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).
Morphogen gradients are thought to create concentration thresholds that differentially position the expression boundaries of multiple target genes. Despite intensive study, it is still unclear how the concentration profiles within gradients are spatially related to the critical patterning thresholds they generate. This study used a combination of quantitative measurements and ectopic-misexpression experiments to examine the transcriptional-repression activities of the Hunchback (Hb) protein gradient in Drosophila embryos. The results define five expression boundaries that are set primarily by differences in Hb concentration and two boundaries that are set by combinatorial mechanisms involving Hb and at least one other repressor. Hb functions as a repressive morphogen, but only within a specific range of concentrations (~40% to ~4.4% of maximum Hb concentration), within which there are at least four distinct concentration thresholds. The lower limit of the range reflects a position where the slope of the gradient becomes too shallow for resolution by specific target genes. Concentrations above the upper limit do not contribute directly to differential-repression mechanisms, but they provide a robust source that permits proper functioning of the gradient in heterozygous embryos that contain only one functional hb gene (Yu, 2008).
This study measured the relative Hb concentrations associated with the positions of seven expression boundaries and tested whether different Hb concentrations can account for the differential positioning of these boundaries along the AP axis of the Drosophila embryo. These experiments lead to the following conclusions. 1. The Hb gradient functions as a bona fide repressive morphogen for five target-gene expression boundaries, eve 3, nub, pdm2, eve 4, and kni, all of which appear to be positioned primarily, if not exclusively, by specific thresholds of Hb concentration. These boundaries move anteriorly in concert with the dynamic changes of the Hb gradient in wild-type embryos, they shift anteriorly in zygotic hb mutants, and their sensitivities to repression by ectopically expressed Hb are consistent with their relative positions in wild-type embryos. Two other boundaries, the anterior boundary of Kr and the anterior boundary of the posterior gt domain, are established by combinatorial mechanisms involving Hb and Gt, and Hb and Kr, respectively. 2. There is a specific concentration range (~40% to ~4.4% [Hb]max) that mediates the major morphogenetic activities of the Hb repression gradient. Within this range, four thresholds were detected, one at ~40% [Hb]max that sets the anterior boundary of eve 3, one at ~12% [Hb]max that sets the anterior boundaries of both nub and pdm2, one at ~8% [Hb]max that sets the anterior boundary of eve 4, and one at ~4.4% [Hb]max that sets the anterior boundary of kni. These results suggest that these five target genes are exquisitely sensitive to small changes in Hb concentration. Hb also acts as a direct repressor to position the anterior boundary of the Hox gene Ultrabithorax (Ubx), which is first activated in late cycle 14 just before the initiation of gastrulation. The anterior Ubx boundary is positioned between the eve 3 and eve 4 boundaries and thus may represent a fifth threshold within the morphogenetic range described in this study. Ventral misexpression of Hb causes a strong repression of Ubx. However, it was not possible to directly compare the sensitivity of Ubx with the other target genes because the patterns of these genes have begun to decay when Ubx is first activated (Yu, 2008).
Previous studies have identified discrete regulatory elements that recapitulate the five expression patterns within the morphogenetic range described in this study. All of these elements contain multiple Hb binding sites, and one attractive model is that differences in sensitivity are determined by the quantity and/or quality of their Hb binding sites. The more sensitive eve 4+6 enhancer seems to contain a stronger cluster of Hb binding sites than the less sensitive eve 3+7 enhancer, which is consistent with this hypothesis. However, in preliminary experiments, it was found that this simple model cannot be applied to the five target genes shown in this study to be differentially sensitive. For example, the kni expression pattern is more sensitive to Hb-mediated repression than either eve 3 or eve 4, but its enhancer sequence does not appear to have a stronger cluster of Hb binding sites than either the eve 3+7 or the eve 4+6 enhancer. Similarly, two enhancer elements have been found to be associated with the pdm locus, which contains both nub and pdm2. When tested in reporter genes, both enhancers drive patterns of gene expression similar to the endogenous nub and pdm2 patterns, but they do not appear to contain similar clusters of Hb sites (Yu, 2008).
If differential sensitivity cannot be linked to differences in the number or affinity of Hb binding sites for this set of regulatory elements, other architectural features may control the level of Hb required for repression. These features may include changes in spacing between adjacent Hb sites, or specific site orientations that affect cooperative binding. Also, specific arrangements between repressor and activator sites may influence the apparent sensitivities. Consistent with this, it has been shown that specific arrangements between Dl and Twi sites are critical for Dorsal-dependent target-gene expression in the prospective neuroectodermal region along the DV axis. A careful analysis of the enhancer elements that respond to Hb-mediated repression will be required to fully understand the molecular rules that govern differential sensitivity (Yu, 2008).
At the low end of the effective morphogenetic range, there is a ~2-fold difference between the Hb concentration at the eve 4 boundary (~8% [Hb]max) and the amount at the kni boundary (~4.4% [Hb]max). Moving farther posteriorly, from the kni boundary to the gt boundary, does not correlate with a significant drop in the relative Hb concentration (~4.4% [Hb]max to ~3.7% [Hb]max). It is proposed that the slope of the gradient in this region is too shallow for differential target-gene positioning. However, by participating in a combinatorial mechanism with Kr, the very low concentrations of Hb in this region can set the gt boundary in a more posterior position than the kni boundary. Hb and Kr both bind to the regulatory element that drives posterior expression of gt, suggesting that these interactions may be direct (Yu, 2008).
Within the morphogenetic range, the anterior-most boundary is that of eve 3, which corresponds to ~40% [Hb]max. Outside this range on the anterior side is the Kr boundary, which was previously shown to expand anteriorly in zygotic hb mutants. In these experiments, Kr appeared to be quite resistant to repression by ectopic Hb, which seemingly contradicts a previous study that showed that high levels of Hb were sufficient for repression. However, in the previous study, ectopic Hb was provided maternally, significantly before the sna-hb transgene used in this study would be activated. Together, the two studies support the idea that the Kr boundary is initially set independently by Hb, and that maintenance of the boundary requires both Hb and Gt activities. The results suggest that maintenance is mediated primarily by Gt, but that Gt is an effective repressor only in the presence of Hb. The potentiating effect of Hb on Gt-mediated repression may involve direct binding of Hb and Gt to the Kr promoter, which contains binding sites for both proteins (Yu, 2008).
One of the most important findings from this study is that the effective range of Hb's morphogenetic activity is between 40% [Hb]max and 4.4% [Hb]max. This range may seem surprising in light of the fact that Hb is expressed at much higher levels throughout the anterior half of the embryo. Previous studies suggest that anteriorly expressed Hb is required for activation of most Bcd-dependent target genes, which are expressed in a variety of anterior patterns, and that the zygotic stripe of Hb expressed at the position of PS4 is required for the activation of the Hox gene Antennapedia. It is proposed that the high level of Hb protein in anterior regions also provides a reservoir, or buffer, that ensures that the repressive gradient, with all of its thresholds, remains intact in individual embryos that vary in their absolute levels of Hb expression. Such a buffering mechanism could explain how heterozygous embryos, which contain roughly half the concentration of Hb, can nonetheless develop normally (Yu, 2008).
It is proposed further that most other morphogens will function via concentration ranges similar to the one measured in this study. The two best-studied morphogens in Drosophila are Bcd and Dorsal (Dl), both of which are viable and fertile in the heterozygous state. In embryos laid by bcd/+ females, there are dramatic shifts in the positioning of target genes in the early embryo, but the order of gene positioning remains unchanged, the embryos survive to adulthood, and the adults are fertile. Survival would not be possible if activation of a critical target gene required a threshold greater than 50% of the maximum concentration of Bcd (Yu, 2008).
During early Drosophila embryogenesis, formation of the anterior-posterior (A/P) axis depends on spatial gradients of maternal morphogens. It is well recognized that positional information is transmitted from these morphogens to the gap genes. However, how this information is being transmitted is largely unknown. The transcriptional Mediator complex is involved in the fine tuning of the signaling between chromatin status, transcription factors and the RNA polymerase II transcription machinery. This study found that a mutation in the conserved subunit of the Mediator complex, dMED31, hampers embryogenesis prior to gastrulation and leads to aberrant expression of the gap genes knirps and Krüppel and the pair-rule genes fushi tarazu and even-skipped along the A/P axis. Expression of the maternal morphogens dorsal and hunchback was not affected in dMED31 mutants. mRNA expression of dMED31 exactly peaks between the highest expression levels of the maternal genes and the gap genes. Together, these results point to a role for dMED31 in guiding maternal morphogen directed zygotic gap gene expression and provide the first in vivo evidence for a role of the Mediator complex in the establishment of cell fate during the cellular blastoderm stage of Drosophila (Bosveld, 2008).
Proper fine tuning of the eukaryotic transcriptome depends on numerous cis and trans acting factors that modulate the chromatin environment of genes and influence the RNA polymerase II (RNAPII) transcription machinery. The Mediator complex is a core processor in the signaling between RNAPII and transcription factors. This complex is an evolutionary conserved protein assembly of 25-30 subunits (Bosveld, 2008).
Support for a specific role during development of Mediator subunits is provided by several studies in Drosophila. These studies describe mostly functions of Mediator subunits during late developmental stages, but a role of the subunits during early embryonic development is largely unknown. The Mediator consists of more then 25 subunits, pointing to a multifaceted role of this complex during metazoa development. Understanding this complexity starts with the identification of the function of each subunit (Bosveld, 2008).
