Krüppel
Two cis-acting control elements, CD1 and CD2, control early expression. CD2 is located at -1000 to -3000 from start of transcription, and CD1 is at -3000 to -4000. The initial expression of Krüppel occurs in a precisely bounded central region of the Drosophila blastoderm embryo. The spatial limits of the Kr
expression domain are controlled by the morphogenetic activities of the anterior organizer gene
bicoid and the anterior gap gene hunchback . A 730 bp KR control element, (CD1) drives gene expression
in the endogenous KR central domain. This cis-acting element, Kr730, is composed of BCD
and HB responsive sequences. They map into regions of multiple HB and BCD protein in vitro binding
sites. A 142 bp core fragment containing one low affinity HB and five medium to strong BCD protein
binding sites drives gene expression in a Kr-like location in the center of the embryo. This fragment represents a target for the redundant activator/repressor system provided
by the anterior morphogens BCD and HB (Hoch, 1991).
The proximal promoter of Krüppel consists of a 44-base pair
fragment containing the RNA start sites with significant promoter activity. This minimal
promoter is flanked both upstream and downstream by binding sites for the GAGA factor. The
GAGA factor is the predominant sequence-specific DNA binding factor that interacts with the KR
promoter region, and the purified protein activates Kr transcription in vitro. However, strong
transcriptional activation of Kr as well as of Ultrabithorax, another GAGA factor-responsive gene,
requires the presence of a DNA binding transcriptional repressor. The GAGA factor is able to
relieve this repression in a binding site-dependent manner, and, thus, these data suggest that the
GAGA factor functions as an antirepressor, rather than an activator, of the Kr gene (Kerrigan, 1991).
High hunchback activity represses Krüppel in the anterior (Schulz, 1994).
Knirps regulates Krüppel by acting as a competitive repressor of Bicoid-mediated activation of Krüppel. Knirps binds at a single site in a 730bp regulatory region located 3.4 kb upstream of Krüppel. The Krüppel site (Kr730) contains a single strong KNI binding site, a 16 base pair element. The KNI binding site overlaps with one BCD binding site (Hoch, 1992 and references).
After the blastoderm stage Kr is under the control of at least 10 different regulatory units, sometime overlapping, up to10kb upstream of the gene. These different regulatory units drive expression in the Malpighian tubules, amnioserosa cells, muscle precursor cells, the developing nervous system, midline precursor cells and the larval eye (Bolwig's organ) (Hoch, 1990).
Knirps regulates krüppel by acting as a competitive repressor of Bicoid-mediated activation of Krüppel. Knirps binds at a single site in a 730bp regulatory region located 3.4 kb upstream of krüppel. The Krüppel site (Kr730) contains a single strong KNI binding site, a 16 base pair element. The KNI binding site overlaps with one BCD binding site (Hoch, 1992 and references).
Early developmental patterning of the Drosophila embryo
is driven by the activities of a diverse set of maternally and
zygotically derived transcription factors, including repressors encoded by gap genes such as Krüppel, knirps, and giant and the mesoderm-specific snail gene. At a molecular level, the mechanism of
repression by gap transcription factors is not well understood. Initial characterization of
these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better
understand the molecular mechanism of short-range repression, the properties of the Giant
gap protein have been investigated. The ability of endogenous Giant to
repress when bound close to the transcriptional initiation site was tested. Tandem 'CD1' Giant binding sites derived from the Krüppel (Kr) promoter were inserted 5' of a transposase basal promoter (55 bp
from the initiation of transcription of the lacZ reporter gene). Strong repression of the lacZ
gene is detected in embryos bearing this transgene; expression
of the lacZ gene in both anterior and posterior Giant regions is strongly attenuated. The repression of lacZ in anterior and posterior regions is relieved when the transgene is assayed in gt mutant embryos, confirming that the Giant protein is mediating the repression. Giant effectively represses a
heterologous promoter when binding sites are located at -55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites
are moved to more distal locations, repression is diminished (Hewitt, 1999).
