indicating
that Odd is capable of repressing some genes wherever and
whenever Odd is expressed, while the ability to repress
others is temporally or spatially restricted (Dréan, 1998).
Two of the seven pair-rule genes tested do not show
significant changes in expression at the stages examined. These
include the genes odd-paired (opa) and, surprisingly, ftz. In odd minus embryos, ftz stripes do not
resolve properly, remaining about 3 cells wide until
well into the process of germ band extension. This suggests that Odd may be a repressor
of ftz. However ectopic Odd does not
repress ftz expression. Also unexpected was the fact that
ectopic Odd has effects on all three of the 'primary' pair-rule
genes. These were previously thought not to be regulated by
Odd. In stage 5 embryos, stripe 1 of hairy is
efficiently repressed by ectopic Odd. The first stripe
of eve is also repressed at this stage.
Repression of h stripe 1 continues in older embryos and is
accompanied by weaker repression of stripes 2-6. These
effects of Odd on h correlate with what appears to be a modest
broadening of h stripes in odd-minus embryos, particularly stripe 1. Early repression of the first stripes of h and eve
likely accounts for the cuticular head defects that arise from
early pulses of ectopic Odd expression.
Interestingly, in odd-minus embryos, the entire 7-stripe pattern of
h appears to expand, both anteriorly and posteriorly. This is
also true of eve and runt stripes. These data provide
no explanation for this, but it may explain the fairly consistent
spacing of h stripes, despite their apparent broadening (Dréan, 1998).
The segmentation gene, runt, is expressed by a subset of the 30 neuroblasts that give rise to each neuromere of the Drosophila embryo. Runt is also expressed in a subset of ganglion mother cells and neurons and its activity has been shown to be necessary for the formation of a subset of even-skipped (eve)-expressing lateral neurons, the EL neurons. There are 8-10 EL neurons per abdominal hemisegment, which originate from neuroblast 3-3. The EL neurons are interneurons that express the zinc-finger transcription factor encoded by eagle. The EL neurons project axons through the anterior commissure across the midline, then turn anteriorly into the longitudinal fascicles. Inactivation of runt during neuroblast delamination, using a temperature-sensitive allele of runt, leads to a loss of eve expression in the EL neurons. Eve expression in the EL neurons is not affected when Runt is inactivated after the neuroblasts have delaminated, suggesting that Runt activity is necessary only at the time of neuroblast delamination for the development of the EL neurons (Dormand, 1998).
runt is a good candidate for a gene that specifies neuroblast identities. To test this, Runt was ectopically expressed in restricted subsets of neuroblasts. Runt is sufficient to activate even-skipped expression in the progeny of specific neuroblasts. Eve is ectopically induced when runt is mis-expressed in all neuroblasts, using the pan neural driver scabrous-GAL4. The average of 9 EL neurons per hemisegment is increased to an average of 16 eve-expressing lateral cells per hemisegment. Ectopic Runt expression causes a severe disruption of the nerve cord, as shown by the abnormal medial eve expression and severe disorganization of the axons. However, Runt is not sufficient to induce eve expression in the progeny of all the neuroblasts. Neuroblast 6-1 and/or neuroblast 6-2 must express another protein that is essential for Runt to activate eve expression. Using the marker Tau-green fluorescent protein to highlight the axons, it was found that the extra Even-skipped-expressing neurons project axons along the same pathway as the EL neurons. Runt is expressed in neuroblast 3-3, supporting an autonomous role for runt during neuroblast specification (Dormand, 1998).
Proteins expressed both by neuroblast 3-3 and by neuroblasts 6-1 or 6-2 are possible candidates for cofactors acting with Runt to induce EL neurons. Neuroblast 6-1 expresses the steroid receptor superfamily member Seven-up and neuroblast 6-2 expresses the zinc-finger transcription factor Ming (Castor) in common with neuroblast 3-3. Although Eve expression is not affected in castor mutants, it would be interesting to investigate whether either Cas or Seven-up contribute to other aspects of the EL neuron fate (Dormand, 1998).
hopscotch is required
maternally for the establishment of the normal array of embryonic segments. In hop mutants,
although expression of the gap genes appears normal, there are defects in the expression patterns
of the pair-rule genes even-skipped, runt, and fushi tarazu. The effect of hop on the expression of these genes is stripe-specific (Binari, 1994).
Pair-rule gene expression is disrupted in Dichaete mutants. Expression of the gap genes Krüppel, knirps, and giant are normal, indicating that Dicaetae acts in parallel or downstream of these gap genes. the so-called primary pair-rule gene even-skipped, Hairy, and runt each show reductions in levels of expression in Dichaete mutants, with variable stripe specific effects on eve, fushi tarazu, hairy and runt. Since the stripes of pair rule genes generally occur in the correct anterior-posterior position in Dichaete mutants, the gene is unlikely to provide key positional information; it is more likely to be required in the maintainance or establishment of appropriate levels of pair-rule gene expression in the central region of the embryo (Russell, 1996 and Nambu, 1996).
An investigation was carried out of the gene regulatory functions of Drosophila Sox box protein 70D (also known as Dichaete or Fish-hook), a high mobility group (HMG) Sox protein that is essential for embryonic
segmentation. The Dichaete HMG domain binds to the vertebrate Sox protein
consensus DNA binding sites, AACAAT and AACAAAG, and this binding induces
an 85 degrees DNA bend. A heterologous yeast system has been used to show that
the NH2-terminal portion of Dichaete protein can function as a transcriptional activator. The HMG and C-terminal regions may partially mask the transcriptional activation function of the N-terminal region. Dichaete
directly regulates the expression of the pair rule gene even-skipped (eve) by binding to
multiple sites located in downstream regulatory regions that direct formation of eve
stripes 1, 4, 5, and 6. Dichaete may function along with the Drosophila POU domain proteins
Pdm-1 and Pdm-2 to regulate eve transcription, since genetic interactions are detected
between Dichaete and pdm mutants. In the blastoderm embryo, pdm-1 and pdm-2 are both expressed in wide posterior bands of cells that are completely contained within the Dichaete expression domain. In double Dichaete/pdm mutants there is a complete loss of eve stripe 5, and fusions between stripes 3 and 4 as well as stripes 6 and 7. This pattern of defects is never observed in mutants for only one or the other of the two genes. The downstream region contains a perfect octamer POU domain consensus binding site. Dichaete protein is expressed in a
dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic
compartments. The protein is first detected in embryos during nuclear cycle 12. At this time Dichaete is present in a wide stripe that encompasses most of the trunk domain, extending from eve stripes 2-7. It is suggested that the DNA-bending properties of Dichaete could enhance or stabilize interactions between regulatory complexes present at distant downstream eve regulatory regions and upstream regulatory complexes including those at the eve promoter. Sox proteins are known to interact with POU domain proteins in vertebrates (Ma, 1998).
dead ringer is required for proper patterning of the abdomen. To test the basis for defects in patterning,
genes required for segment formation in the
Drosophila embryo were examined. Expressions of the axis patterning gene, bicoid;
the gap genes hunchback, Krüppel, knirps and giant; the
primary pair-rule genes even-skipped, hairy and runt, and the
segment polarity genes wingless and engrailed were examined
in embryos lacking germline and zygotic dri function. Most of
these genes are expressed normally with respect to their role
in segment formation. The variable disruption to abdominal
segment formation correlates with a variable
reduction in expression of engrailed and wingless (wg) in
stripes 9-14. The most consistent
effect on expression of the segmentation genes in the dri
maternal and zygotic mutant embryos is a disruption to the
expression of even-skipped (eve)
stripe 4, observed in nearly all embryos lacking both
maternal and zygotic dri product. Specifically, the ventrolateral
portion of eve stripe 4, although initiated appropriately is not maintained in dri
mutant embryos, leading to the subsequent aberrant appearance of wg stripes 7 and 8 and disruption to the
parasegment 4 ventrolateral setal
belts (Shandala, 1999).
Activation of fushi tarazu andeven-skipped expression in ganglion mother cells requires prospero function. Repression of deadpan and asense in ganglion mother cells requires prospero function (Doe, 1991).