Drosophila MED31 was identified by bioinformatics analysis (Boube, 2000) and its presence in the Mediator complex was confirmed in purified complexes from embryos and cells (Park, 2001; Gu, 2002). In a pull down assay, the Mediator (containing dMED31) complex binds to the transcription factors Bicoid (Bcd), Krüppel (Kr), Fushi tarazu (Ftz), Dorsal (Dl) and HSF, but not Twist (Twi), Hunchback (Hb) and Even-skipped (Eve) (Park, 2001). Moreover, the Mediator complex is required for in vitro transcription from developmentally important promoters regulated by these transcription factors. Despite these in silico and in vitro results, to date the functional role of MED31 in eumetazoa remains elusive. This study reports the identification of the highly conserved Drosophila Mediator subunit dMED31 as a novel maternal-effect gene necessary for proper segment specification during early embryogenesis. dMED31 mutant females have fecundity defects and embryos deposited by homozygous mothers display severe defects along the anterior-posterior (A/P) axis when gastrulation is initiated. Whereas expression of maternal morphogens is not affected, alterations in gap and pair-rule gene expression during the proceeding blastoderm stage correlate with these defects observed in dMED31 mutant embryos. Remarkably, a small percentage of the progeny of homozygous mutant females escape from embryonic death and develop into adults. These escapers have defects in their abdominal segmentation pattern, a phenotype enhanced by mutations in dMED13. These findings provide the first in vivo evidence for a specific role of dMED31 in establishing cell fate in the cellular blastoderm and point to a role for the Mediator in guiding maternal morphogen directed zygotic gap gene expression (Bosveld, 2008).
These findings identify a component of the conserved eukaryotic transcriptional Mediator complex, dMED31, that is required for normal initiation of zygotic gene expression during the blastoderm stage of Drosophila embryogenesis. Female flies that carry a mutation in the dMED31 gene suffer from fecundity defects and the embryos deposited by these females display abnormal embryogenesis due to aberrant cell migration events upon gastrulation. Impaired embryogenesis coincided with changes in kni, Kr, ftz and eve expression along the A/P axis. Furthermore, adult flies derived from embryos that escaped from embryonic death displayed severe defects in their abdominal segmentation. Because mRNA production was hampered in dMED311/1, these abdominal defects were likely the result of abnormal maternal and zygotic dMED31 mRNA production. A mutation in the Mediator subunit dMED13 also caused segmentation defects and this mutant enhanced the dMED31 mutant maternal effect phenotype. Therefore, these data indicate that the Mediator complex directs zygotic gene expression upon egg deposition to establish cell fate in the embryonic blastoderm (Bosveld, 2008).
In order to accomplish cell fate determination, cells gain a transcriptional poised state during early embryogenesis that is maintained throughout development and requires many cis and trans acting factors that modulate the chromatin environment of the genes involved. In Drosophila, cell identity along the A/P is established in the blastoderm stage when the pair-rule genes are expressed. A/P polarity is controlled by the maternal morphogenes cad, nos, bcd and hb whose activity results in the spatio-temporal expression of the gap genes gt, kni, tll and Kr. These gap genes are the first genes expressed along the A/P axis and encode transcription factors that in turn govern patterned expression of the pair-rule genes. Pair-rule gene expression occurs in distinct stripes and is accompanied by cellularization. Thus when cellularization takes place, large clusters of cells gain an imprint that defines the primordial segments. Cell identity is fine tuned when expression of the segment polarity and Hox genes is activated. Although this cascade of maternal, gap, pair-rule and segment polarity genes is well studied, much remains unknown how the maternal morphogens regulate RNAPII activity at their cognate promoters in order to establish regional domains of gap gene expression (Bosveld, 2008).
Because segmentation defects in escaper flies derived from dMED311/1 mothers were restricted to the abdomen, it is possible that the bithorax complex (BX-C) is abnormally expressed. This complex contains the homeotic genes Ultrabithorax, abdominal A and Abdominal B, which control the identity of the posterior two-thirds of the fly. Mutations in hb, Kr, tll and kni affect expression of BX-C and result in homeotic transformation. No complete homeotic transformations of entire parasegments were observed, suggesting an indirect effect of dMED311 on Hox activation. Since segment identity is established during early embryogenesis, this implies that only groups of cells and not whole primordial segments gained abnormal imprinting. Regional errors in cellular imprinting are supported by the variety of the abdominal defects observed in adult flies. Moreover, defects in embryogenesis were accompanied by cell loss at the embryonic poles and aberrant migratory behavior of cells upon gastrulation, processes which occur prior to the activation of the segment polarity and the Hox genes. Finally, early developmental defects coincided with abnormal expression of the gap genes kni and Kr and subsequently the pair-rule genes ftz and eve. These genes are expressed prior to Hox gene expression and are required for activation/repression of the Hox cluster. Although it is possible that the abdominal region is preferentially sensitive for a mutation in dMED31, it is more likely that random defects during formation of the abdomen are tolerated, whereas defects in other regions of the embryo are incompatible with adult viability and these adults never eclose (Bosveld, 2008).
Several intriguing questions remain: why is the embryonic phenotype so variable (>90% of the mutant embryos die, while a small percentage of embryos is able to reach the adult stage), why are mainly embryos affected by a mutation in dMED31 and what is the primary embryonic defect caused by a mutation in dMED31? Answers to these questions can be derived from studies of the Mediator in yeast in combination with the current data. The budding yeast MED31 protein is part of the Mediator transcription initiation complex. Although a mutation in yeast MED31 affects gene expression, mutants display no sensitivity to transcriptional inhibition by 6-azauracil and MED31 is not essential for growth. However, yeast MED31 mutants have a synthetic growth defective phenotype when combined with mutations in genes encoding for the two largest subunits of RNAPII (RPB1, RPB2) and the transcription initiation factors TFIIB and TFIIS. As in yeast, depletion of dMED31 in Drosophila SL2 cells by RNAi does not interfere with the Mediator composition and no growth alterations have been reported. Thus, Drosophila MED31, like yeast MED31, might not be essential for RNAPII activity per se, but could be an auxiliary factor involved in the signaling between specific transcription factors and the RNAPII machinery. Together, these findings and the current data suggest that dMED31 is not required for transcription in general, but is merely required for the fine tuning of transcription of specific genes (Bosveld, 2008).
Based on studies in yeast, it has been proposed that the Mediator functions as a platform that allows rapid regulation of transcription at (re)initiation. Fast regulation and (re)initiation of transcription might be key during the interphase periods of the final syncytial cell cycles when zygotic transcription is initiated, while such large scale, strict and 'fast' control over transcription would not be essential during subsequent stages of development and thus may explain why dMED31 function is essential during early embryogenesis. The observation that a small percentage of embryos derived from dMED311/1 mothers is able to develop into an adult, while the majority of the embryos displayed severe defects during embryogenesis, might also be attributed to such auxiliary function(s) of the dMED31 protein. Minor differences in dMED31 protein levels, due to the hypomorphic dMED311 allele, may result in subtle changes in the expression of the gap and pair-rule genes and allow embryos to progress throughout embryogenesis, but with the formation of segmentation defects. On the other hand, in the majority of embryos, more severe changes in gap and pair-rule patterning occur, which results in embryonic death (Bosveld, 2008).
In summary, this study demonstrated that dMED31 is essential to establish regional domains of expression of cell fate determinants kni, Kr, ftz and eve. mRNA expression of dMED31 peaks exactly between maternal morphogen and gap gene expression and it has been demonstrated that the Mediator complex is able to bind to several maternal transcription factors. Together this indicates that the Mediator complex constitutes an interface between the maternal morphogens and the RNAPII machinery to guide zygotic gene expression of cell fate determinants that specify primordial segment identity. These findings provide the first in vivo evidence for a role of the Mediator complex in establishing cell fate during early embryogenesis and since MED31 resembles one of the most conserved subunits within the Mediator complex this protein could serve a crucial role in the control of RNAPII activity during early developmental processes in all higher eukaryotes (Bosveld, 2008).
Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).
The establishment and maintenance of gene expression patterns in
development is regulated in part at the level of chromatin modification
through the concerted actions of the Polycomb and trithorax family of genes
(PcG/trxG). In Drosophila, Polycomb and Trithorax response elements
(PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb
protein complexes and maintain active or silent states, presumably in a
heritable manner. In mammalian cells however, such PRE/TREs have not been
conclusively identified. Polycomb and Trithorax gene products function by
methylating specific histone lysine residues, yet how these complexes
recognize individual loci in a temporal and tissue specific manner during
development is unclear. Recently, a novel protein, PTIP (also
known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins
(Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax
DNA-binding site. These data suggest that Pax proteins, and other similar
DNA-binding proteins, can provide the locus and tissue specificity for HMT
complexes during mammalian development (Fang, 2009).
In mammals, the PTIP protein is found within an HMT complex that includes
the SET domain proteins ALR (GFER) and MLL3, and
the accessory proteins WDR5, RBBP5 and ASH2. This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip-/- mouse embryos and conditionally inactivated Ptip-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP
protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to
phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals,
PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a
time when the mesoderm becomes compartmentalized into axial, intermediate and
lateral plate. These data suggest that PTIP provides a link between tissue
specific DNA-binding proteins that specify cell lineages and the H3K4
methylation machinery (Fang, 2009).
To extend these finding to a non-mammalian organism and address the
evolutionary conservation of Ptip, it was asked whether a Drosophila
ptip homolog could be identified and if so, whether it is also an
essential developmental regulator and part of the epigenetic machinery. The
mammalian Ptip gene encodes a novel nuclear protein with two
amino-terminal and four carboxy-terminal BRCT domains, flanking a
glutamine-rich sequence. Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning
defects and developmental arrest, whereas zygotic null mutants developed to
the third instar larval stage but also exhibited anterior/posterior (A/P)
patterning defects. In cell culture, depletion of Polycomb-mediated repression
activates developmental regulatory genes, such as the homeotic gene
Ultrabithorax (Ubx). This derepression is dependent on trxG
activity and also requires PTIP. Microarray analyses in cell culture of
Polycomb and polyhomeotic target genes indicate that many,
but not all, require PTIP for activation once repression is removed. The
activation of PcG target genes is coincident with PTIP binding to promoter
sequences and increased H3K4 trimethylation. These data argue for a conserved
role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).
Embryonic development requires epigenetic imprinting of active and inactive
chromatin in a spatially and temporally regulated manner, such that correct
gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic
development. In larval development, ptip
coordinately regulates the methylation of histone H3K4 and demethylation of
H3K27, consistent with the reports that mammalian PTIP complexes with HMT
proteins ALR and MLL3, and the histone demethylase UTX. In wing
discs, ptip is required for appropriate A/P patterning by affecting
morphogenesis determinant genes, such as en and ci. These
data demonstrate in vivo that dynamic histone modifications play crucial roles
in animal development and PTIP might be necessary for coherent histone coding.