It is probable that more distally located repressor sites (gt-110 and
gt-160, located respectively 110 and 160 bp from the start of transcription) may be effective only at higher
concentrations of Giant because the binding sites may not be
filled effectively. A partially filled site may still be effective at
close range because when Giant is close to the promoter, chance interactions between Giant and its target will occur more frequently, obviating the need for saturation of the
binding site by Giant. At a greater distance, these chance
interactions would be less frequent, leading to weak repression.
Partial filling of binding sites may simply be an indication that
the levels of Giant protein are below the Kd for the binding site,
so the site is empty for a fraction of the time. Alternatively,
even identical sites may not bind Giant protein equally well;
Giant may bind cooperatively to its cognate sites with a target protein, so that moving the sites farther from the target may break repressor-target cooperative interactions. Raising
the level of Giant protein (moving up the gradient) would
suffice to give greater occupancy of Giant sites, and reestablish
repression. Quantitation of relative Giant protein levels in
embryos suggests that differences in repressor protein
levels of less than two fold is sufficient to switch a gene from
an active to an inactive state. Similar cooperative effects have
been reported for activators, including the Bicoid activator. If Giant is
located close to the promoter, chance interactions between
Giant and its target will occur more frequently because of the
short diffusion distance, obviating the need for saturation of
the binding site by Giant. The mechanism whereby endogenous promoters might
differentially respond to different concentrations of repressors
is not known, but the results obtained from this study suggest
that exact placement of short-range repressors with respect to
other promoter elements and the number of binding sites
might suffice to endow a promoter with high or low
sensitivity. Additional factors, such as binding site affinity, may
also contribute to differential promoter sensitivity toward
repression. For example, a binding site located within the eve
stripe 2 enhancer was found to bind Giant protein less well than a site in the Kr promoter: consistent with this finding, eve has been found to be less sensitive to Giant repression than Kr. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient (Hewitt, 1999 and references).
A chimeric Gal4-Giant protein lacking the basic leucine zipper domain can specifically
repress reporter genes, suggesting that the Giant effector
domain is an autonomous repression domain. The three Giant
sites in the even-skipped promoter cover large regions and do not closely
resemble the compact sites from the Kr promoter.
Giant is likely to interact as a homodimer with the Kr promoter, and
as a heterodimer with the complex eve stripe 2 enhancer sites,
using an as-yet unidentified partner basic leucine zipper protein. Such a gene product may be
encoded by a locus identified on the left arm of chromosome
2, mutations in which can cause a gt-like phenotype. Consistent with the possibility that Giant
protein repression may involve other factors, recent experiments suggest that eve might be co-regulated by a Giant partner protein localized to the anterior portion of the embryo
(Wu, 1998). However, the activity of the Gal4-Giant protein on a variety of activators shows Giant can act as a homodimer on reporter genes (the Gal4 DNA binding domain binds to its cognate site as a dimer). If Giant
binds to DNA as a heterodimer in the embryo, the partner protein may serve to modulate DNA binding rather than to effect transcriptional repression (Hewitt, 1999 and references).
The analysis of transgenes with successively greater repressor-promoter
spacing demonstrates that short-range repression can act on more than an all-or-nothing basis, however,
the activity of these repressors is sharply attenuated over
relatively short distances. It has been suggested that short-range
repression may directly target adjacent transcription factors in a process akin to quenching; in this case, Giant might
alternately quench transcription activators or the basal
machinery, depending on the location of the binding site. Such lack of
specificity may be consistent with quenching acting through
chromatin modification on an extremely local level, as has been
reported for Ume6-mediated repression in yeast. Alternatively, repression by direct targeting of the basal transcription machinery is possible, as has been reported for the Krüppel and Even-Skipped proteins. Cofactors such as dCtBP are implicated in the activity of some short-range repressors, such as Knirps,
Krüppel and Snail, but apparently not Giant. Further molecular characterization of Giant repression activity will be necessary to distinguish between these alternatives (Hewitt, 1999 and references).