In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. miranda, which encodes a new protein that co-localizes with Prospero in
mitotic neuroblasts, tethers Prospero to the basal cortex of mitotic neuroblasts, directing Prospero into
the GMC, and releases Prospero from the cell cortex within GMCs. miranda thus creates intrinsic
differences between sibling cells by mediating the asymmetric segregation of a transcription factor into
only one daughter cell during neural stem-cell division. The expression of even-skipped was followed in embryos mutant for six miranda alleles. A stereotyped pattern of GMCs and neurons express eve. The well characterized aCC/pCC, RP2, CQ, U, and EL neurons all express eve. In prospero mutant embryos, the aCC/pCC and RP2 neurons fail to express eve and most U and CQ neurons also fail to express eve. It was expected that all miranda alleles would show reduced Prospero activity in the GMC either because Prospero inappropriately segregates into both neuroblasts and GMCs, or because Prospero fails to translocate efficiently into the nuclei of GMCs. This predicts that the EVE CNS phenotype of miranda mutant embryos might resemble the Eve CNS phenotype of prospero mutant embryos, should miranda exert its effect through its ability to bind, segregate and release Prospero. This is the case for two catagories of miranda mutants. For the five alleles in which Prospero falls off the cortex (the inner surface of the cell membrane) of neuroblasts, there is an observed reduction of about one-half, in the number of RP2, aCC/pCC, U and CQ neurons expressing eve. Consistent with a decrease in the level of Prospero protein distributed into GMCs, this phenotype resembles a weak prospero phenotype. A one-half reduction in the number of eve-expressing EL neurons is observed; in prospero mutants all EL neurons form normally. This additional eve phenotype may result from the ectopic expression of Pros in neuroblasts or from defects in the partition of other factors dependent on Miranda function. This study raises some interesting questions. Miranda is itself asymmetrically localized: (1) what proteins tether it to the basal cortex of neuroblasts? (2) What proteins regulate miranda so that it releases Prospero in the GMC once cytokinesis is complete? (Ikeshima-Kataoka, 1997).
To some degree, eyelid affects the expression of pair-rule genes. For example, slight defects are seen in the expression pattern of even-skipped: stripes 3 and 4 often appear weaker than normal, stripe 2 wider, and stripes 5 and 6 closer together. This pattern is similar to that of eve during its early expression in wild-type embryos, suggesting a failure of refinement. Clearly eld functions in embryogenesis before wingless is expressed, suggesting that Eld function is not restricted to the Wingless signaling pathway (Treisman, 1997).
T-related gene expression is activated by tailless, but
Trg does not regulate itself. Trg expression in the hindgut and anal pad primordia is required
for the regulation of genes encoding transcription factors (even-skipped, engrailed, caudal,
AbdominalB and orthopedia) and cell signaling molecules (wingless and decapentaplegic). In
Trg mutant embryos, the defective program of gene activity in these primordia is followed by
apoptosis (initiated by reaper expression and completed by macrophage engulfment),
resulting in severely reduced hindgut and anal pads. Early eve expression is unaffected in Trg mutants. By stage 8, however, eve expression in the anal pad primordium is almost entirely absent from Trg mutants (Singer, 1996).
Huckebein is required for even-skipped expression in neuroblasts.
Huckebein regulates aspects of GMC and neuronal identity required for proper motoneuron axon pathfinding
in the NB 4-2 lineage. It is expressed in a subset of Drosophila CNS
precursors, including the NB 4-2/GMC 4-2a/RP2 cell lineage. In huckebein mutant embryos,
GMC 4-2a does not express the cell fate marker eve; conversely, huckebein
overexpression produces a duplicate eve-positive GMC 4-2a. Loss of huckebein does not
affect the number, position, or type of neurons in the NB 4-2 lineage; however, all motoneurons
show axon pathfinding defects and never terminate at the correct muscle (Chu-LaGraff, 1995).
Four genes, ming, even-skipped, unplugged and achaete, are expressed in specific neuroblast
sublineages. These neuroblasts can be identified in embryos lacking both neuroblast
cytokinesis and cell cycle progression (string mutants) and in embryos lacking only neuroblast
cytokinesis (pebble mutants). unplugged and achaete genes are expressed
normally in string and pebble mutant embryos, indicating that temporal control is independent of
neuroblast cytokinesis or counting cell cycles. In contrast, neuroblasts require cytokinesis to
activate sublineage castor (expression, while a single, identified neuroblast requires cell cycle
progression to activate even-skipped expression. This suggests that neuroblasts have an
intrinsic gene regulatory hierarchy controlling unplugged and achaete expression, but that cell cycle-or cytokinesis-dependent mechanisms are required for castor and eve CNS expression (Cui, 1995).
even-skipped expression still
occurs if the first neuroblast division is delayed, but not if the division is prohibited. Moreover,
even-skipped expression is also dependent on progression through S phase which follows
immediately after the first division. However, cytokinesis during the first NB division is not required
for even-skipped expression as revealed by observations in pebble mutant embryos. The coupling of eve expression to cell cycle progression,
presumably functions to prevent a premature activation of expression by a positive regulator of eve which is
produced already in the neuroblast during G2 and segregated asymmetrically into the ganglion
mother cell during mitosis (Weigmann, 1995).
Polycomb group genes regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other PC-G
genes show gap, pair rule, and segment polarity segmentation defects.
Posterior sex combs and polyhomeotic interact with Krüppel and
enhance embryonic phenotypes of hunchback and knirps; polyhomeotic enhances
even-skipped. Flies that are
heterozygous for mutations of even-skipped, carrying duplications of extra sex combs, are extremely subvital. Embryos and surviving
adults of this genotype show strong segmentation defects in even-numbered segments (McKeon, 1994).
even-skipped is also expressed in heart precursor cells in the mesoderm, and is involved in the process of mesodermal segmentation. Expression of eve depends on Wingless, supplied either endogenously from mesodermal cells, or exogenously, from overlying ectodermal cells. even-skipped is expressed in clusters even when wingless is uniformly expressed, suggesting that Wingless is acting here in a permissive role rather than an instructive one (Lawrence, 1995).
Germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several
germline-specific markers. These defects have been traced back to the syncytial blastoderm stage. Pole cells in nos minus
embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped
are ectopically activated in nos minus germ cells. nos minus germ cells are unable to attenuate the cell cycle and instead continue dividing. Unexpectedly, removal
of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos minus germ cells. Supporting the conclusion that Sxl is an important target for nos
repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity (Deshpande, 1999).
Soon after formation, wild-type pole cells in Drosophila downregulate RNA polymerase II transcription until they have been incorporated into the primitive gonad. The premature activation of these germline-specific genes is likely to reflect a more general defect in transcriptional regulation that arises early in embryogenesis, soon after the pole cells are formed. Instead of shutting off RNA polymerase II transcription, nos- pole cells inappropriately transcribe several somatic genes. Why do nos- germ cells fail to regulate RNA polymerase II transcription? The only known regulatory target for nos in the embryo is the hb transcription factor. Nos together with the Pumilio protein is thought to bind to maternally derived hb mRNA and block its translation. Since Hb protein is produced throughout much of the posterior in the absence of Nos, one possibility is that this gap gene protein activates transcription in the pole cells. However, this explanation does not seem likely. Although hb regulates eve and ftz in the soma, it is not clear that the ectopic expression of only the Hb protein would be sufficient to activate either of these genes in the absence of other factors. A more likely possibility is that nos- germ cells have a defect in the system responsible for attenuating RNA polymerase II activity (Deshpande, 1999).
Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and
axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each
hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. A P-element
induced mutation is described that alters the expression pattern of Eve in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was
mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf (FlyBase name: Alhambra). Like its human counterparts,
Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia.
In Dalf mutant embryos, the RP2 motoneuron no longer maintains Eve expression. The effect of the Dalf mutation on Eve expression is
RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are
retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4
driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal
growth (Bahri, 2001).
The Dalf embryonic expression pattern suggests possible
widespread defects in mutant embryos. To address this
issue, a collection of neuronal/glia markers
(22c10, anti-ELAV, BP102, 1D4, anti-REPO), and the
muscle marker, anti-MHC, were examined but no
obvious gross abnormalities were observed in the mutant embryo.
The effect of Dalf mutation on EVE expression is RP2-specific. Removal of Dalf does not affect other markers
normally expressed by RP2, nor does it affect Eve expression in other Eve-expressing neurons. In RP2, Dalf is not
required for the onset of Eve expression, but is necessary
for its maintenance in later embryonic development. This
phenotype largely resembles the effect of removing 3'-UTR
sequences of eve. The neural expression of eve in GMC4-2a and 1-1a, and neurons derived from
them (RP2, aCC/pCC), has been attributed to a single 3'
regulatory element (RP2 + aCC/pCC element). However,
unlike endogenous Eve, the expression under the
RP2 1 aCC/pCC element at stage 15 is quite severely
reduced relative to the earlier stage. The
3'-UTR of eve that is not included in the RP2 + aCC/pCC
element construct is responsible for the maintenance of Eve
expression in later stages. Since the 3'-UTR causes no
change in mRNA levels, it was suggested that the mechanism would seem to involve translational control (Bahri, 2001 and references therein).
DALF has the potential, through its putative DNA binding and protein dimerization motifs, to influence eve expression. Furthermore, the LAP domain of DALF can bind the
eve regulatory region in vitro. Whether
the in vivo Dalf effect on eve is mediated through the 3'-UTR or other eve sequences, or whether its effect is on the
transcriptional or translational level, remains to be identified. In addition, since DALF is ubiquitously expressed, but
its effect on Eve expression is specific to RP2, it seems
likely that DALF may function in collaboration with other
factors that are specific to this neuron (Bahri, 2001).