In addition, ptip is required for the activation of a broad array of
PcG target genes in response to derepression in cultured fly cells. These data
are consistent with a role for ptip in trxG-mediated activation of
gene expression patterns (Fang, 2009).
Early development requires ptip for the appropriate expression of
the pair rule genes eve and ftz. The characteristic
seven-stripe eve expression pattern is regulated by separate enhancer
sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The
characteristic 14 stripes of en expression depends on the correct
expression of pair rule genes, which are clearly affected in ptip
mutants. However, the maintenance of en expression at later stages
and in imaginal discs is regulated by PREs and PcG proteins.
If ptip functions as a trxG cofactor, then expression of en
along the entire A/P axis in the imaginal discs of ptip mutants might
be due to the absence of a repressor. This might explain the surprising
presence of ectopic en in the anterior halves of imaginal discs from
zygotic ptip mutants. This ectopic en expression is likely
to result in suppression of ci through a PcG-mediated mechanism. Yet,
it is not clear how en is normally repressed in the anterior half,
nor which genes are responsible for derepression of en in the
ptip mutant wing and leg discs (Fang, 2009).
The direct interaction of PTIP protein with developmental regulatory genes
is supported by ChIP studies in cell culture. Given the structural and
functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG
target genes that are activated upon loss of PC and PH activity. Consistent
with the interpretation that a PTIP trxG complex is necessary for activation
of repressed genes, mPTIP only bound to DNA upon loss of Pc and
ph function. In the Kc cells, suppression of both Pc and
ph results in the activation of many important developmental
regulators, including homeotic genes. A recent report details the genome-wide
binding of PcG complexes at different developmental stages in Drosophila and
reveals hundreds of PREs located near transcription start sites.
Strikingly, most of the genes found to be activated in the Kc cells after PcG
knockdown also contain PRE elements near the transcription start site (Fang, 2009).
In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to
promote assembly of an H3K4 methyltransferase complex. The tissue and locus
specificity for assembly may be mediated by DNA-binding proteins such as PAX2
(Patel, 2007) or SMAD2 (Shimizu, 2001), which
regulate cell fate and cell lineages in response to positional information in
the embryo. In flies, recruitment of PcG or trxG complexes to specific sites
also can require DNA-binding proteins such as Zeste,
DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG
complexes. Purification of a trxG complex capable of histone acetylation
(TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By
contrast, the mammalian MLL/ALL proteins are components of large multi-protein
complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).
Mammalian PTIP is also thought to play a role in the DNA damage response
based on its ability to bind to phosphorylated p53BP1. PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases. Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex. Both
budding and fission yeast contain multiple BRCT domain proteins that are
involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All
of these yeast proteins have mammalian counterparts. However, neither the
fission nor budding yeast genomes encodes a protein with six BRCT domains and
a glutamine-rich region between domains two and three, whereas such
characteristic PTIP proteins are found in Drosophila, the honey bee,
C. elegans and all vertebrate genomes. These comparative genome
analyses suggest that ptip evolved in metazoans, consistent with an
important role in development and differentiation (Fang, 2009).
In summary, Drosophila ptip is an essential gene for early
embryonic development and pattern formation. Maternal ptip null
embryos show early patterning defects including altered and reduced levels of
pair rule gene expression prior to gastrulation. In cultured cells PTIP
activity is required for the activation of Polycomb target genes upon
derepression, suggesting an important role for the PTIP protein in
trxG-mediated activation of developmental regulatory genes. The conservation
of gene structure and function, from flies to mammals, suggests an essential
epigenetic role for ptip in metazoans that has remained
unchanged (Fang, 2009).
Individual neurons adopt and maintain defined morphological and
physiological phenotypes as a result of the expression of specific
combinations of transcription factors. In particular, homeodomain-containing
transcription factors play key roles in determining neuronal subtype identity
in flies and vertebrates. dbx belongs to the highly divergent H2.0
family of homeobox genes. In vertebrates, Dbx1 and Dbx2
promote the development of a subset of interneurons, some of which help
mediate left-right coordination of locomotor activity. This study identifies and
shows that the single Drosophila ortholog of Dbx1/2
contributes to the development of specific subsets of interneurons via
cross-repressive, lineage-specific interactions with the motoneuron-promoting
factors eve and hb9 (exex). dbx is
expressed primarily in interneurons of the embryonic, larval and adult central
nervous system, and these interneurons tend to extend short axons and be
GABAergic. Interestingly, many Dbx+ interneurons share a sibling
relationship with Eve+ or Hb9+ motoneurons. The
non-overlapping expression of dbx and eve, or dbx
and hb9, within pairs of sibling neurons is initially established as
a result of Notch/Numb-mediated asymmetric divisions. Cross-repressive
interactions between dbx and eve, and dbx and
hb9, then help maintain the distinct expression profiles of these
genes in their respective pairs of sibling neurons. Strict maintenance of the
mutually exclusive expression of dbx relative to that of eve
and hb9 in sibling neurons is crucial for proper neuronal
specification, as misexpression of dbx in motoneurons dramatically
hinders motor axon outgrowth (Lacin, 2009).
As beautifully illustrated by Ramon y Cajal, the nervous
system is remarkable for its diversity of cellular phenotypes. In fact, recent
physiological and expression studies suggest the presence of thousands of
distinct neuronal subtypes in the mammalian brain. The
genetic and molecular basis through which individual or small groups of
neurons adopt and maintain specific, often unique, morphological and
physiological characteristics (neuronal specification) remains poorly
understood (Lacin, 2009).
Studies in mice, Drosophila and C. elegans
highlight the importance of a large and growing number of transcription
factors, which act in a combinatorial manner to govern neuronal specification. Most of these transcription factors are expressed in complex, partially overlapping
patterns of neurons, with the specific differentiated phenotype of a neuron
being largely dictated by the precise complement of transcription factors it
expresses. As detailed below, much of this work has focused on the
specification of distinct motoneuron subtypes due in part to the relative ease
of distinguishing individual or groups of motoneurons from each other based on
axonal trajectory. Less is known about the factors that govern the
specification of interneuron subtypes, even though interneurons outnumber
motoneurons and can also be grouped based on morphology. For example,
interneurons in the Drosophila central nervous system (CNS) outnumber
motoneurons by about 10-fold, and can be roughly divided into intersegmental
interneurons, which extend projections that span more than one segment, and
local interneurons, which terminate their axons within the segment of
origin (Lacin, 2009).
Regulatory interactions, often cross-repressive in nature, between
transcription factors that govern neuronal specification help ensure that
individual neurons adopt the appropriate cellular phenotype. For example, in
vertebrates cross-repressive interactions between the LIM-homeodomain (LIM-HD)
proteins LIM-1 (Lhx1: Mouse Genome Informatics) and ISLET1 (Isl1: Mouse
Genome Informatics) establish and maintain the non-overlapping expression of
these proteins in lateral and medial neurons of the lateral motor column,
respectively. Lhx1 and Isl1 direct their respective groups of motoneurons
to extend axons dorsally or ventrally into the limb mesenchyme in part by
regulating the expression of the repulsive guidance receptor, Epha4.
Mutually exclusive expression of two sets of transcription factors also
defines distinct motoneuron subtypes in the Drosophila CNS. Here, all
motoneurons that project axons to dorsal muscle targets express the
homeodomain protein even-skipped (eve). By
contrast, most motoneurons that project axons to ventral muscles co-express
the LIM-HD proteins Lim3 and Islet (Tailup: FlyBase), and the homeodomain
proteins Hb9 (Exex: FlyBase) and Nkx6 (HGTX: FlyBase).
Cross-repressive interactions between eve and hb9/nkx6 help
maintain these mutually exclusive expression patterns, and these genes in turn
help direct the projection patterns of their respective motoneurons along
different routes (Lacin, 2009).
Functional studies are beginning to tease apart the
transcriptional regulatory networks that govern the specification of
postmitotic neurons. However, such analysis is complicated, at least in
Drosophila, by the context-dependent nature of many of these
regulatory interactions. For example, eve and hb9 are each
expressed in about a handful of distinct groups of neurons per hemisegment,
yet eve is necessary to repress hb9 expression in only two
of the roughly 20 neurons that normally express eve.
Similarly, hb9 is required to inhibit eve expression in only
one or two of its expressing neurons and is sufficient to repress eve
in only a subset of Eve+ neurons in the CNS (Lacin, 2009).
Context-dependent regulatory interactions may reflect the underlying
organization of the Drosophila CNS. Essentially all cells in the CNS
derive from one of a limited set of stem-cell-like precursors, called
neuroblasts. Thirty neuroblasts develop per hemisegment, with each
neuroblast dividing in a stem-cell-like manner to produce a largely invariant
family or clone of neurons. Many different transcription factors are expressed
within the neurons of any one lineage, with such factors exhibiting
overlapping or mutually exclusive expression in the lineage in a
transcription-factor-dependent manner. In addition, most transcription factors
that govern neuronal specification are expressed in multiple different groups
of neurons, with each group of neurons likely to derive from a different
neuroblast lineage. Thus, context-dependent regulatory interactions between
two such transcription factors may often reflect lineage-specific
interactions, with these interactions preferentially occurring in lineages in
which both factors are expressed versus lineages in which one or the other is
expressed. It is presently difficult to test this model, as in general the
individual lineages from which distinct groups of neurons marked by the
expression of a given transcription factor arise have not been delineated (Lacin, 2009).
In the vertebrate neural tube, Dbx1 and Dbx2, two
paralogous genes that encode homeodomain-containing proteins of the H2.0
class, are expressed within the p0, p1 and pD6 progenitor domains, with
Dbx1 expression nested within that of Dbx2. The
ventral limits of Dbx1 and Dbx2 expression are maintained
via cross-repressive interactions with Nkx6-2 and Nkx6-1, respectively.