The Giant protein is a short-range transcriptional repressor that refines the expression pattern of gap and pair-rule genes in the Drosophila blastoderm embryo. Short-range repressors including Knirps, Krüppel, and Snail utilize the CtBP cofactor for repression, but it is not known whether a functional interaction with CtBP is a general property of all short-range repressors. Giant repression activity was studied in a CtBP mutant and it has been found that this cofactor is required for Giant repression of some, but not all, genes. While targets of Giant such as the even-skipped stripe 2 enhancer and a synthetic lacZ reporter show clear derepression in the CtBP mutant, another Giant target, the hunchback gene, is expressed normally. A more complex situation is seen with regulation of the Krüppel gene, in which one enhancer is repressed by Giant in a CtBP-dependent manner, while another is repressed in a CtBP-independent manner. These results demonstrate that Giant can repress both via CtBP-dependent and CtBP-independent pathways, and that promoter context is critical for determining giant-CtBP functional interaction. To initiate mechanistic studies of the Giant repression activity, a minimal repression domain within Giant has been identified that encompasses residues 89-205, including an evolutionarily conserved region bearing a putative CtBP binding motif (Stunk, 2001).
What characteristics of a regulatory region dictate CtBP-dependent or CtBP-independent repression? In considering which features of a gene determine CtBP-dependence or -independence, the structure of the basal
promoter cannot be the deciding factor, for the same Kr
promoter is regulated by distinct elements, some that
exhibit CtBP-dependence and some that show CtBP-independence.
Similarly, the eve gene is repressed by Knirps
via CtBP-dependent and CtBP-independent regulatory elements. While the eve enhancers in question are kilobases apart, the Kr regulatory elements
driving anterior and central domain (CD) expression are closely intertwined, and
appear to share at least some of the same activator binding
sites, suggesting that subtle differences in enhancer architecture
or differences in levels of regulatory proteins interacting
with those elements may dictate CtBP dependence. The Giant
binding site in the Kr CD2 enhancer site was shown to be of
higher affinity than the gt1 site in the eve stripe 2 enhancer. Thus, there may be a correlation between Giant binding site affinity and the requirement for
CtBP, with elements containing Giant sites of lower affinity
showing CtBP-dependence. A consensus has been derived for the
Giant protein by aligning binding sites for Giant from eve,
Kr, and the recently identified abdA iab-2 enhancer site. The consensus features an extended half-site inverted repeat TNTTAC, consistent
with the dimeric nature of basic zipper proteins, and a
central ACGT core common to recognition motifs for many
basic zipper proteins. The higher affinity sequences from the CtBP-independent
Kr CD element are closer to the consensus than those of the CtBP-dependent eve stripe 2 enhancer. Weaker sites may only be partially occupied, resulting in an overall lower level of Giant mediated repression. A loss of
CtBP might further depress repression activity below a critical threshold, leading to the derepression observed. Repression of the lacZ reporter containing the giant CD1 site from Kr is CtBP-dependent, a result that contrasts with the CtBP independence of the CD itself, but this particular site may not be optimal, since it contains two mismatches. Full Giant activity may also be mediated on the native CD element through the additional high-affinity CD2 site (Stunk, 2001).
Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).
HB is autonomously
capable of activating the target gene Krüppel at low concentrations and repressing it at high
concentrations (Schulz, 1994).
Bicoid and Hunchback activate Krüppel expression (Hoch, 1991), while Knirps and Tailless repress Krüppel in the posterior end of the embryo (Hoch, 1992). However, Krüppel is activated by Tailless, Huckebein and Forkhead in the posterior Malpighian tubules domain (Gaul, 1990 and Schulz, 1994).
Kruppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the
embryo. The interactions of Kruppel with other terminal genes has been studied. The gap genes tailless and huckebein, which
repress Kruppel in the central segmentation domain, activate Kruppel expression in the posterior Malpighian tubule domain. The
opposite effect on the posterior Kruppel expression is achieved by the interposition of forkhead,
which is not involved in the control of the central domain. In addition, Kruppel activates different genes in the Malpighian tubules
than in the central domain. Thus, both the regulation and the function of Kruppel in the Malpighian tubules differ strikingly from its
role in segmentation (Gaul, 1990). Kr activity determines the neural fate of tip cells by acting as a
direct downstream target of proneural basic helix-loop-helix (bHLH) proteins that are restricted in response to Notch signalling. Forkhead interacts with a 400 bp cis active element of Krüppel directing Kr expression in Malpighian tubules. There are two FKH binding sites. This element also contains functional binding sites for the restricted proneural bHLH factors (Hoch, 1998).
giant, ectopically expressed, represses the expression of both the Krüppel and knirps segmentation gap genes. An analysis of the interactions between Krüppel, knirps and giant reveals
a network of negative regulation. The apparent positive regulation of knirps by Krüppel is mediated by the negative effect of Krüppel on giant and a negative effect of Giant
on knirps (Capovilla, 1992).
hindsight may regulate Krüppel. Krüppel, which accumulates in wild-type embryos in the nuclei of amnioserosal cells, is absent from most but not all of these cells in stage 11 hnt mutants (Yip, 1997).
As the germ band shortens in Drosophila melanogaster embryos, cell shape changes cause segments to narrow anteroposteriorly and to lengthen dorsoventrally. One
of the genes required for this retraction process is the hindsight (hnt) gene. hnt encodes a nuclear Zinc-finger protein that is expressed in the extraembryonic
amnioserosa and the endodermal midgut prior to and during germ band retraction. Through analysis of hnt genetic mosaic embryos, it has been shown that hnt activity in the amnioserosa, particularly in those cells that are adjacent to the
epidermis, is necessary for germ band retraction. In hnt mutant embryos the amnioserosa undergoes premature cell death. Prevention of premature apoptosis in hnt mutants does not rescue retraction. Thus, failure of this process is not
an indirect consequence of premature amnioserosal apoptosis; instead, hnt must function in a pathway that controls germ band retraction (Lamka, 1999).
The Kruppel
gene is activated by hnt in the amnioserosa while the Drosophila insulin receptor (INR) functions downstream of hnt in the germ band. Kr protein is first detected in the amnioserosa of wild-type embryos during germ band extension; however, in hnt mutants, Kr is clearly absent from most amnioserosal cells by stage 11. Loss of Kr in hnt mutants is not a result of premature apoptosis of the amnioserosa. Rather the Kr gene resides downstram of Hnt in the hnt genetic hierarchy in the amniserosa. A specific role for Kr in germ band retraction remains to be defined. Using a heat shock insulin receptor transgene to overexpress the wild-type insulin receptor, nearly complete rescue of germ band retraction is observed in hnt mutants. The extent of rescue depends on the strengtth of the hnt used. The fact that high levels of the Inr can rescue germ band retraction in hnt mutants is consistent with the possibility that the Inr functions downstream of hnt in a germ band retraction pathway. Evidence against a
physical model in which the amnioserosa 'pushes' the germ band during retraction is presented. Rather, it is likely that the amnioserosa functions in production, activation, or
presentation of a diffusible signal required for retraction (Lamka, 1999).
Mutations in several Polycomb (Pc) group genes cause maternal-effect or zygotic segmentation
defects, suggesting that Pc group genes may regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other Pc group
genes show gap, pair rule, and segment polarity segmentation defects.
Posterior sex combs and polyhomeotic interact with Krüppel (McKeon, 1994).