It is only in late embryonic development that the Dalf
mutation causes Eve loss in RP2, which does not affect the
axonal projection of RP2 to its target dorsal muscle. This is
not surprising since similar results have been reported using
a temperature-sensitive allele of eve. This study shows that Eve function is only required in motoneurons (but not their targets) prior to or during the
early phase of ISN formation so that their axons can grow
dorsally to reach the target muscle. The later removal of eve
function rarely affects formation of the ISN and axonal
projections (Bahri, 2001).
Dalf mutants have retarded growth and do not molt, a
phenotype commonly seen in several mutations, including
those affecting the ecdysone pathway. However, the failure to
rescue the molting of mutant larvae with ecdysone argues against
an upstream involvement of Dalf in this hormonal pathway.
Evidence from the rescue experiment indicates that Dalf is
specifically required in neuronal tissues for normal growth
to occur. In this regard, most neurons are known to express
the hormone receptors and require their activity for development and maturation. Furthermore, DALF is ubiquitously expressed in the nervous system and
it contains a putative motif with nuclear receptor binding
potential. Therefore, based on its expression pattern, protein
structure and mutant phenotype, it is possible for Dalf to
function as a downstream co-regulator/effector in the
hormonal pathway in neurons. Alternatively, Dalf could
be involved in hormone-independent regulatory pathways
that lead to functional neurons (Bahri, 2001).
The retarded growth of Dalf homozygotes suggests that
Dalf may modulate cell proliferation. However, Dalf
expression in the embryo is mainly found in differentiated
tissues, and the overexpression of Dalf in a wide range of
cells and organs during embryogenesis and larval stages
showed no obvious defects. For example, as judged by staining for Eve, no neuronal aberrations
were seen in late transgenic embryos overexpressing full
length Dalf or its cysteine-rich N-terminal domain only.
Moreover, the transgenic animals were viable and normal
under these conditions. These results suggest that Dalf
cannot generally override normal growth control mechanisms, and it may rather indirectly influence cell proliferation
through a neuronal-dependent mechanism (Bahri, 2001).
dHb9 (FlyBase designation: Extra-extra [Exex]), the Drosophila homolog of vertebrate Hb9, encodes a factor central to motorneuron (MN) development. Exex regulates neuronal fate by restricting expression of Lim3 and Even-skipped The mutually exclusive expression patterns of Eve and Exex and the ability of Exex to repress Eve led to an investigation of whether Eve exhibits a reciprocal ability to repress Exex. Whether eve represses exex was tested by following Exex in eve1D mutant embryos. This temperature-sensitive allele allowed the circumvention of the early requirement for eve during embryonic segmentation. On average, two ectopic Exex-positive neurons were observed in each hemisegment of eve mutant embryos. The position of these neurons identifies one as RP2 and the other as likely aCC or pCC. Therefore, eve exhibits a reciprocal ability to repress exex in a subset of dorsally projecting MNs (Broihier, 2002).
During segmentation, Eve has been shown to act as a transcriptional repressor and contains two domains with repressive capability -- one dependent on the corepressor Groucho (Gro) and one Gro independent. To determine whether Eve requires Gro to repress Exex in the CNS, Exex expression was assayed in eve null embryos that contain an eve transgene deleted for the Gro-interaction domain. In these embryos, Exex is derepressed in RP2 and one of the corner cells. Since this phenotype is essentially identical to that of eve1D mutants, it is concluded that Eve represses Exex in a Gro-dependent manner. These results demonstrate that Eve/Evx proteins act through Gro to regulate cell fate in the CNS (Broihier, 2002).
To investigate if Eve is also sufficient to repress Exex, Eve was misexpressed in all postmitotic neurons. In these embryos, Exex expression is abolished, demonstrating that Eve is a potent repressor of Exex expression in the CNS. Thus, Eve is both necessary and sufficient to repress Exex. Taken together, these genetic studies demonstrate crossrepressive interactions between exex and eve function to delimit the expression of Exex to ventral and lateral MNs—and Eve to dorsal MNs. Since both Exex and Eve are key cell fate determinants, this mutually repressive relationship likely helps to consolidate distinct MN fates (Broihier, 2002).
exex mutant embryos display several ectopic Eve-positive neurons. Using the protein-positive ExexJJ154 allele, it was found that these ectopic Eve cells arise from cells that normally express Exex, suggesting that Exex represses Eve cell autonomously. The nonoverlapping expression patterns of Exex and Eve further indicate that Exex acts operationally as an Eve repressor in the CNS. To investigate whether Exex is sufficient to repress Eve, Exex was misexpressed in all postmitotic neurons, and it was found that Exex represses Eve in all Eve-positive neurons except the EL neurons. By late stage 14, only one or two weakly Eve-positive neurons remain in the positions normally occupied by the U, RP2, a/pCC, and fpCC neurons, while the cluster of Eve-positive EL interneurons appears normal. Thus, Exex expression is sufficient to repress Eve expression in all dorsally projecting MNs. The inability of Exex to repress Eve expression in the ELs suggests that the relative ability of Exex to repress Eve is controlled by factors expressed specifically in different neuronal types (Broihier, 2002).
The homeodomain protein Nkx6 is a key member
of the genetic network of transcription factors that specifies
neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9/ExEx to specify ventrally projecting motoneuron fate and to repress
dorsally projecting motoneuron fate. While Nkx6
acts in parallel with hb9 to regulate motoneuron fate, Nkx6 plays a distinct role to promote axonogenesis; axon growth of Nkx6-positive
motoneurons is severely compromised in Nkx6 mutant
embryos. Furthermore, Nkx6 is necessary for the expression of the neural
adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work
demonstrates that Nkx6 acts in a specific neuronal population to
link neuronal subtype identity to neuronal morphology and
connectivity (Broihier, 2004).
In many model systems, MNs that extend axons along common trajectories
express similar sets of transcriptional regulators, which in turn
regulate key aspects of the differentiation of these MN subtypes. Drosophila MNs are classified by the location of
the body wall muscles they innervate. MNs that innervate dorsal body wall
muscles in Drosophila express the homeodomain (HD) transcription
factor Even-skipped (Eve). Furthermore, genetic analyses indicate that Eve is a key determinant of the fate of dorsally projecting MNs. Eve engages in a cross-repressive interaction with the HD protein Hb9, a determinant of ventrally projecting MNs (Broihier, 2004 and references therein).
Ventrally projecting MNs also express the HD transcription factors Lim3 and Islet. Functional analyses have demonstrated that these three HD factors are
required for proper axon guidance of ventrally projecting MNs. The
genetic hierarchy governing the fate of ventrally projecting neurons has,
however, remained elusive as Lim3, Islet, and Hb9 are expressed independently
of each other (Broihier, 2004 and references therein).
To explore the genetic networks behind neuronal diversification in
Drosophila, the role of the Drosophila Nkx6
homolog in regulating distinct MN fates was investigated. Genetic interactions
were characterized between Nkx6 and factors essential for neuronal fate acquisition. Evidence that Nkx6 collaborates with hb9
(exex – FlyBase) to regulate the fate of distinct neuronal
populations. This analysis of hb9 Nkx6 double mutant embryos indicates
that ventrally projecting MNs fail to develop properly in these embryos, while
expression of eve, a key determinant of dorsally projecting MN
identity, expands. In addition, Nkx6 promotes
axonogenesis of Nkx6-positive neurons. Consistent with a direct regulatory
role in this process, Nkx6 activates the expression of the neural
adhesion molecule Fasciclin III in ventrally projecting motoneurons. These
data suggest that Nkx6 is a primary transcriptional regulator of
molecules essential for axon growth and guidance in a specific neuronal
population (Broihier, 2004).
The findings that Nkx6 has roles in both the specification and differentiation of ventrally projecting MNs places Nkx6 in the regulatory circuit that specifies distinct postmitotic neuron fates in the Drosophila CNS. In the mouse, Nkx6 protein function in MN progenitors regulates Hb9 expression in postmitotic MNs. Drosophila Nkx6 is expressed in neural precursors and postmitotic neurons while Hb9 expression is nearly exclusive to postmitotic neurons. However, in contrast to the linear relationship of Nkx6.1/2 and Hb9 in vertebrates, Nkx6 and hb9 were found to act in parallel to specify neuronal fate
in Drosophila. Nkx6 and
hb9 act in concert both to repress expression of the dorsal MN
determinant Eve and to promote expression of Lim3 and Islet in ventrally
projecting RP MNs. It will be of interest to extend this genetic analysis to
other groups of ventrally projecting MNs. It will be also be important to
examine the directness of these genetic interactions. Both Nkx6 and
hb9 contain conserved TN domains that in vertebrate HD proteins have
been shown to interact with the Groucho co-repressor, suggesting that
Nkx6 and hb9 function as transcriptional repressors. This
raises the possibility that Nkx6 and hb9 bind to sequences
in the eve enhancer and directly repress its transcription. In
addition, Nkx6 and hb9 activate lim3/islet
gene expression within ventrally projecting MNs, raising the possibility that
they do so by repressing an unidentified repressor of ventrally projecting MN
identity (Broihier, 2004).
eve represents an appealing candidate for the unidentified
repressor in this model. Ectopic Eve expression in RP MNs in hb9 Nkx6 double mutants may repress Lim3 and Islet. Consistent with this, though it was not possible to unambiguously identify the ectopic Eve neurons in hb9 Nkx6 mutants, many of them are situated close to the midline, suggesting they may represent mis-specified RP MNs. Furthermore, pan-neuronal
eve expression represses Lim3 and Islet expression in the RP MNs
demonstrating that Eve can repress Lim3 and Islet (Landgraf, 1999). A
direct test of this model will require resolving the identity of the ectopic
Eve neurons in hb9 Nkx6 mutant embryos (Broihier, 2004).