Moreover, whereas Dbx1 and Dbx2 are expressed in neural
progenitors but not postmitotic neurons, Dbx+ progenitors give rise
to v0, v1 and dl6 interneurons, with a subset of V0 interneurons expressing
Evx, the vertebrate ortholog of eve, in a
Dbx1-dependent manner. Functional analysis of V0 interneurons reveals
that a subset of Evx-negative commissural interneurons makes inhibitory
connections with contralateral motoneurons that innervate hindlimb muscles;
Dbx function is required in these interneurons to regulate left-right
alternation of motoneuron firing, required for proper walking movements
(Lanuza, 2004; Lacin, 2009 and references therein).
This paper reports the identification and characterization of the
Drosophila dbx gene. Lineage tracing reveals that many
Dbx+ neurons share a sibling relationship with Eve+ or
Hb9+ motoneurons. The cellular phenotype of these pairs of sibling
neurons is strikingly distinct - Dbx+ interneurons are small and
extend short axons; Eve+ or Hb9+ motoneurons are large
and extend long axons. Notch/Numb-mediated asymmetric divisions establish the
non-overlapping expression of dbx and eve, or dbx and hb9, within each pair of sibling neurons. Cross-repressive interactions between dbx and eve, and dbx and hb9, then help maintain the complementary expression profiles of these transcription factors in the relevant sibling neurons, a process crucial for the ability of these neurons to adopt and maintain their distinct differentiated phenotypes (Lacin, 2009).
dbx is expressed in a dynamic pattern of neuroblasts, intermediate precursors termed ganglion mother cells (GMCs), and postmitotic cells. dbx is first expressed in a few neuroblasts
and GMCs in gnathal segments during early stage 11. Shortly
thereafter, dbx expression initiated in multiple cells within each
segment as well as in the brain; based on size, relative sub-epidermal position and marker gene expression, Dbx+ cells appeared to identify a stereotyped subset of neuroblasts, GMCs and postmitotic cells. After stage 12, most Dbx+ cells are
postmitotic, with some cells expressing dbx transiently and others
retaining dbx expression throughout embryogenesis. At the end of
embryogenesis, approximately 33 Dbx+ cells resided per thoracic
hemisegment and about 20 Dbx+ cells per abdominal hemisegment. Gnathal segments contained even more Dbx+ cells. Such
segment-specific differences in dbx expression probably reflect
homeotic gene input (Lacin, 2009).
Many CNS neurons retain dbx expression into larval stages,
during which time additional Dbx+ neurons arise in the nerve cord. Dbx+ neurons are also found in the CNS of
adults. A few Dbx+ are observed in neuroblasts in thoracic segments of third instar larvae. These Dbx+
neuroblasts appeared to bud off Dbx+ GMCs and neurons, as small
clusters of Dbx+ neurons resided immediately adjacent to these
neuroblasts. Thus, many neurons
maintained dbx expression for extended periods of time in larvae,
pupae and adults, consistent with dbx helping to maintain the
specific differentiated phenotype of these neurons (Lacin, 2009).
Double-label studies with molecular markers that label all neurons (ELAV), all glia (Repo) or most motoneurons (Eve/Hb9) revealed that essentially all postmitotic Dbx+ cells are interneurons. For
example, by the end of embryogenesis all postmitotic Dbx+ cells
express ELAV but not Repo. In addition, no
Dbx+ neurons express eve or hb9, which together mark
most embryonic motoneurons, identifying Dbx+ neurons as
interneurons. Together with the data detailed above, these results indicate
that as in vertebrates, dbx expression labels progenitor cells of
interneurons, and in contrast to vertebrates many interneurons maintain
dbx expression in flies (Lacin, 2009).
Dbx+ neurons identify a largely uncharacterized population of
interneurons; little if any co-expression is observed between dbx
and markers of different subsets of interneurons, such as engrailed,
dachshund, nmr-1 (H15 - FlyBase), nmr-2 (mid -
FlyBase) and eagle. However, many Dbx+ interneurons are GABAergic; significant co-expression occurs between Dbx and glutamic acid decarboxylase (GAD), a marker of GABAergic neurons. By contrast, no Dbx+ interneurons appeared to be seratonergic, dopaminergic or glutamatergic by the first larval instar stage, and only one Dbx+ interneuron is cholinergic. Since GABAergic interneurons are inhibitory in Drosophila as in vertebrates, it is inferred that many embryonic Dbx+ cells are inhibitory GABAergic interneurons (Lacin, 2009).
dbx expression thus marks a poorly defined population of
interneurons. To characterize Dbx+ interneurons in greater detail a modified version of the FLP/FRT system was used to map their lineage. Random clones of
tau-myc-GFP+ cells were generated in otherwise wild-type embryos, and then GFP+ lineage clones were screened for that contain Dbx+ neurons. Comparison of the
morphology and location of such clones relative to the location and morphology
of identified neuroblast lineages as determined by Dil-labeling enabled
mapping of essentially all Dbx+ neurons in abdominal segments to
five neuroblast lineages. This approach also revealed that at least two additional neuroblasts, preliminarily identified as NBs 2-4 and 6-4, produced
Dbx+ neurons in thoracic segments, and that many Dbx+
neurons exhibited at most short axonal projections (Lacin, 2009).
The NB4-2 lineage produces four small Dbx+ interneurons in
abdominal segments and seven Dbx+ neurons in thoracic segments.
This lineage contains the CoR motoneurons, which project axons out of the
segmental nerve (SN), and the Eve+ RP2 motoneuron, which projects
its axon out of the inter-segmental nerve (ISN). The four Dbx+ neurons included RP2 sib and at least two CoR sibs. These Dbx+ neurons extended at most short axons, consistent with previous observations that all interneurons in this lineage are local interneurons (Lacin, 2009).
The NB5-2 lineage produces two medial Dbx+ neurons within a
large family of over 20 cells. Axons from this clone cross the midline via
the anterior and posterior commissures, with a single
motoneuron, the AC motoneuron, projecting its axon across the midline and out
of the SNb nerve branch (Lacin, 2009).
The NB6-1 lineage produces two Dbx+ neurons in abdominal
segments and four Dbx+ neurons in thoracic segments at the end of
embryogenesis. Abdominal NB6-1 clones analyzed at stage 13 included three additional Dbx+ neurons as well as dbx expression in NB6-1. Thus, some cells expressed dbx transiently in this lineage. The neurons that retain dbx expression appear to be late-born neurons; they resided at the extreme ventral surface of the NB6-1 family of neurons (Lacin, 2009).
The NB6-2 lineage contains three Dbx+ neurons in abdominal
segments and five Dbx+ neurons in thoracic segments. Axonal
projections from these clones cross the midline as two bundles through the
posterior commissure; these bundles arch anteriorly, mirroring the
morphology of NB6-2 clones. These neurons expressed dbx at levels below those observed in other lineages (Lacin, 2009).
The NB7-1 lineage produces eight small Dbx+ neurons in thoracic
and abdominal segments, with many of these neurons residing next to the
well-characterized and lineally related Eve+ U motoneurons, the
close proximity of U motoneurons and Dbx+ neurons reflect sibling
relationships in multiple cases. In accord with the previous observation that
all interneurons in this lineage are local interneurons, all Dbx+ neurons in this lineage extended at most short axons (Lacin, 2009).
Expression and additional lineage-tracing studies confirmed that the
juxtaposition of Dbx+ interneurons next to Eve+ or
Hb9+ motoneurons reflects sibling relationships in many cases. For
example, double-labeling studies in wild-type embryos revealed transient
co-expression of eve and dbx in RP2 sib and in the siblings
of the U1-U3 motoneurons immediately after their birth from Eve+
GMCs. Whereas eve expression is quickly extinguished in these cells, dbx
expression is maintained in RP2 sib until the end of embryogenesis, and in
the U1-U3 sibs for an extended period of time (the presence of eight
Dbx+ neurons in the 7-1 lineage rendered it difficult to follow
individual Dbx+ neurons throughout embryogenesis) (Lacin, 2009).
To ascertain if all Eve+ U motoneurons share a sibling
relationship with Dbx+ neurons, two-cell clones were generated
marking each U motoneuron and its sibling. Each U motoneuron
can be unambiguously identified based on its relative position. Thus, this approached revealed that, like RP2, the U1 and U5 sibs expressed dbx from shortly after their birth until the end of embryogenesis. Similarly, the U2 and U3 sibs expressed dbx from their birth until stage 14, at which point they begin to downregulate dbx expression. Although the U4 sib did not express dbx after stage 15, two-cell U4 clones were not obtained before stage 15. Thus, the U4 sib may transiently express dbx. It is concluded that most of the sibling interneurons of the RP2 and U motoneurons express dbx transiently or continuously during embryogenesis (Lacin, 2009).
The generation of many two-cell clones within the NB4-2 lineage that
contained one Hb9+ CoR motoneuron and one Dbx+
interneuron confirmed sibling relationships between Dbx+
interneurons and Hb9+ motoneurons. In contrast to U
motoneurons, individual CoR motoneurons could not be identified unambiguously
by position. Thus, the clones may not have marked all CoR motoneurons.
However, four five-cell clones were identified that contain three
Hb9+ CoR motoneurons and two Dbx+ interneurons, pointing to a one-to-one sib relationship between at least two CoR motoneurons and
Dbx+ interneurons. The 5-2 lineage is the only other lineage in
abdominal segments that contained Dbx+ interneurons and an
Hb9+ motoneuron. Although it was not possible to create two-cell clones containing this motoneuron, sublineage clones are consistent with a sibling relationship between this motoneuron and a Dbx+ interneuron. Thus, lineage and expression studies reveal that dbx expression labels
the sibling interneuron of at least seven motoneurons in each abdominal
segment; all of these interneurons are local interneurons that extend short axons or no axons (Lacin, 2009).