Genetic experiments and a targeted misexpression approach have been combined to examine the role of the gap gene giant (gt) in patterning anterior regions of the Drosophila embryo. The results suggest that gt functions in the repression of three target genes, the gap genes Kruppel (Kr) and hunchback (hb), and the pair-rule gene even-skipped (eve). The anterior border of Kr, which lies 4-5 nucleus diameters posterior to
nuclei that express GT mRNA, is set by a threshold repression mechanism involving very low levels of Gt protein. The gap gene Kr is activated in a broad central region of
precellular embryos. Midway through cleavage cycle 14, this domain extends from 41-59% egg
length. The initial positioning of the anterior border of this
domain is thought to be controlled by repression involving a
combination of maternal and zygotic hunchback transcripts. To test whether gt
is also involved in setting or maintaining this border, the Kr expression pattern was analyzed in embryos containing the st2-gt transgene, a modified version of the 480 bp eve stripe 2
enhancer. These embryos show no changes in the
initial positioning of the Kr expression domain early in
cleavage cycle 14, but slightly later there is a dramatic
retraction of the anterior Kr border. The delay in the observed repressive effect on the Kr anterior border is probably due to the fact that the Kr domain is expressed earlier
than the st2-gt transgene. Higher levels of ectopic gt result in a more severe retraction, suggesting that Kr transcription is very sensitive to repression by gt. To test whether gt affects Kr expression during normal development, Kr expression was examined in embryos that carry
a strong hypomorphic gt allele. The initial Kr expression
pattern was correctly established in these gt hypomorphic embryos. However, slightly later, a significant anterior expansion (from 59% to 65% egg length) is observed,
suggesting that gt-mediated repression is essential for
maintaining the position of the anterior border of the Kr domain (Wu, 1998).
giant activity is required, but not sufficient, for the formation of the anterior border of eve
stripe 2, which lies adjacent to nuclei that express GT mRNA. It is proposed that gt's role in forming this border is to potentiate repressive interaction(s) mediated by other factor(s) that are also localized to anterior regions of the early embryo. It is not clear whether gt is sufficient for repression of the in vivo eve stripe 2 response. To test this, eve expression was examined in embryos
containing the st2-gt transgene, which extends the gt domain so that it overlaps the position of eve stripe 2. Surprisingly, the ectopic gt causes only a weak transient reduction of the stripe early in cycle 14. Later the stripe recovers to full strength, but expands toward the posterior by about two nucleus diameters. Double in situ hybridization experiments show that the timing and the extent of the expansion correlates well with the retraction of the Kr domain, suggesting
that the expansion of eve stripe 2 is indirectly caused by relief from Kr repression. Doubling the ectopic gt expression levels still does not cause a significantly stronger repression,
suggesting that eve stripe 2 is quite insensitive to gt repression. To test whether the effects of ectopic gt on eve stripe 2 are controlled by the early or late regulatory elements, the expression of lacZ reporter genes was examined in embryos containing st2-gt transgenes. It
is likely that the posterior expansion of endogenous eve stripe
2 caused by the st2-gt transgene is mediated through the early acting enhancer (Wu, 1998).
The recalcitrance of the eve stripe 2 response to ectopic gt
expression led to a reexamination of the eve expression pattern in gt
mutant embryos. Early in cycle 14, these mutants show a
derepression in the interstripe region between stripes 1 and 2. However, later in cycle 14, gt mutants show a dramatic reduction in stripe 2 expression levels, suggesting a
role for gt in maintaining the stripe. Since Kr has been
previously implicated as the repressor that forms the stripe 2
posterior border, it is possible that the stripe 2 reduction in gt
mutants is indirectly caused by Kr, which expands anteriorly to completely overlap the diminishing stripe. The repression of eve stripe 2 observed in gt mutants can be relieved
by reducing Kr levels. These results suggest that a major
function of the anterior gt domain is to prevent Kr from
expanding anteriorly, thus permitting the expression of eve
stripe 2. Furthermore, since gt repression maintains the position
of the anterior Kr border in wild-type embryos, it indirectly
defines the position of the posterior border of eve stripe 2 (Wu, 1998).