There are at least three short-range gap repressors in the precellular Drosophila embryo: Krüppel, Knirps, and Giant. Krüppel
and Knirps contain related repression motifs, PxDLSxH and PxDLSxK, respectively, which mediate interactions with the dCtBP corepressor protein. Giant might also interact with dCtBP. The misexpression of Giant in ventral regions of transgenic embryos results in the selective repression of eve stripe 5. A stripe5-lacZ transgene exhibits an abnormal staining pattern in dCtBP mutants that is consistent with attenuated repression by Giant. The analysis of Gal4-Giant
fusion proteins has identified a minimal repression domain that contains a sequence motif, VLDLS, which is conserved in at least two other sequence-specific
repressors. Removal of this sequence from the native Giant protein does not impair its repression activity in transgenic embryos. It is proposed that
Giant-dCtBP interactions might be indirect and mediated by an unknown bZIP subunit that forms a heteromeric complex with Giant (Nibu, 2001).
The minimal Giant repression domain spans amino acid residues 60-133. Alignment of this sequence with the Drosophila
database identifies significant homology with the zinc finger repressor, Odd-skipped (Odd). Odd represses the
expression of engrailed within the even-numbered parasegments and thereby defines which of the Ftz-expressing cells activate
engrailed. Giant and Odd share the following sequence: VLDLSxxxxSxExP. A third transcriptional repressor in the early
embryo, Tailless, also contains the VLDLS motif. Tailless is important for repressing segmentation gene expression in the
anterior and posterior poles. It is unclear whether this sequence participates in Giant-dCtBP interactions, even though it is related to the dCtBP motif
(PxDLSxR/K/H). Perhaps VLDLS helps recruit an unknown corepressor protein that mediates the residual repression activity of Gal4-Giant fusion proteins
in dCtBP mutants (Nibu, 2001).
The low levels of Giant produced by an twi-giant transgene are sufficient to repress the endogenous eve stripe 5 pattern but not stripe 2. The failure to
repress stripe 2 is consistent with previous studies, which suggested that Giant might interact with a localized 'partner' in anterior regions of the early embryo. It is also possible that stripe 2 regulation depends on high concentration of the Giant protein. There are two alternative explanations for the sufficiency
of low levels of Giant to repress stripe 5. First, the stripe 5 enhancer might contain optimal high-affinity Giant operator sites. Alternatively, Giant might
interact with an unknown bZIP subunit, X, that is broadly expressed in the early embryo (Nibu, 2001).
The second possibility, whereby Giant-X heterodimers regulate stripe 5 expression, is favored. Putative Giant operator sites in the stripe 5 enhancer lack
obvious dyad symmetry, which might be expected for Giant-Giant homodimers. Moreover, the VLDLS motif is essential for the repression activity of
Gal4-Giant fusion proteins but is dispensable in the context of the twi-giant transgene. For example, a deletion that removes the entire
minimal repression domain (amino acids 60-133) does not significantly impair the ability of a twi-giant transgene to repress eve stripe 5 and hairy stripes 3, 4, and 5. Presumably, Gal4-Giant fusion proteins function as homomultimers, so that mutations in the repression domain attenuate or eliminate activity. In contrast, the same mutations might not disrupt the activities of a heterodimeric Giant-X complex because of the ability of subunit X to recruit dCtBP. Future studies will focus on the identification of subunit X and the corepressor(s) that interact with the conserved VLDLS motif (Nibu, 2001).
Heartless is required for the differentiation of a variety of mesodermal tissues in the Drosophila embryo, yet the identity of its ligand(s) has remained a mystery over the years. Two FGF genes, thisbe (ths; FGF8-like1) and pyramus (pyr; FGF8-like2), have been identified which probably encode the elusive ligands for this receptor. The two genes were named for the 'heartbroken' lovers described in Ovid's Metamorphoses because the genes are linked and the mutant phenotype exhibits a lack of heart. The genes exhibit dynamic patterns of expression in epithelial tissues adjacent to Htl-expressing mesoderm derivatives, including the neurogenic ectoderm, stomadeum, and hindgut. Embryos that lack ths+ and pyr+ exhibit defects related to those seen in htl mutants, including delayed mesodermal migration during gastrulation and a loss of cardiac tissues and hindgut musculature. The misexpression of Ths in wild-type and mutant embryos suggests that FGF signaling is required for both cell migration and the transcriptional induction of cardiac gene expression. The characterization of htl and ths regulatory DNAs indicates that high levels of the maternal Dorsal gradient directly activates htl expression, whereas low levels activate ths. It is therefore possible to describe FGF signaling and other aspects of gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004).
Although only one FGF ligand has been identified, Drosophila contains two FGF receptors, Breathless and Heartless (Htl). The Htl receptor is essential for the development of various mesoderm lineages, including cardiac tissues, hindgut visceral musculature, and the body wall muscles. Htl is initially expressed throughout the mesoderm of early embryos, and its activation is thought to trigger the spreading of the mesoderm across the internal surface of the neurogenic ectoderm. The mesoderm cells that come into contact with the dorsal ectoderm receive an inductive signal, Dpp, which triggers the expression of genes such as tinman (tin) and even-skipped (eve) that are required for the differentiation of cardiac and pericardial tissues, respectively. However, the mechanism of Htl activation is uncertain. It has been suggested that localized FGFs emanating from the neurogenic ectoderm might be responsible for Htl activation and provide an instructive cue that guides the migration of the mesoderm. An alternative view is that Htl plays a permissive role in migration by rendering the mesoderm competent to respond to an unknown localized signal (Stathopoulos, 2004).
Htl may be required both for the spreading of the mesoderm and the subsequent specification of cardiac tissues. The misexpression of Dpp throughout the ectoderm, in both dorsal and ventral regions, causes widespread activation of tin expression within the mesoderm. However, eve expression is not expanded, and it has been suggested that its activation depends on both Dpp signaling (normally achieved through spreading) and a second dorsally localized signal, possibly FGF. The analysis of the hindgut visceral musculature provides evidence for this dual role of FGF signaling in movement and specification. The activation of Htl is required for the initial spreading of the visceral mesoderm around the hindgut, as well as the subsequent differentiation of the hindgut musculature (Stathopoulos, 2004).
To investigate the function of FGF signaling in the early embryo, Htl ligands, which have eluded intensive genetic screens, have been identified. This study identified two closely linked genes, thisbe (ths) and pyramus (pyr), which encode FGF signaling molecules that appear to function in a partially redundant fashion to activate Htl. Ths and Pyr are most closely related to the FGF8/17/18 subfamily, which controls gastrulation as well as heart and limb development in vertebrates. Both ths and pyr are expressed in the neurogenic ectoderm during the spreading of the internal mesoderm in gastrulating embryos. These two genes also exhibit dynamic expression in the stomadeum, hindgut, and muscle attachment sites of older embryos. These sites of expression closely match the genetic function of htl described in previous studies. Moreover, a small deletion that removes both ths and pyr causes a variety of patterning defects, including delayed spreading of the mesoderm during gastrulation, the loss of cardiac tissues and hindgut visceral musculature, and abnormal patterning of the body wall muscles. These defects are similar to those seen for htl mutants. The ectopic expression of Ths in the early mesoderm of gastrulating embryos causes an expansion in the domain of Htl activation and a corresponding expansion in the eve expression pattern. These observations suggest that Htl controls both the spreading of the mesoderm and (along with Dpp and Wingless) the specification of pericardial cells. Computational methods were used to identify a mesoderm-specific enhancer for htl that is directly activated by peak levels of the maternal Dorsal gradient. Because ths is directly activated by low levels of the gradient, it is possible to describe gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004).