Analysis of dbx, eve and hb9 expression in embryos
homozygous mutant for numb or spdo - two genes that exert
opposite effects on Notch/Numb-mediated asymmetric divisions - confirmed the
observed sibling relationships between Dbx+ neurons and
Eve+ or Hb9+ motoneurons. For example, loss of
numb function led to reciprocal effects on eve and
dbx expression in the 4-2 and 7-1 lineages, with duplication of
Eve+ U motoneurons occurring at the expense of Dbx+
interneurons in the 7-1 lineage, whereas RP2 sib (Dbx+) was
duplicated at the expense of the Eve+ RP2 in the 4-2 lineage. Removal of spdo function elicited the reciprocal effect on dbx and
eve expression in these lineages. Similarly, loss of numb
(or spdo) function led to reciprocal effects on dbx and
hb9 expression in the 4-2 and 5-2 lineages. The spdo and
numb mutant phenotypes each displayed essentially 100% penetrance and
expressivity with respect to the groups of neurons assayed. It is concluded that
Notch/Numb-mediated asymmetric divisions direct the expression of dbx
and eve (or dbx and hb9) to opposite siblings
within multiple sib pairs, and in so doing establish the non-overlapping
nature of dbx and eve, and dbx and hb9
expression in different pairs of sibling neurons (Lacin, 2009).
The transient co-expression of dbx and eve in RP2 sib and U sib interneurons raises the possibility that negative regulatory interactions between dbx and eve help maintain the non-overlapping nature of the expression profiles of these factors in distinct sets of sibling neurons. To assay for regulatory interactions between dbx, eve and hb9, a null allele of dbx was created via imprecise P element excision, and a full-length UAS-dbx transgene was also prepared. Systematic loss of function and misexpression studies uncovered cross-repressive interactions between dbx and eve, and dbx and hb9, that were largely specific to those lineages that produce sib pairs of Dbx+/Eve+ or Eve+/Hb9+ neurons
(Lacin, 2009).
In embryos homozygous mutant for dbxδ48 inappropriate retention of eve expression was observed in the normally
Dbx+ RP2 sib. A majority of thoracic, but a minority
of abdominal (21%), segments exhibited the RP2 sib phenotype. Conversely, elav-Gal4-mediated misexpression of dbx in all postmitotic neurons was sufficient to repress eve expression in the RP2 and U motoneurons but not in aCC/pCC or the EL neurons, the other Eve+ neurons in the CNS. Thus, dbx is necessary and sufficient to repress eve in the RP2/RP2 sib pair of sibling neurons, and sufficient but not necessary to repress eve expression in U motoneurons, revealing that the ability of dbx to repress eve is restricted to those lineages that produce Eve+ and Dbx+ sibling neurons (Lacin, 2009).
In reciprocal experiments, embryos that lacked eve function
specifically in the CNS displayed normal dbx expression. By
contrast, eve misexpression throughout the CNS specifically repressed
dbx expression in RP2 sib (and other neurons in the 4-2 lineage) and
the U sib neurons (and other neurons of the 7-1 lineage); dbx expression in all other lineages was grossly normal. Thus, the ability of eve to repress dbx also appears restricted to those lineages that normally produce Dbx+ and Eve+ sibling neurons (Lacin, 2009).
Analogous tests revealed similar cross-repressive interactions between
dbx and hb9. Loss of dbx or hb9 function
had no effect on hb9 or dbx expression, respectively.
However, dbx misexpression repressed hb9 expression in the
CoR motoneurons of the 4-2 lineage as well as in the Hb9+ neurons
of the 5-2 lineage. Also reduced hb9 expression was observed in other neurons; however, the lineages to
which these cells belong are unknown. Conversely, generalized hb9
misexpression repressed dbx expression in the RP2 sib and CoR sibs of
the 4-2 lineage as well as the Dbx+ neurons of the 5-2 and 7-1
lineages. Thus, dbx
and hb9 also exhibit cross-repressive interactions that are largely
restricted to those lineages that produce Dbx+ and Hb9+
sibling neurons. Together these functional studies suggest that negative
regulatory interactions between dbx and eve, and
dbx and hb9, help maintain the mutually exclusive expression
patterns of these factors in different pairs of sibling neurons (Lacin, 2009).
Since many Dbx+ neurons extend short axons, it was asked whether
dbx regulates axonal growth. Although axonal projections are grossly
normal in embryos homozygous mutant for dbx, dbx misexpression in all
postmitotic neurons led to a decrease in the ability of many neurons to extend
axons. For example, 93% of hemisegments exhibited a significant decrease in motor axon projection into the periphery, with thinning or loss of the ISN nerve
(within which U and RP2 motoneurons project axons), the SNc nerve (CoR
motoneurons) and the SNb nerve (U and AC motoneurons). Within the nerve cord
thin and broken longitudinal connectives were observed, at 100% penetrance, between neuromeres as well as an occasional increase
in the apparent size of the axonal scaffold within neuromeres, raising the
possibility that dbx misexpression induces neurons that normally
project axons into the periphery or between segments to project axons locally.
It is concluded that dbx expression must be strictly repressed in
Hb9+ and Eve+ motoneurons for these neurons to extend
axons to their appropriate targets. Note dbx misexpression is
unlikely to convert motoneurons into interneurons, since expression of the
vesicular-Glutamate receptor, a relatively specific motoneuron marker, is
grossly normal in the face of generalized dbx expression (Lacin, 2009).
The ability of dbx misexpression to inhibit motor axon outgrowth
in the ISN, SNb and SNc branches generally correlates with its ability to
inhibit eve or hb9 expression in specific motoneurons that
extend axons in these branches. However, loss of hb9 or eve
function elicits significantly weaker motor axon phenotypes than that observed
upon generalized dbx misexpression. nkx6 has been shown to
promote axon outgrowth in flies, and dbx and nkx6 exhibit
cross-repressive interactions in the vertebrate neural tube. Thus,
the effect of dbx misexpression on nkx6 expression was assayed, and it was found that dbx was sufficient to reduce nkx6
expression in all, and to eliminate expression in some, Nkx6+
neurons, supporting the model that dbx limits axon growth at least in part by repressing nkx6. However, in contrast to vertebrates, loss of dbx function had no obvious effect on nkx6 expression, and neither loss of nkx6 function nor generalized Nkx6 misexpression grossly disrupted
dbx expression (Lacin, 2009).
Homozygous mutant dbxδ48
adult flies are viable, fertile and exhibit locomotor phenotypes. For example,
dbx mutant flies are sessile and uncoordinated, and perform poorly
in simple climbing assays, and although they can initiate the flight
response, they can not maintain flight. It is inferred that Dbx+ neurons function within neural circuits crucial for specific locomotor functions. Of note, in vertebrates (Lanuza, 2004), Dbx+ neurons are known to regulate proper left-right alternation of motoneuron firing required for proper walking movements (Lacin, 2009).
In conclusion, many Dbx+ interneurons share a sibling relationship with Eve+ or Hb9+ motoneurons, and the cellular phenotypes of these sibling neurons are highly disparate: Dbx+ interneurons
extend short axons; Eve+ or Hb9+ motoneurons extend long
axons. This work suggests a model for the establishment and maintenance of
distinct cellular phenotypes between sibling neurons. Initially,
Notch-mediated asymmetric divisions establish distinct transcription factor
expression profiles in sibling neurons, demonstrated in this study by the ability of such asymmetric divisions to direct dbx expression to one neuron and
eve (or hb9) expression to its sibling in multiple pairs of
sibling neurons. Once sibling neurons establish distinct transcription factor
expression profiles, cross-repressive interactions between these factors help
maintain gene expression differences between sibling neurons, implied in this study by the lineage-specific, cross-repressive regulatory relationships observed
between dbx and eve, and dbx and hb9. Such
cross-repressive interactions are crucial for proper neuronal differentiation,
since inappropriate dbx expression in motoneurons impairs motor-axon
projections. Transcription factors, such as dbx, eve and
hb9, which partake in cross-repressive interactions, also contribute
more directly to neuronal differentiation via regulation of downstream
effector genes. For example, eve upregulates the netrin receptor,
Unc-5, in RP2 and other dorsal motoneurons, which in turn helps guide the
motor-axons of these neurons to their appropriate targets (Lacin, 2009).
Genetic redundancy is a common theme between transcription factors that
exhibit cross-repressive regulatory interactions during neuronal
specification, and may in fact increase the robustness of this process. For
example, loss of function in dbx, eve or hb9 yields subtle
effects on the expression of the other two genes, whereas generalized
misexpression of each gene leads to clear changes in the expression of the
other two. In addition, because loss of dbx function
induces inappropriate retention of eve expression in about 50% of the
normally Dbx+ RP2 sib neurons, other transcription factors must
partially compensate for loss of dbx within RP2. Such factors might
also compensate for loss of dbx function during axon growth.
Moreover, hb9 and nkx6, which are expressed in nearly
identical patterns of CNS neurons, also act redundantly to repress
eve in a specific set of neurons. Within
the bristle lineage, the Su(H) and Sox15 transcription factors act redundantly
in the socket cell to repress the expression of Shaven, a Pax-family
transcription factor, thus restricting shaven expression to the
sibling shaft cell. This event is crucial for socket cell differentiation, since derepression of shaven in the socket cell transforms socket cells towards shaft cells (Lacin, 2009).
This work also highlights the lineage-specific nature of regulatory
relationships between transcription factors that govern neuronal
specification. For example, dbx is competent to inhibit eve
expression only in the two lineages that produce Dbx+ and
Eve+ sib neurons. Similarly, eve can inhibit dbx
expression in the same two lineages, but not in the three other lineages that
produce Dbx+ but not Eve+ neurons. Similar
lineage-specific, cross-repressive regulatory interactions occur between
transcription factors expressed in neurons born at different times within the
same lineage. The Hb9+ CoR and Eve+ RP2 motoneurons
probably arise from sequentially born GMCs within the 4-2 lineage, with
loss-of-function studies indicating that eve represses hb9
in RP2, consistent with hb9 repressing eve in the CoR
motoneurons. Thus, cross-repressive interactions between individual
transcription factors during neuronal specification often reflect
lineage-specific, rather than CNS-wide, regulatory relationships (Lacin, 2009).