Once mesoderm spreading is complete, the leading edge of the mesoderm comes into contact with Dpp-expressing cells in the dorsal ectoderm. Dpp signaling might be sufficient for the activation of some of the target genes required for the patterning of the visceral mesoderm, such as tin and bap during stage 10. However, Dpp is insufficient for other inductive events such as the activation of tin and eve in different heart precursors. The loss of eve expression in ths;pyr and htl mutants does not appear to be due to a breakdown in mesoderm spreading. Although this spreading is delayed in the mutants, it does ultimately occur. The late activation of the Htl receptor may be essential for the induction of eve expression and the specification of pericardial tissues. Previous studies suggest that Dpp works together with another signal that may be localized in the dorsal ectoderm. This second signal appears to trigger Ras signaling because the expression of a constitutively activated form of Ras causes expanded expression of eve. Evidence that the second signal might be FGF stems from the analysis of a dominant-negative Htl receptor, which blocks the full expression of cardiac and pericardial gene markers after the mesoderm has spread. The present study considerably strengthens the case that FGF is the second signal that patterns the dorsal mesoderm. The misexpression of Ths in the mesoderm causes a substantial expansion in the dorsal mesoderm and the number of Eve-expressing cells. Moreover, ths and pyr are expressed in specific 'spots' within the dorsal ectoderm that are adjacent to the internal mesoderm where eve is activated. Thus, the simplest interpretation of the results is that FGF signaling controls both the spreading and patterning of the dorsal mesoderm (Stathopoulos, 2004).
The spreading and subsequent subdivision of the mesoderm into distinct dorsal and ventral lineages can be viewed as direct readouts of the Dorsal gradient. The identification of mesoderm enhancers for htl and dof/hbr/smsf based on clustering of Dorsal-binding sites (and associated sequence motifs) suggests that these genes are directly activated by high levels of the Dorsal gradient. Htl-dependent signaling is triggered by Ths and Pyr, which are selectively expressed in the neurogenic ectoderm in response to low levels of the Dorsal gradient. After spreading, dorsal mesoderm cells comes into contact with Dpp-expressing cells in the dorsal ectoderm, and are thereby induced to form dorsal lineages such as cardiac tissues. The same low levels of the Dorsal gradient that activate ths and pyr also activate sog expression and repress dpp. The Sog inhibitor ensures that Dpp signaling is restricted to the dorsal ectoderm. Thus, the differential regulation of Htl and its ligands determines the precise limits of mesoderm-ectoderm germ-layer interactions during gastrulation (Stathopoulos, 2004).
Drosophila segmentation is governed by a well-defined gene regulation network. The evolution of this network was investigated by examining the expression profiles of a complete set of segmentation genes in the early embryos of the mosquito, Anopheles gambiae. There are numerous differences in the expression profiles as compared with Drosophila. The germline determinant Oskar is expressed in both the anterior and posterior poles of Anopheles embryos but is strictly localized within the posterior plasm of Drosophila. The gap genes hunchback and giant display inverted patterns of expression in posterior regions of Anopheles embryos, while tailless exhibits an expanded pattern as compared with Drosophila. These observations suggest that the segmentation network has undergone considerable evolutionary change in the dipterans and that similar patterns of pair-rule gene expression can be obtained with different combinations of gap repressors. The evolution of separate stripe enhancers in the eve loci of different dipterans is discussed (Goltsev, 2004).
In Drosophila, different levels of the Hunchback and Knirps gap repressor gradients define the limits of eve stripes 3, 4, 6, and 7, while Giant and Kruppel establish the borders of stripes 2 and 5. In situ hybridization probes were prepared for Anopheles orthologues of all four of these gap genes, as well as a fifth gap gene, tailless. hunchback displays a broad band of expression in the anterior half of the Anopheles embryo, encompassing both the presumptive head and thorax. This pattern is similar to that observed in Drosophila, although there are a few notable deviations: (1) there is no obvious maternal expression seen in early Anopheles embryos, whereas maternal hunchback mRNAs are strongly expressed throughout early Drosophila embryos; (2) there is a significant change in the posterior staining pattern. The Drosophila gene displays a strong posterior stripe of expression that is comparable in intensity to the anterior staining pattern. In Anopheles, this staining is significantly weaker than that of the anterior domain, and the posterior pattern is shifted anteriorly into the presumptive abdomen (Goltsev, 2004).
The Kruppel and knirps staining patterns are similar in Anopheles and Drosophila embryos. In both cases, the principal sites of expression are seen in the presumptive thorax and abdomen, respectively. However, the remaining two gap genes, giant and tailless, exhibit distinctive staining patterns. In Anopheles, giant exhibits a continuous band of staining in anterior regions, whereas the Drosophila gene is excluded from the anterior pole. Moreover, there is a prominent band of staining in the presumptive abdomen of Drosophila embryos that is not seen in Anopheles. Finally, tailless is expressed in a narrow stripe in the posterior pole of Drosophila embryos, whereas Anopheles embryos display a dynamic pattern that (transiently) extends throughout the presumptive abdomen (Goltsev, 2004).
These observations document significant changes in the expression patterns of maternal determinants and gap genes in flies and mosquitoes, although the dynamic eve pattern is quite similar in the two systems. The most notable differences were seen for the gap genes hunchback and giant. Additional in situ hybridization assays were done in an effort to obtain a more comprehensive view of these changing patterns; hunchback is initially expressed in the anterior half of Anopheles embryos, with no staining detected in posterior regions. Weak posterior staining is detected by the onset of gastrulation, but expression appears to be localized within the presumptive abdomen rather than the posterior pole as seen in Drosophila. This shift was confirmed by costaining with eve. In Drosophila, the anterior hunchback pattern is lost except for a stripe of staining in the thorax, and this stripe persists along with the posterior pattern during gastrulation. In Anopheles, the early hunchback expression pattern gives way to localized expression in the presumptive serosa. Drosophila lacks a comparable staining pattern, although similar patterns have been documented in Tribolium, and mothmidges. It is conceivable that the late hunchback pattern is responsible, directly or indirectly, for the repression of eve stripes in the presumptive serosa (Goltsev, 2004).
As seen for hunchback, there is no detectable expression of giant in posterior regions of early Anopheles embryos. Weak staining appears in the posterior pole by the onset of gastrulation. This staining is clearly posterior to the hunchback pattern in the presumptive abdomen. Thus, the posterior hunchback and giant patterns are reversed in Anopheles as compared with Drosophila. The anterior giant pattern encompasses the entire anterior half of Anopheles embryos and extends into the anterior pole. The staining pattern is refined at gastrulation, including the loss of expression in the presumptive serosa and the formation of discrete bands. Nonetheless, unlike the situation in Drosophila, expression persists in the anterior pole, thereby raising the possibility that different mechanisms are used to establish the anterior border of eve stripe 2 in flies and mosquitoes (Goltsev, 2004).
The altered patterns of hunchback and giant expression in posterior regions raise the possibility that different combinations of gap repressors are used to establish eve stripes 5, 6, and 7 in Anopheles and Drosophila. It is unlikely that Giant establishes the posterior border of eve stripe 5 and that Hunchback delimits the posterior border of stripe 7, as seen in Drosophila. The expression profiles of additional gap genes were analyzed in an effort to identify potential repressors for these stripe borders. The most obvious candidates are huckebein and tailless, since both are expressed in the posterior pole of Drosophila embryos. No expression of huckebein was seen in early embryos, although strong staining appears after germband elongation (Goltsev, 2004).
The gap gene tailless is initially detected at the anterior and posterior poles, with roughly equivalent levels of staining at the two sites. At slightly later stages, the anterior domain is lost, and the posterior pattern expands throughout the presumptive abdomen. The tailless transcripts detected in posterior regions exhibit a graded distribution, with peak levels at the posterior pole and progressively lower levels in more anterior regions. During cellularization, staining is reduced in posterior regions and reappears near the anterior pole. This broad and dynamic staining pattern is consistent with the possibility that the Tailless repressor specifies the posterior borders of one or more posterior eve stripes (Goltsev, 2004).
Torso signaling was examined in the Anopheles embryo in an effort to understand the basis for the expanded tailless expression pattern. In Drosophila, tailless is activated by the Torso signaling pathway, which can be visualized with an antibody against diphospho (dp)-ERK. The antibody detects localized staining in the terminal regions of early Drosophila embryos. A similar staining pattern is detected in Anopheles, although staining may be somewhat broader in Anopheles than Drosophila. It is therefore conceivable that the expansion of the posterior tailless expression pattern seen in Anopheles might be due to an expanded activation of the Torso signaling pathway (Goltsev, 2004).
The combinations of gap repressors that define the borders of eve stripes 2 to 7 are known in Drosophila. Stripes 2 and 5 are formed by the combination of Giant and Kruppel repressors, while distinctive borders for stripes 3, 4, 6, and 7 are established by the differential repression of the stripe 3/7 and stripe 4/6 enhancers in response to distinct concentrations of the Hunchback and Knirps repressor gradients. Double-staining assays provide immediate insights into the likely combination of gap repressors that are used for any given stripe. For example, the giant and Kruppel expression patterns abut the borders of eve stripes 2 and 5. Double-staining assays were done to determine the potential regulators of the Anopheles eve stripes. These experiments involved the use of digoxigenin-labeled hunchback, Kruppel, knirps, and giant hybridization probes along with an FITC-labeled eve probe. Different histochemical substrates were used to separately visualize the two patterns (Goltsev, 2004).
The anterior hunchback pattern extends through eve stripe 2 and approaches the anterior border of stripe 3. While the posterior pattern extends through stripes 6 and 7, this pattern is quite distinct from the posterior hunchback pattern seen in Drosophila, which abuts the posterior border of eve stripe 7. The anterior giant pattern extends from the anterior pole to eve stripe 2, while the posterior pattern abuts the posterior border of eve stripe 7. In Drosophila, the posterior giant pattern extends from eve stripe 5 to stripe 7. The Kruppel pattern may be somewhat narrower in Anopheles than Drosophila. It encompasses eve stripe 3 in Anopheles but includes both stripes 3 and 4 in Drosophila. Finally, knirps exhibits the same limits of expression in Anopheles as Drosophila. In both cases, the staining pattern extends from eve stripes 4 to 6. In Anopheles, the anterior knirps pattern straddles the anterior border of eve stripe 1. Some of the eve stripes are associated with the same combinations of gap repressors in flies and mosquitoes (e.g., stripes 2, 3, and possibly 4), whereas others show distinctive combinations of gap repressors (e.g., stripes 5, 6, and 7 (Goltsev, 2004).
The systematic comparison of segmentation regulatory genes in Anopheles and Drosophila suggests that the segmentation gene network has undergone considerable evolutionary change among dipterans despite highly conserved patterns of eve expression. Three particular changes in the network are discussed: the localization of maternal determinants, the formation of the anterior border of eve stripe 2, and the formation of the posterior borders of eve stripes 5, 6, and 7 (Goltsev, 2004).
In Drosophila, hunchback contains two promoters, and the maternal promoter leads to the ubiquitous distribution of hunchback mRNAs throughout early embryos. No hunchback mRNAs were detected in early Anopheles embryos. This apparent absence of maternal transcripts raises the possibility that localized Nanos products are not required for inhibiting the synthesis of Hunchback proteins in posterior regions of Anopheles embryos. In Drosophila, the embryonic lethality caused by nanos mutants can be suppressed by the removal of maternal Hunchback products. This nanos-hunchback interaction is ancient and probably operates in basal insects, and possibly basal arthropods. However, the potential absence of this interaction in Anopheles is consistent with the idea that nanos has an additional essential function. Indeed, a recent study suggests that Nanos is required for maintaining stem cell populations of germ cells in Drosophila (Goltsev, 2004).
Anopheles lacks bicoid and contains a lone Hox3 gene that is more closely related to zen and specifically expressed in the serosa. How is hunchback activated in the presumptive head and thorax in Anopheles? The homeobox gene orthodenticle can substitute for bicoid in Tribolium. However, orthodenticle does not appear to be maternally expressed in Anopheles, but instead, staining is strictly zygotic and restricted to anterior regions, similar to the pattern seen in Drosophila. Sequential patterns of orthodenticle, giant, and hunchback expression are established by differential threshold readouts of the Bicoid gradient in Drosophila. It is possible that an unknown maternal regulatory gradient emanating from the anterior pole is responsible for producing similar patterns of expression in Anopheles. It is proposed that this unknown regulatory factor may be localized to the anterior pole by Oskar. Oskar coordinates the assembly of polar granules and is essential for the localization of Nanos in the posterior plasm. It might also localize one or more unknown determinants in anterior regions of Anopheles embryos (Goltsev, 2004).
The eve stripe 2 enhancer is the most thoroughly characterized enhancer in the segmentation gene network. It can be activated throughout the anterior half of the embryo by Bicoid and Hunchback, but the Giant and Kruppel repressors delimit the pattern and establish the anterior and posterior stripe borders, respectively. Removal of the Giant repressor sites within the stripe 2 enhancer in cis or removal of the repressor in trans causes an anterior expansion of the stripe 2 pattern. However, ectopic expression does not extend to the anterior pole, suggesting that an additional anterior repressor regulates the stripe 2 enhancer. Recent studies identified Sloppy-paired as the likely anterior repressor. The limits of the giant and Kruppel expression patterns seen in Anopheles suggest that they might define the eve stripe 2 borders, just as in Drosophila. However, at the critical time when eve stripe 2 is formed in Anopheles, the giant staining pattern extends to the anterior pole, while the corresponding Drosophila gene is repressed in these regions. It is therefore possible that Giant is sufficient to form the anterior border in Anopheles and that repression by Sloppy-paired represents an innovation in Drosophila (Goltsev, 2004).
There are numerous differences in the patterns of gap gene expression in Drosophila and Anopheles. In Drosophila, the posterior stripe of hunchback expression is the source of a repressor gradient that specifies the posterior borders of eve stripes 6 and 7. Anopheles exhibits a distinct posterior staining pattern, with expression extending through stripes 6 and 7. It is therefore unlikely that Hunchback regulates these stripes as seen in Drosophila. Instead, the location of the posterior hunchback pattern suggests that it regulates the posterior border of eve stripe 5 in Anopheles. In Drosophila, this border is formed by Giant, but in Anopheles, the posterior giant expression pattern is restricted to the posterior pole where it abuts stripe 7. Thus, a combination of Kruppel and Giant defines the eve stripe 5 borders in Drosophila, whereas Kruppel and Hunchback might be used in Anopheles (Goltsev, 2004).
In Drosophila, eve stripes 6 and 7 are regulated by different concentrations of Knirps and Hunchback. Low levels of Knirps define the anterior border of stripe 7, while higher levels are needed to repress eve stripe 6. Conversely, low levels of Hunchback establish the posterior border of eve stripe 6, while higher levels regulate stripe 7. The position of the knirps expression pattern is consistent with the possibility that it defines the anterior limits of stripes 6 and 7, just as in Drosophila. However, the posterior borders of these stripes are probably not regulated by Hunchback. The expanded pattern of tailless expression seen in Anopheles might permit it to establish the posterior border of eve stripe 6 and possibly stripe 7. An alternative candidate for the posterior stripe 7 border is giant, which is expressed in a tight domain within the posterior pole. Consistent with this possibility is the observation that the posterior giant pattern comes on relatively late, and the posterior stripe 7 border is the last to form among the seven eve stripes. The reversal of the posterior hunchback and giant expression patterns, along with the expanded tailless pattern, strongly suggests that different combinations of gap repressors are used to define eve stripes 5, 6, and 7 in Drosophila and Anopheles (Goltsev, 2004).
An implication of the preceding arguments is that each of the seven eve stripes is regulated by a separate enhancer in Anopheles. Only five enhancers regulate eve in Drosophila since four of the seven stripes (3, 4, 6, and 7) are regulated by just two enhancers (3/7 and 4/6) that respond to different concentrations of the opposing Hunchback and Knirps repressor gradients. The change in the posterior hunchback pattern virtually excludes the use of this strategy in Anopheles. Thus, stripes 3 and 7 are probably regulated by separate enhancers since different combinations of gap repressors appear to define the stripe borders. Similar arguments suggest that stripes 4 and 6 are also regulated by separate enhancers (Goltsev, 2004).
Why do some enhancers generate two stripes, while others direct just one? Consider the eve stripe 2 and stripe 3/7 enhancers in Drosophila. The stripe 3/7 enhancer is activated by ubiquitous activators, including dSTAT, and the two stripes are 'carved out' by the localized Hunchback and Knirps repressors. Knirps establishes the posterior border of stripe 3 and anterior border of stripe 7, while Hunchback establishes the anterior border of stripe 3 and posterior border of stripe 7. The stripe 2 enhancer directs just a single stripe due to the localized distribution of the stripe 2 activators, particularly Bicoid. In principle, a ubiquitous activator would cause the stripe 2 enhancer to direct two stripes, stripes 2 and 5. Opposing Giant and Kruppel repressor gradients would carve out the borders of the two stripes, similar to the way in which Hunchback and Knirps regulate the stripe 3/7 and stripe 4/6 enhancers. Presumably, the eve stripe 5 enhancer directs a single stripe of expression because it is regulated by a localized activator, possibly Caudal (Goltsev, 2004).
It is suggested that ancestral dipterans contained an eve locus with separate enhancers for every stripe. Anopheles eve might represent an approximation of this ancestral locus. The consolidation of enhancers that generate multiple stripes was made possible by cross-repression of gap gene pairs. In Drosophila, there are mutually repressive interactions between Hunchback and Knirps, as well as between Giant and Kruppel. The use of these interacting gap pairs along with ubiquitous activators permits the formation of two stripes from a single enhancer. It is possible to envision two ways in which mutual cross-repression of these gap genes helps to establish the precise patterns of pair-rule gene expression: (1) it ensures that there are zones free of repressor activity on both sides of Kruppel (for the Kruppel and Giant pair) and Knirps (for the Knirps and Hunchback pair) domains; (2) it protects the patterns of pair-rule gene expression from mutations that could potentially shift the domains of gap gene expression. For example, a mutation that could shift the expression of Kruppel would simultaneously shift the expression of Giant always leaving a repressor-free zone where Eve stripes would be established. Therefore, the evolution of the eve locus depends on the changes in the preceding tier of the segmentation network: refinement in gap gene cross-regulatory interactions (Goltsev, 2004).