Notch/Numb-mediated asymmetric divisions also exhibit lineage-specific effects on dbx and eve expression. During these asymmetric divisions, high-level Notch signaling in one daughter cell induces it to adopt Notch-dependent 'A' fate, whereas the absence of Notch signaling in the other daughter permits it to adopt the 'B' fate. In the 4-2 lineage Notch signaling promotes the development of Dbx+ interneurons and inhibits the formation of the Eve+. By contrast, in the 7-1 lineage Notch signaling inhibits the formation of Dbx+ neurons and promotes the development Eve+ U motoneurons. Thus, the presence/absence of Notch signaling exerts opposite effects on dbx and eve expression in a lineage-specific manner (Lacin, 2009).
In contrast to dbx and eve, Notch signaling inhibits the
formation of nearly all Hb9+ motoneurons. Notch signaling also
inhibits the motoneuron fate during the asymmetric divisions that produce the
RP2, aCC and three VUM motoneurons. The U motoneurons, which require Notch
activity to develop, are the only exception to Notch-mediated inhibition of
the motoneuron fate in flies. These observations are interesting in light of a
previous model that speculated that vertebrate motoneurons share a common
evolutionary ancestry with Drosophila Hb9+ motoneurons,
based on the common expression of hb9, lim3 and islet in
most vertebrate motoneurons and all fly motoneurons that project to ventral
body wall muscles. Might Notch signaling generally inhibit the motoneuron fate
in vertebrates? Recent work in zebrafish reveals that Notch inhibits the
motoneuron fate during the asymmetric divisions of some pMN progenitors that
yield sibling motoneuron and interneurons. However, the fraction of pMN progenitors that divide asymmetrically in this manner remains unclear. Thus, whether Notch signaling strictly inhibits the motoneuron fate during asymmetric divisions in vertebrates awaits further investigation (Lacin, 2009).
This work on dbx, eve and hb9 indicates that the
transcriptional networks that control neuronal specification are organized in
a lineage-specific manner. Support for this model comes from the
identification of three largely lineage-specific enhancers in the eve
regulatory region: one enhancer drives expression in the U neurons, one drives
expression in the lineally related EL neurons and a third drives expression in
the RP2 and a/pCC neurons. Might Dbx, Hb9 and the Notch pathway exert their
lineage-specific effects on eve directly via the appropriate
cis-regulatory module? Preliminary data support this notion, as consensus and
evolutionarily conserved Dbx-binding sites (Noyes, 2008) reside in the RP2 and U motoneurons enhancers. More generally, are the
regulatory regions of dbx, hb9 and other similarly functioning genes
also organized in a lineage-specific manner? And, do the regulatory regions of
the effector genes through which transcription factors such as Dbx, Hb9 and
Eve regulate neuronal differentiation reflect a similar organization? Future
work that addresses these questions should help clarify the cis-regulatory and
transcriptional logic that governs neuronal specification and differentiation
in Drosophila (Lacin, 2009).
In contrast to most transcription factors that govern neuronal subtype
identity, dbx is not an essential gene. Adult flies that lack
dbx function exhibit defects in flight and ambulatory movement. In
vertebrates, interneurons derived from Dbx+ progenitors coordinate
left-right alternation of motoneuron firing required for proper walking
movements via direct, probably inhibitory synaptic input to motoneurons
(Lanuza, 2004). The sibling relationship between Dbx+ interneurons and many motoneurons suggests that Dbx+ interneurons perform similar functions in Drosophila. Such speculation is supported by work in other insects, where small axonless interneurons modulate motoneuron function. In this light, the non-essential nature of dbx in flies may facilitate charting of
the neural circuits through which Dbx+ neurons regulate distinct
locomotor functions in Drosophila (Lacin, 2009).
Transcriptional repression is essential for establishing
precise patterns of gene expression during development. Repressors governing early Drosophila segmentation can be classified as short- or long-range factors based on
their ranges of action, acting either locally to quench adjacent
activators or broadly to silence an entire locus. Paradoxically,
these repressors recruit common corepressors, Groucho and CtBP, despite their different ranges of repression. To reveal the mechanisms underlying these two
distinct modes of repression, chromatin analysis was performed using the prototypical long-range repressor Hairy and the short-range repressor Knirps. Chromatin immunoprecipitation and micrococcal nuclease mapping studies reveal
that Knirps causes local changes of histone density and
acetylation, and the inhibition of activator recruitment,
without affecting the recruitment of basal transcriptional
machinery. In contrast, Hairy induces widespread histone
deacetylation and inhibits the recruitment of basal machinery without inducing chromatin compaction. This study provides detailed mechanistic insight into short- and long-range repression on selected endogenous target genes and suggests that the transcriptional corepressors can be differentially deployed to mediate chromatin changes in a context-dependent manner (Li, 2011).
To directly compare functional aspects of Hairy- and Knirps- mediated
repression in the Drosophila embryo,
these proteins interactions were studied with two segmentally expressed
pair-rule genes. Hairy directly represses fushi tarazu (ftz),
a secondary pair-rule gene expressed in the blastoderm
embryo in a seven-stripe pattern. ftz is regulated by both
regionally acting gap genes and the segmentally expressed
hairy pair-rule gene. Chromatin immunoprecipitation
(ChIP) experiments have revealed dense clusters of peaks
around the ftz gene for key transcription factors active in the
blastoderm embryo, including Caudal, Hunchback, Knirps,
Giant, Huckebein, Krüppel, and Tailless. These transcription
factors bind to the promoter-proximal Zebra element, the
stripe 1+5 enhancer located 3' of ftz, and a presumptive 5'
regulatory region located between 23 kbp and 28 kbp. Hairy has been found to bind in vivo to all of these regions. This repressor is expressed in a striped
pattern in the blastoderm embryo; therefore, the ftz gene is
active in some nuclei and repressed in others. In order to
obtain a homogeneous population of nuclei for chromatin
studies, Hairy protein was overexpressed in embryos using
a heat-shock driver, which results in complete repression of
ftz. This repression requires the recruitment of
the Groucho corepressor, because a mutant version of Hairy
that does not bind to Groucho fails to repress ftz (Li, 2011).
Interestingly, a titration of heat-shock induction resulted in
a nonuniform, progressive loss of specific ftz stripes, with
stripe 4 being the most sensitive and stripe 1+5 the least. This result points to the intriguing possibility that Hairy can act locally on specific enhancers, at least very transiently, although the end result of Hairy repression is
complete silencing of all enhancer elements. The asynchronous
repression of the ftz locus also suggests that Hairy-mediated
long-range repression does not act solely by direct
targeting the basal promoter, as suggested by a previous
model for this class of repressor, because this mechanism
should cause uniform inhibition of stripe elements.
Similar to ftz, the pair-rule gene even skipped (eve) is also
expressed in a seven-stripe pattern and is regulated by
multiple modular enhancers. eve is a well-characterized
target of the short-range repressor Knirps, which sets
posterior boundaries of eve stripe 3 and 4 and anterior borders
of eve stripe 6 and 7. After substantial overexpression
of Knirps (20 min heat-shock induction), the repressor is able
to repress all of the eve stripe enhancers except for the stripe
5 enhancer. When the induction is titrated, Knirps
represses individual enhancers in a stepwise manner, with
the most sensitive enhancers downregulated earliest, at
a low dose of Knirps. Together, these experiments indicate
that Hairy can initially act locally but ultimately acts in a globally
dominant fashion, whereas Knirps acts in a restricted manner (Li, 2011).
To compare the effects of repression by Hairy and Knirps, chromatin changes associated with repression of ftz
and eve were studied via ChIP. No significant change of
histone H3 occupancy were detected at regions sampled throughout the ftz
locus after Hairy overexpression (although some
regions showed modest differences. In contrast, Knirps
repression of eve resulted in significantly increased histone H3
density, particularly in two of the three regions corresponding
to the Knirps-sensitive enhancers, namely stripe 4+6 and
stripe 2. Little change was noted in the promoter region, transcribed region, or the stripe 1 and 5 enhancers, which are not readily repressed by Knirps. An
apparent increase in histone H3 density on the repressed
stripe 3+7 enhancer, although of low statistical significance, correlates with other alterations common to repressed enhancers, noted below (Li, 2011).
To provide a more detailed picture of chromatin structure, a micrococcal nuclease (MNase) mapping protocol
used in yeast and cultured cells was adapted for Drosophila embryos. MNase mapping showed that Hairy repression had little effect on chromatin accessibility throughout the ftz locus, whereas Knirps induced a significant
increase in MNase insensitivity specifically at the eve stripe
3+7, 2, and 4+6 enhancers and a minor increase in stripe 1
protection. The promoter and the eve stripe 5 enhancer were little changed, mirroring the patterns noted for overall histone H3 occupancy. The changes noted
for the eve locus appear to be specific, because Knirps did not
induce any change of a nontargeted intergenic site on the third
chromosome. Hairy also had no effect at this locus.
The similar results from overall histone H3 density and
MNase mapping suggest that Hairy-mediated long-range
repression does not involve a general compaction of chromatin
on the ftz locus. In contrast, repression by Knirps is
associated with an increase in the histone density of targeted
enhancer regions, which may result either from Knirps recruitment
of factors that mediate chromatin condensation or the
blocking of proteins responsible for loosening of chromatin.
Recruitment of Groucho by other repressor proteins is also
associated with distinct effects: Runt-dependent repression
of slp1 does not involve changes in H3 density, but Brinker
repression of the vgQ enhancer does. The distance
dependence of these repressors has not been established, but in light of the current results, it is apparent that the Groucho corepressor can be involved in distinct effects depending on the context of recruitment (Li, 2011).