Finally, it is easy to imagine that certain dipterans have a single enhancer for stripes 2 and 5, rather than the separate enhancers seen in Drosophila. Perhaps, the symmetric repression of Giant and Kruppel is a relatively recent occurrence, only now creating the opportunity for consolidated expression of stripes 2 and 5 (Goltsev, 2004).
Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the
same machinery and RNA signals drive specific accumulation of maternal RNAs in
the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos;
interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor
complex that drives transcript localization in a variety of tissues (Bullock, 2001).
Asymmetric RNA localization is evident during zygotic
development, especially in the unicellular syncytial blastoderm
embryo. At this stage, several transcripts including those of the
pair-rule and wingless (wg) segmentation genes lie exclusively
apically of the layer of several thousand peripheral nuclei. Localization of these transcripts seems to be mediated by signals within their
3' untranslated regions (UTRs), and to be driven on microtubules by the minus-end-directed molecular motor, dynein. The linkers and other factors that provide the cargo
specificity are unknown. Nor is it clear if transcript localization in
blastoderm embryos relates to that in other types of cells (Bullock, 2001).
There is a rapid apical localization of fluorescently
labelled fushi tarazu ( ftz) pair-rule transcripts injected into the basal cytoplasm of the cycle 14 blastoderm embryo. Although these
experiments indicated a requirement for nuclear proteins fluorescein, labelling compromizes the structure of the transcripts, and pair-rule
[even-skipped, hairy (h), ftz, paired and runt] and wg transcripts
labelled with several other fluorochromes localize apically within 5-8 min without the need for exogenous protein. Indeed, injected unlabelled transcripts also
localize apically. The protein-free assay
retains specificity for apical transport, since transcripts that are
normally unlocalized [Krüppel (Kr), huckebein] or enriched in the basal cytoplasm (string) are not transported apically
and instead diffuse away from the site of injection (Bullock, 2001).
Blastoderm localization signals can
drive transcript transport during oogenesis. This view is supported
by more detailed analysis of maternally expressed pair-rule transcripts. The injection assay reveals a minimum region between
positions 1,374 and 1,579 in ftz that is necessary and sufficient for
localization in blastoderm embryos. A similar region of ftz seems to be required for localization
of transcripts into the oocyte. Furthermore, h and
runt transcripts, driven maternally by the Hsp70
promoter, also accumulate specifically in the oocyte and later reside
at its anterior cortex, whereas Kr or truncated h transcripts lacking most of the 3' UTR fail to localize either in
blastoderm embryos or during oogenesis (Bullock, 2001).
Whether Egl and BicD are present in early
embryos was examined. Both proteins are supplied maternally to the embryo.
They are noticeably enriched apical to the nuclei at blastoderm
stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the
proteins is present in the basal cytoplasm (Bullock, 2001).
Whether endogenous Egl and BicD can associate
with injected localizing transcripts, as might be expected if they are
components of the RNA localization machinery, was tested. Injection of h transcripts leads to marked enrichment of Egl and BicD protein
levels at the sites of RNA localization. Similar results are
found on injection of the other tested maternal and zygotic localizing transcripts ( ftz, bcd, grk, K10, nos, osk and w). Both proteins accumulate basally at the site of injection within 1-2 min. Protein recruitment is not inhibited in
embryos preincubated with colcemid, showing that it is not dependent on intact microtubules. Thus, the proteins are
recruited locally before transport and are transported together
apically with transcripts (Bullock, 2001).
Whether BicD and Egl are required for apical
localization in blastoderm embryos was examined. Strong BicD alleles block oogenesis early, and weaker mutant mothers that lay fertilized
eggs (BicDHA40/BicDR26
and BicDH3/BicDR26) retain
sufficient BicD activity for a normal apical distribution of endogenous pair-rule transcripts. However, the
reduced BicD activity in these embryos no longer supports efficient
transport of injected transcripts: 62% of BicDHA40
/BicDR26
and 73% of BicDH3/BicDR26 embryos show no or weak localization 5-8 min
after injection, compared with 10% of wild-type embryos.
Moreover, an antibody against BicD blocks RNA
transport. Preinjection into the basal cytoplasm of anti-BicD antibody 4C2 strongly inhibits the localization of injected h, ftz, grk and stg-K10TLS transcripts in
70%-75% of embryos. The microtubule
cytoskeleton is not obviously affected by the brief (~20 min) antibody treatment, indicating that the effects on RNA transport
are probably direct. Injection of anti-BicD antibody prevents
apical localization of endogenous pair-rule transcripts, also leading to
anteroposterior smearing of their distribution. Thus, apical
transcript localization seems to be important in restricting the range
of activity of pair-rule genes, and allowing their combinatorial
control of Drosophila segmentation (Bullock, 2001).
Injecting blastoderm embryos with anti-Egl also inhibits apical
localization of both exogenous and endogenous pair-rule transcripts, without overtly disrupting the microtubule network. Moreover, its effect is more potent in embryos from mothers containing only a single copy of the egl gene, indicating that the antibody disrupts RNA
localization by inhibiting the activity of Egl. Egl and BicD are
probably also involved in transporting other cargoes. The arrangement of peripheral nuclei is disrupted after injection of antibodies
to either of the two proteins, consistent with data showing
a requirement for BicD in nuclear migration in eye imaginal
disc cells. Embryos injected with either antibody undergo abnormal morphogenesis, which is also indicative of Egl
and BicD transporting additional cargoes (Bullock, 2001).
These results indicate that Egl and BicD are principal elements of a
complex that transports RNA in blastoderm embryos. Egl and
BicD appear to be present as pools of excess cytoplasmic protein
that associate selectively with localizing transcripts and are transported together apically. Protein recruitment occurs before transport and does not require microtubule integrity; rather, transport
depends on Egl and BicD activity. Egl and BicD probably act directly
to mediate RNA transport associated with establishment and maintenance of the oocyte. Thus, mutant transcripts that are
defective in export from nurse cells into the oocyte fail to recruit
Egl or BicD in blastoderm embryos. grk transcripts are also
recognized by the Egl-BicD-microtubule transport pathway,
which is consistent with the hypothesis that nurse cells are a
source of these transcripts for the early oocyte and that they do
not derive exclusively from the oocyte nucleus (Bullock, 2001).
Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of
protein/RNA co-transport. The complex may have an additional
role in anchoring transcripts at their destination. Alternatively,
maintenance of localized transcripts might not depend on an
independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).
Dhc, Egl and BicD have markedly similar distributions during
oogenesis and in blastoderm embryos, and seem to function
together in specifying oocyte identity. It is proposed that an Egl/BicD complex links specific RNAs to dynein and the microtubules. The same machinery may operate elsewhere in
Drosophila. For example, inscuteable transcripts, which localize
asymmetrically in neuroblasts, also localize apically when
injected into blastoderm embryos. Indeed, germline transcripts localize
apically when expressed in follicle cells. Egl and BicD homologs
have been identified in Caenorhabditis elegans and
mammals, and might comprise part of an evolutionarily
conserved cytoskeletal system for transporting transcripts and
other cargoes (Bullock, 2001).
Establishment of segmental pattern in the Drosophila syncytial
blastoderm embryo depends on pair-rule transcriptional regulators. mRNA
transcripts of pair-rule genes localise to the apical cytoplasm of the
blastoderm via a selective dynein-based transport system and signals within
their 3'-untranslated regions. However, the functional and evolutionary
significance of this process remains unknown. Subcellular
localisation of mRNAs from multiple dipteran species has been analyzed both in situ and by injection into Drosophila embryos. Transcript localisation was assayed in four species that can be cultured in the laboratory. Two of them, Episyrphus (Syrphidae) and Megaselia (Phoridae), are cyclorrhaphan flies (i.e. higher dipterans) but, unlike Drosophila, belong to basal branches of this taxon; the other two, Coboldia (Scatopsidae) and Clogmia (Psychodidae), belong to different branches of lower Diptera. Although localisation
of wingless transcripts is conserved in Diptera, localisation of
even-skipped and hairy pair-rule transcripts is
evolutionarily labile and correlates with taxon-specific changes in
positioning of nuclei. In Drosophila localised pair-rule
transcripts target their proteins in close proximity to the nuclei and
increase the reliability of the segmentation process by augmenting gene
activity. These data suggest that mRNA localisation signals in pair-rule
transcripts affect nuclear protein uptake and thereby adjust gene activity to
a variety of dipteran blastoderm cytoarchitectures (Bullock, 2004).
Apical localisation of pair-rule mRNAs in Drosophila syncytial
blastoderm embryos was first noted 20 years ago, but the developmental and
evolutionary significance of this process has remained unclear. Apical pair-rule mRNA localisation is conserved in cyclorrhaphan species that
diverged over 145 million years ago, indicating that this process has a
significant developmental role under natural conditions. Likewise, the
widespread maintenance of wg transcript localisation in Diptera
supports the importance of this process on a phylogenetic scale, even though,
in Drosophila, wg appears to be less sensitive than pair-rule genes
to a reduction in endogenous transcript localisation (Bullock, 2004).