Histone acetylation is dynamically regulated on transcribed
genes in eukaryotes, with histone acetylation generally correlated
with active loci. The histone deacetylase Rpd3 is
a component of both Hairy and Knirps corepressor
complexes; therefore, histone acetylation levels
were assayed across the eve and ftz genes before and after repression. Hairy repression resulted in widespread histone H4
deacetylation throughout the ftz locus. The ectopically expressed Hairy protein itself was not observed to spread but remained restricted to regions
of the gene previously observed to bind endogenous Hairy. Using anti-H3-acetylation antibodies, similar widespread H3 deacetylation was also noted. This
distributed effect on the ftz locus correlates with prior observations
that Hairy-mediated long-range repression might involve a Groucho-mediated 'spreading' mechanism. By this means, Rpd3 may be delivered to extensive areas of
a gene. To test whether a spreading of histone deacetylation
might correlate with the successive inhibition of ftz enhancers, histone acetylation levels were investigated across ftz after a brief 5 min heat shock followed by immediate fixing, before the entire complement of enhancers
can be repressed. In this setting, deacetylation was mostly
concentrated around the stripe 1+5 enhancer and the immediate
5' regulatory region, areas that show Hairy occupancy
in vivo. More distal 5' regulatory regions and the transcription
unit itself showed little initial change, consistent with a spreading action of this repressor during the more extensive repression period (Li, 2011).
A different picture emerged from studies of Knirps acting on
eve. Here, repression led to selective decreases in H3 and H4
acetylation levels, concentrated over the eve stripe 4+6 and
stripe 2 enhancers, with lesser decreases noted at stripe 3+7
and stripe 1 enhancers. A local change in acetylation was also noted near the transcriptional initiation site, but not immediately 5' and 3' of this area. The reductions in histone acetylation levels seen on both eve and ftz are
consistent with Hairy and Knirps recruiting deacetylases to
their target genes. However, it is striking that the broad
deacetylation mediated by Hairy on ftz is not associated with
dramatic changes in histone density or resistance to nuclease
accessibility, whereas increased histone density and resistance
to nuclease digestion are associated with Knirps repression
on eve. It is possible that in addition to inducing deacetylation,
Knirps triggers additional histone modifications or
interacts with nucleosome-remodeling complexes to further
alter chromatin at the enhancers. H3 lysine 27 methylation is
one chromatin signature associated with silenced genes;
however, no significant change in this modification was noted
at ftz or eve upon repression (Li, 2011).
Previous studies indicated that Hairy can effectively
repress a reporter gene without displacing the activators. Attempts were made to test whether this was the case on an endogenous
gene, ftz, by examining occupancy by Caudal, a transcription
factor that also activates eve. Caudal activates the
posterior stripes of both ftz and eve, and it was found that Caudal
binds the ftz 5' regulatory region and the promoter-proximal
Zebra element. Repression of the locus by Hairy did not affect the Caudal
binding pattern, similar to the results obtained with a Hairy-regulated reporter gene. In contrast, Knirps repression decreased Caudal occupancy specifically at
the eve 3+7 and 4+6 enhancers,
bringing overall protein occupancy down to near baseline
levels. This decrease is not an effect of global decrease of
Caudal occupancy, because the Caudal binding peak at the
eve promoter was not affected. A similar decrease in Caudal
occupancy was also observed on a hunchback enhancer after
repression by Knirps. Interestingly, Bicoid
occupancy of the eve stripe 2 and stripe 1 enhancers was
not altered by Knirps, although these enhancers were repressed. Clearly, loss of transcription factor occupancy is not required for short-range repression of
a cis-regulatory element. It is possible that different transcriptional
activators exhibit differential sensitivity to chromatin
changes induced during repression (Li, 2011).
New insights have suggested that many developmental genes,
including those regulated by short-range repressors such as
Snail, feature RNA polymerase paused in the promoter region
even in their inactive state, suggesting postrecruitment levels
of regulation. Components of the core
machinery were analyzed before and after repression by Hairy and Knirps.
Upon Hairy repression, a marked decrease of RNA polymerase
II (Pol II) occupancy was observed at the ftz locus. The same
trend was observed for the preinitiation, initiation, and elongation
forms of Pol II. These results suggest that Hairy
directly or indirectly blocks recruitment of Pol II. Similar decreases were noted with levels of TATA box-binding protein (TBP) at the promoter (Li, 2011).
In contrast, induction of Knirps did not change Pol II occupancy
at the eve transcription unit, even under condition where
most enhancers were repressed. (Under conditions
tested in this study, over three-quarters of the embryos had shut down
expression of all but stripe 1 and/or 5.) Similarly, TBP occupancy
remained at a comparable level before and after Knirps repression. The constant level of RNA polymerase on
the eve transcription unit was a surprise in light of the sharp
reduction in mRNA production as measured by in situ hybridization.
However, there is precedence for this effect: Runt repression
of slp1 appears to act through elongation control, which
causes no change of the concentration of Pol II on slp1.
Knirps may produce a similar effect by inducing a slower transit
rate of Pol II on the repressed eve locus. Similar observations
have been made at the hsp70 gene upon depletion of elongation
factors such as Spt6 or Paf1 (Li, 2011).
The differential distance dependence of short- and long- range
repressors such as Hairy and Knirps has been observed
in many contexts. However, the mechanisms by
which these proteins function have been poorly understood.
With the recent demonstration that transcriptional factors
considered to be short- and long-range repressors utilize
shared cofactors, namely CtBP and Groucho, there has been
a question of whether long-range repression is actually functionally distinct from short-range repression (Payankaulam, 2009). The current study
provides evidence that the chromatin states associated with
long- and short-range repressors are distinct in several ways.
It is not yet knowm whether the effects seen on ftz are
observed for all Hairy targets, although the similarity of changes
observed on the lacZ reporter subject to Hairy repression
suggests that they are conserved (Martinez, 2008). Similarly, the reproducibility
of Knirps-induced changes at different eve enhancers
indicates that this protein can effect related chromatin changes
on cis-regulatory modules bound by different activators. Snail,
another short-range repressor, also appears to mediate localized
deacetylation and activator displacement; thus, this mechanism
may be a common feature of this entire class of repressors
(Qi, 2009; Y. Nibu, personal communication to Li, 2011). It will be
interesting to determine how general are the observations
made in this study for long- and short-range repression, a question
that can be approached using genome-wide methods. In
any event, the highly divergent activities of Knirps and Hairy
demonstrated in this study not only underscore the fact that
these proteins can mediate biochemically divergent events
but also raise interesting questions about how similar cofactors
can participate in such distinct effects in a context-dependent
manner. It is possible that the corepressors adopt distinct
conformations when recruited by different repressors, or the
corepressor may form distinct complexes with unique activities. In addition to determining how cis- and trans-acting factors affect repression pathways, these mechanistic insights will provide important contextual information for interpretation of genome-wide transcription factor binding and chromatin
modifications and will inform quantitative modeling of cis-regulatory elements for the aim of understanding the activity and evolution of enhancers (Li, 2011).
Cis-regulatory modules (CRMs) are defined by unique combinations of transcription factor-binding sites. Emerging evidence suggests that the number, affinity, and organization of sites play important roles in regulating enhancer output and, ultimately, gene expression. This study investigated how the cis-regulatory logic of a tissue-specific CRM responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two E-twenty-six (ETS) members: the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, it is suggested that Yan and Pnt have distinct syntax preferences. Not only does Yan prefer high-affinity sites, but an overlapping pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the efficient Yan recruitment promoted by this high-affinity ETS supersite not only biases Yan-Pnt competition at the specific CRM but also organizes Yan-repressive complexes in three dimensions across the eve locus. Taken together, these results uncover a novel mechanism by which differential interpretation of CRM syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions (Boisclair Lachance, 2018).
Development of a multicellular organism relies on tissue-specific gene expression programs to establish distinct cell fates and morphologies. The requisite patterns of gene expression must be both spatiotemporally precise and robust in the face of genetic and environmental variation; this is achieved through the action of transcription factors (TFs), whose activating and repressive inputs are integrated at the cis-regulatory modules (CRMs) or enhancers of their target genes. Consequently, the sequence of each CRM provides a physical blueprint for a combinatorial regulatory code that translates upstream signaling information into downstream gene expression. While significant advances have been made in the ability to distinguish regulatory elements from background noncoding genomic DNA and identify consensus TF-binding motifs within them, understanding of how the intrinsic logic of the cis-regulatory syntax (namely, the number, affinity, position, spacing, and orientation of binding sites) organizes the necessary set of protein-protein and protein-DNA interactions remains poor. Because single-nucleotide polymorphisms in TF-binding sites are being increasingly correlated with altered gene expression and disease susceptibility, the ability to deduce the regulatory logic of an enhancer based on its sequence is important (Boisclair Lachance, 2018).
The tendencies for TFs to cluster into superfamilies and for cells to coexpress multiple nonredundant members of the same superfamily imply that enhancer syntax must enable TFs with very similar DNA-binding preferences to compete, cooperate, and discriminate between binding sites to achieve appropriate gene expression output. Recent insight into these behaviors has come from studies of Hox family TFs. The emerging model suggests a specificity-affinity trade-off such that low-affinity sites are best discriminated, while high-affinity sites can be bound by many different Hox factors. Clustering multiple low-affinity Hox sites permits the cooperative and additive interactions needed for robust gene activation responses without compromising specificity. Whether analogous syntax rules apply to other TF superfamilies is not known, and how transcriptional repressors solve the specificity-affinity problem remains to be tested (Boisclair Lachance, 2018).
The ETS superfamily includes both activators and repressors, all of which recognize the same core DNA sequence, 5'-GGAA/T-3'. ETS TFs are found across metazoan phyla and play key roles in regulating the gene expression programs that direct many aspects of normal development and patterning. Exemplifying this, the Drosophila transcriptional activator Pointed (Pnt) and the repressor Yan operate downstream from receptor tyrosine kinase (RTK) signaling pathways to orchestrate numerous cell fate transitions. Much of the current understanding of Yan and Pnt stems from studying their regulation of even-skipped (eve) expression during cardiac muscle precursor specification at stage 11 of embryogenesis and prospero (pros) expression during R7 photoreceptor specification in the developing eye. Abrogating Pnt-mediated activation or Yan-mediated repression of eve or pros leads to respective loss or ectopic induction of the associated cell fate. Gel shift assays using probes from eve or pros CRMs revealed that most Yan-bound ETS sites are also bound by Pnt, and subsequent high-throughput assays confirm their preferences for very similar sequences. Because none of the in vitro biochemistry has been done with full-length proteins, how accurately the results will predict the outcome of Yan-Pnt competition for ETS sites in CRMs in vivo is uncertain (Boisclair Lachance, 2018).
Hints that binding site syntax might influence Yan recruitment come from in vitro binding studies with TEL1, the human counterpart of Drosophila Yan, and from mathematical modeling of Yan's ETS site occupancy. TEL1 and Yan, unlike Pnt or its mammalian counterpart, ETS1, self-associate via their sterile α motifs (SAMs), and this homotypic interaction is essential for transcriptional repression in both flies and humans. Using gel shift assays, SAM-SAM interactions were shown to mediate cooperative binding of TEL1 at paired ETS sites. A recent theoretical analysis of TEL1/Yan occupancy at equilibrium explains how such cooperative SAM-SAM interactions might promote preferential recruitment to tandem ETS-binding sites. Because neither study examined repressive output, the question of whether preferential or cooperative binding of Yan to closely apposed ETS sites might bias Yan-Pnt competition to permit more complex discrimination of CRM syntax than current models assume remains pressing (Boisclair Lachance, 2018).
To evaluate how CRM syntax organizes the opposing repressive and activating inputs from Yan and Pnt to dictate precise transcriptional output, this study assessed the impact of mutating the eight putative ETS-binding sites identified in the eve muscle heart enhancer (MHE) that drives eve expression in 10 segmentally arrayed clusters of pericardial and muscle cells. This study found that sites with strong affinity best discriminate between Yan and Pnt, with paired sites showing the strongest bias. Thus, mutating a pair of overlapping, conserved, high-affinity ETS sites significantly elevated or expanded MHE reporter expression, consistent with compromised repression. Using CRISPR/Cas9 gene editing of the endogenous MHE, it was shown that mutation of this high-affinity ETS supersite reduced Yan recruitment to not only the MHE but also two other CRMs across the eve locus. Mesodermal Eve expression was elevated, consistent with compromised Yan recruitment, resulting in inadequate repression. In this compromised background, environmental and genetic stresses that would normally be buffered against were now sufficient to induce specification of ectopic Eve-positive (Eve+) cells and reduce survival. It is concluded that the conserved high-affinity ETS pair within the MHE plays a unique and pivotal role in not just recruiting Yan-repressive complexes to the isolated enhancer but also longer-range coordination of transcriptional complex organization and function across the locus (Boisclair Lachance, 2018).
Focusing on ETS-binding motifs within a conserved regulatory module, the eve MHE, this study identified a simple syntax that allows for robust qualitative and quantitative control of enhancer output. Based on extensive mutagenesis of this enhancer, it is proposed that Yan's and Pnt's respective preferences for high- and low-affinity ETS sites provide a mechanism for integrating their competing repressive and activating inputs at individual CRMs. In particular, it was found that the use of paired strong-affinity sites appears critical to the assembly of repressive complexes that dampen eve expression in newly specified cardiac precursors where Yan levels are low and prevent ectopic eve induction in the surrounding mesoderm where levels of activating TFs such as Twi are high. CRISPR/Cas9-mediated mutation of the endogenous MHE confirmed the importance of such optimized syntax for precise Eve expression levels and uncovered an unexpected role of the ETS2,3 dual Yan-binding supersite in longer-range organization of Yan complexes across the locus. It is speculated that efficient Yan recruitment to high-affinity supersites not only influences short-range interactions at the specific enhancer but also fosters longer-range communication across multiple CRMs (Boisclair Lachance, 2018).
The unique repressive contribution of the ETS2,3 pair may reflect an unconventional form of cooperative recruitment that provides novel regulatory capabilities to Yan-repressive complexes. Specifically, even though the two sites in the ETS2,3 pair are probably too close to permit simultaneous occupancy, their immediate juxtaposition may significantly increase the probability of stable Yan binding. For example, although nothing is known about the kinetics and dynamics of Yan-DNA interactions, the presence of two overlapping high-affinity binding sites could promote stable occupancy by increasing the chance that a newly dissociated Yan molecule would immediately rebind. The syntax could also support a more organized dynamic in which the two molecules of a Yan dimer toggle back and forth rapidly between bound and unbound states at the two sites. Even more speculatively, because SAM-mediated dimerization is required for Yan-mediated repression, if the configuration of the ETS2,3 pair ensured that one molecule of the dimer was always bound, this could leave the second free to interact with either an adjacent nonspecific sequence, as was modeled previously (Hope, 2017); another high-affinity ETS site elsewhere in the MHE (for example, site 8); or, even more speculatively, an ETS motif in the D1 or D2 CRM. It is also possible that the in vivo mechanism by which full-length Yan contacts DNA is different from that suggested by the in vitro assays. For example, interactions with other TFs and cofactors might somehow mitigate the steric clash to allow simultaneous occupancy by a Yan dimer. In this case, higher-order Yan complexes (for example, trimers or tetramers) could mediate the requisite longer-range contacts. Regardless of specific mechanism, the idea that high-affinity 'supersites' might be used to anchor longer-range TF-TF and TF-DNA interactions will be an interesting direction for future investigations (Boisclair Lachance, 2018).
Previous work exploring the in vivo functionality of a Yan protein in which the SAM-SAM interface has been mutated to prevent self-association further supports the importance of SAM-mediated repressor cooperativity. Specifically, it was found that although Yan monomers are recruited to enhancers genome-wide in a pattern close to that of wild-type Yan, adequate repression does not occur, and phenotypes consistent with yan loss of function ensue (Webber, 2013a). This work also noted the prevalence of clustered high-affinity ETS sites across a number of Yan ChIP targets, suggesting that the mechanisms uncovered in the dissection of MHE ETS site syntax might be broadly applicable. Focusing on eve, it is suspected that at the resolution of individual ETS sites, in the absence of SAM-mediated cooperativity, Yan occupancy of the ETS2,3 tandem would be insufficiently stable to either compete appropriately with Pnt at the MHE or organize the necessary 3D communication across the locus (Boisclair Lachance, 2018).
A parallel is noted between the consequences of mutating the high-affinity ETS2,3 supersite in the endogenous eve locus and the findings of an earlier analysis in which three different Yan-bound CRMs were deleted within a genomic Eve-YFP BAC transgene (Webber, 2013b). In this earlier study, while deleting the pattern-driving MHE almost completely ablated mesodermal Eve-YFP induction, deleting a 'repressive' Yan-bound element (referred to as the D1) increased Eve-YFP expression ∼1.5-fold and led to the specification of extra Eve+cells. Additionally, deletion of either the MHE or the D1 in the BAC transgene reduced Yan occupancy at not only the deleted element but also the remaining intact CRMs. This study reports a comparable loss of Yan occupancy across the eve locus upon mutation of the MHE ETS2,3 supersite but only a modest increase in Eve levels and no cell fate specification defects. The discrepancy between reduced Yan occupancy and increased Eve levels in the eveMHEmut2,3 mutant relative to the D1 deletion mutant suggests that deleting an entire CRM not only compromises Yan occupancy across the locus but also disrupts additional repressive inputs. Consistent with this interpretation, the eveMHEmut2,3 background appeared highly sensitized, with the increase in Eve levels and Eve+ cell fate specification associated with a twofold increase in pnt dose almost exactly matching the effects of deleting an entire 'repressive' CRM. Further exploration of how high-affinity ETS pairs organize Yan repression at and between CRMs and how this coordinates the competing and collaborating inputs from other TFs will be needed to test these ideas at eve and, more broadly, other target genes (Boisclair Lachance, 2018).
To conclude, a working model is proposed in which Yan's and Pnt's differential interpretation of ETS syntax adds a 'dimmer' capability to the classic on/off switch, thereby refining its sensitivity and tunability. Focusing on eve as an example, prior to the onset of RTK-induced cardiac cell fate specification or in cells subject to submaximal signaling, it is suggested that Yan's bias for high-affinity sites ensures an effectively 100% probability of occupancy at the ETS2,3 supersite and hence stable repression. In this regime, Yan could also outcompete Pnt at the lower-affinity sites to occupy fully the CRM, or, if Yan levels are limiting, as the data suggest, its preference for high-affinity sites and relative 'distaste' for lower-affinity sites could offer Pnt an opportunity to occupy the latter and perhaps influence Yan repression. In contrast, if Yan and Pnt had identical ETS-binding preferences, a less tuned response to RTK signaling would be expected; indeed, when the high-affinity 2,3 pair was removed and hence the strong bias toward Yan occupancy and repression at the MHE, stochastic ectopic expression was induced in the surrounding mesoderm where RTK levels are submaximal. Thus, their distinct preferences ensure that only maximal RTK activation will trigger the necessary shift in Yan-Pnt occupancy and activity to activate eve expression. Furthermore, while previous models assumed a complete switch from total Yan occupancy to total Pnt occupancy as Eve+ cell fates are specified, this work suggests that the ETS2,3 supersite still recruits Yan-repressive input even in Eve+ cells with very low Yan concentration. It is speculated that the ability to apply continued Yan-repressive input after cell fate induction may contribute to the robustness of certain developmental transitions by stabilizing the newly acquired cell fate. In agreement with this, in the context of the endogenous eve locus, disruption of the ETS2,3 pair sensitized eve to both fluctuations in upstream signaling and environmental conditions (Boisclair Lachance, 2018).
More broadly, it is speculated that the interplay between the cis-regulatory logic of a CRM and the unique biophysical parameters of different TFs permits evolution to fine-tune gene expression output to a specific threshold depending on each cell's developmental requirement. In the case of Yan-Pnt-regulated genes, the interplay between the degree of Yan SAM-mediated self-association and ETS syntax enables this repressor-activator pair to discriminate between ETS sites with unexpected precision. Furthermore, instead of RTK activation inducing a complete switch from Yan occupancy to Pnt occupancy as cell fates are induced, the cooperative recruitment of Yan to supersites may enable newly differentiating cells with lower Yan:Pnt ratios to sustain the Yan-repressive influence needed to ensure precision and robustness of the gene expression patterns. It is suggested that these ideas provide an interesting new vantage point for considering how single-nucleotide polymorphisms in TF-binding sites may heighten susceptibility to disease by compromising the robustness of gene regulatory networks (Boisclair Lachance, 2018).
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