Unlike wg transcripts, pair-rule mRNAs do not localise in some
branches of lower Diptera, and the phylogenetic occurrence of this process
provides interesting insights into its functional significance. Enrichment of
pair-rule transcripts in the apical cytoplasm correlates with the position of
blastoderm nuclei: efficient apical localisation of pair-rule gene transcripts
is found in species which retain an asymmetric apical position of nuclei
throughout the blastoderm stage (Drosophila, Megaselia); less
efficient localisation is seen when the nuclei move from an apical to a more
central position during blastoderm stages (Episyrphus), and no apical
enrichment of transcripts is seen in species where blastoderm nuclei are
surrounded uniformly by a thin layer of cytoplasm (Coboldia,
Clogmia). Localisation signals are also found in several pair-rule
transcripts of the lower dipteran Anopheles. Like Cyclorrhapha, but
unlike many other lower Diptera and most other insects, this culicid species
has evolved a thickened blastoderm with apically positioned nuclei, probably
to allow rapid development as an adaptation to ephemeral larval habitats: columnar cells that emerge from thickened blastoderms can enter gastrulation directly, whereas cuboidal cells that emerge from thin blastoderms still have to
elongate prior to undergoing the requisite cell shape changes (Bullock, 2004).
In Drosophila, pair-rule proteins are enriched in the
apical cytoplasm prior to import into the nuclei in wild-type blastoderms,
whereas they are detected basally in egl mutant embryos, in which
transcript localisation is inefficient. The apical accumulation of pair-rule
proteins under normal circumstances is consistent with the observation that
apical RNA targeting restricts diffusion of cytoplasmic ß-galactosidase.
Apically targeted protein is most likely confined by the cellularisation
process, in which the plasma membrane invaginates between the nuclei and
encloses the apical compartment first (Bullock, 2004).
It has been speculated that mRNA localisation prevents
pair-rule proteins from moving into inter-stripe regions, where they would
cause dominant patterning defects. However, when pair-rule mRNA localisation
is compromised, either by interfering with the localisation machinery or the
RNA signals, no expansion of RNA or protein stripes or ectopic
phenotypic effects are found. Rather, a reduction of pair-rule activity is seen in their
domains of expression in these experiments, indicating that transcript
localisation augments gene function. Pair-rule mRNA localisation does not
appear to be obligatory for protein activity in Drosophila but makes
the segmentation process more reliable: egl mutants, in which
transcripts localise very inefficiently, have a mild increase in segmentation
defects and are acutely sensitive to the reduction of pair-rule gene dose (Bullock, 2004).
By what mechanism does pair-rule mRNA localisation augment the activity of
their transcription factor products? For h it is demonstrated that
suppression of transcript localisation reduces nuclear levels of its protein.
Pair-rule proteins could be specifically modified in the apical cytoplasm, or
localising transcripts could be translated more efficiently. However, given
the diffuse distribution of pair-rule proteins in the basal cytoplasm when RNA
localisation is disrupted in egl mutants and the correlation between
cytoarchitecture and pair-rule transcript localisation in Diptera, a
third possibility is favored, namely that apical mRNA localisation increases nuclear uptake of their proteins by targeting translation in close proximity to the nuclei. Proteins from
non-localising mRNAs would not be available at high levels in the immediate
vicinity of the nuclei, which would result in a decreased nuclear uptake. Such
a role for apical pair-rule mRNA localisation would be redundant in lower
Diptera with only a thin layer of cytoplasm surrounding the nuclei, which
provides little room for diffusion of pair-rule proteins prior to nuclear
import. A mechanism for perinuclear protein targeting might be particularly
significant for nuclear proteins with short half-lives, such as those encoded
by pair-rule genes. Interestingly, localisation of mRNA in the vicinity of the
nucleus to aid import of nuclear proteins has also been reported in cultured
mammalian cells and may be a widespread mechanism to efficiently exploit a
limited pool of transcripts in cells that are polarised or have a high
cytoplasmic:nuclear ratio (Bullock, 2004).
The relationship between cytoarchitecture and apical pair-rule transcript
localisation does not appear to be absolute because a signal is detected in
eve, but not h, from Haematopota, which has
retained the ancestral, cuboidal blastoderm morphology
and because no localisation signal was detected in Anopheles-eve.
Although the developmental context in which these
signals are used cannot yet be discerned (in situ hybridisation is currently not possible in these
species because of egg shells that are difficult to remove and because of
difficulties in obtaining embryos)
these data raise the possibility that,
within a single species, the differential ability of transcripts to be
recognised by the localisation machinery is used to fine-tune transcriptional
control of target genes in the blastoderm by modulating the nuclear
concentration of pair-rule proteins (Bullock, 2004).
The ability of eve and h pair-rule transcripts to use the
localisation machinery varies in Diptera. A range of localisation
efficiencies is observed in situ that is mirrored in all of 11 cases, upon injection into
Drosophila embryos. Thus, differences in localisation efficiency
appear to reflect changes in the respective localisation signals, rather than
alterations in the specificity of the protein machinery. These findings are
consistent with previous studies with artificial variants of the
Drosophila hairy localisation signal, which suggest that the character of
localisation signals modulates the efficiency of localisation by determining
the kinetics of both the initiation of transport and the transport process
itself. Localisation efficiency appears to be determined by multiple RNA:protein
interactions, the sum of which affects the stability and/or activity of the
RNA:motor complex. Therefore, the efficiency of the localisation process can
be modified gradually during evolution by the addition, loss or modification
of individual recognition sites within mRNAs (Bullock, 2004).
It seems that localisation signals in pair-rule genes have emerged multiple
times within Diptera. For example, although the possibility
that localisation signals in h have been lost in multiple different
lineages of lower Diptera cannot be ruled out, the most parsimonious explanation for the phylogenetic distribution of signals in this transcript is that they evolved independently in response to changes in cytoarchitecture in the lineages
leading to Cyclorrhapha and Culicomorpha. Injection of transcripts from
additional species into Drosophila will determine whether
eve localisation signals emerged independently in the lineages
leading to Haematopota and Cyclorrhapha, or were lost in the lineage
leading to Empis (Bullock, 2004).
Work in mammalian cells has provided insights into how localisation signals
might initially appear. These studies suggest that non-localising mRNAs can also
interact with a motor complex, albeit with a comparatively small probability,
and undergo short movements on microtubules. Localisation signals appear to
augment these interactions and lead to the net translocation of an RNA
population along a polarised cytoskeleton by increasing the frequency and
duration of directed transport. The localisation machinery in Diptera may also have a general, weak affinity for mRNAs because a small proportion of particles of injected non-localising transcripts are transported over short distances in
Drosophila embryos. Asymmetric
accumulation of a population of transcripts may therefore evolve gradually as
a result of selection for increased interaction between a specific transcript
and the localisation machinery (Bullock, 2004).
Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions, muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search were performed for direct Ladybird targets. The data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, bric-a-brac 2 gene was identified as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells (Junion, 2007).
Uncovering how the cell fate-specifying genes exert their functions and determine unique properties of cells in a tissue is central to understanding the basic rules governing normal and pathological development. To approach the cell fate determination process at a whole genome level a search was performed for transcriptional targets of the homeobox transcription factor Lb known to be evolutionarily conserved and required for specification of a subset of cardiac and muscular precursors. To this end the targeted expression profiling and the novel ChIP-on-chip method ChEST were combined. The data revealed an unexpectedly complex gene network operating downstream from lb, which appears to act not only by regulating components of the cell identity code but also as a modulator of pan-muscular gene expression at fiber-type level. Of note, the role of Drosophila lb in regulating segment border muscle (SBM) founder motility appears reminiscent of the role of its vertebrate ortholog Lbx1, known to control the migration of leg myoblasts (Vasyutina, 2005) in mouse embryos (Junion, 2007).
Earlier genetic studies revealed that within the same competence domain the cell fate specifying factors acted as repressors to down-regulate genes determining the identity of neighboring cells. Consistent with this finding, lb was found to repress msh and kruppel (kr) during diversification of lateral muscle precursors and even skipped (eve) within the heart primordium. This study found that additional identity code components are regulated negatively by lb. In the lateral muscle domain lb acts as a repressor of the MyoD ortholog nau and the NK homeobox gene slou, both known to be required for the specification of a subset of somatic muscles. This suggests that a particularly complex network of transcription factors (Ap, Msh, Kr, Nau, Slou) controls the specification of individual muscle fates in the lateral domain. Interestingly, none of these factors is coexpressed with lb in the SBM, which appears to be a functionally distinct muscle requiring a specific developmental program. Besides factors with well-documented roles in diversification of muscle fibers, the global approach identified a few novel potential players in the muscle identity network. Among those expressed in somatic muscle precursors are the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).
Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).
The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).
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