In principle, the preceding experiments support the hypothesis that gt acts as a concentration-dependent repressor to set the anterior borders of the Kr and eve stripe 2 expression domains in different positions. Ectopic gt is an effective repressor of Kr,
but has little effect on the activation of eve stripe 2. In situ
hybridization experiments indicate that endogenous gt levels
are significantly higher than the ectopic gt driven by even the
strongest st2-gt transgenic lines. Perhaps these higher endogenous levels are required for
effectively setting the anterior border of eve stripe 2. If this is
the case, the early expansion of eve stripe 2 toward stripe 1
detected in gt mutants should not be affected in
embryos in which the endogenous gt gene is replaced by the
st2-gt misexpression domain. To test this, eve expression was examined in gt mutants that contained the st2-gt5 transgene. Surprisingly, a sharp anterior eve stripe 2 border is formed
in these embryos, with a clear interstripe between eve stripes
1 and 2. Furthermore, the st2-gt domain rescues eve
stripe 2 to full strength, with a posterior expansion that is
probably due to repression of the anterior Kr border.
The relatively low levels of ectopic gt driven by the st2-gt construct overlap the endogenous gt domain and extend 4-5 nucleus diameters posteriorly. The fact that a sharp
anterior eve stripe 2 border is formed in embryos containing only
this domain argues against a simple concentration-dependent
mechanism for setting this border. Rather, it is proposed that other
factor(s) are involved along with gt in defining the anterior
border of eve stripe 2 in vivo. Thus, gt may act as a potentiator of repression
mediated by these localized factors. Since gt
encodes a putative leucine zipper (b-ZIP) protein, one possibility is that this activity is also a b-ZIP protein that can heterodimerize with gt as part of an effective repressor
complex. Repressive function in the absence of gt would be
provided by a homodimer of this protein. Alternatively, since the
gt site deletions tested in previous experiments removed relatively
long sequences (14-43 bp), it is possible that these deletions may
have removed or interrupted binding sites for other protein(s) (Wu, 1998).
gt is required for repression of zygotic hb expression in more anterior
regions of the embryo. Zygotic expression of hb is initially activated by
the bcd and maternal hb gradients in a broad domain that spans the
anterior half of the embryo. This expression is then rapidly refined
during nuclear division cycle 14, leaving a secondary pattern that
includes a variable head domain, a stripe at the position of
parasegment 4 (PS4), and a posterior stripe. The PS4 stripe overlaps
the anterior border of the Kr domain. By examining
hb expression in gt mutants, significant changes in
this secondary pattern were detected. Initially, hb expression at the
position of PS4 is greatly reduced, possibly because of the
anterior expansion of the Kr domain in gt mutants. High levels of hb expression persist in more anterior regions of gt mutant embryos. The persistent hb
expression domain appears very similar in shape to the
normal gt domain, suggesting that gt may
act as a repressor to clear hb expression from this part of the
embryo during wild-type development. To test whether
endogenous gt levels were required for this repression, hb expression was examined in gt mutants that also contained the st2-gt transgene. hb expression is repressed normally by a single copy of the st2-gt5 transgene, suggesting that relatively low levels of ectopic gt can replace this function of the endogenous gene. Since gt seems to be involved in repression of hb in anterior regions, it is possible that this repression is important for
setting the anterior border of the hb PS4 stripe during wild-type
development. To test this, hb expression was examined in
embryos containing the st2-gt transgene. The position of the anterior border of the hb PS4 stripe appears unchanged in these embryos, suggesting that the levels of
ectopic gt tested here are not sufficient to repress hb PS4
expression. However, a slight posterior expansion of this stripe
could be detected in embryos with high levels of misexpression, which is probably caused by the retraction of the Kr domain. This supports the hypothesis that Kr activity is
important for setting the posterior PS4 stripe border, and further demonstrates the importance of gt-mediated restriction of Kr expression to central regions of the embryo (Wu, 1998).
Localized gene expression patterns are critical for establishing body plans in all multicellular animals. In Drosophila, the gap gene hunchback is expressed in a dynamic pattern in anterior regions of the embryo. Hb protein is first detected as a shallow maternal gradient that prevents expression of posterior gap genes in anterior regions. HB mRNA is also expressed zygotically, first as a broad anterior domain controlled by the Bicoid morphogen, and then in a stripe at the position of parasegment 4 (PS4). The PS4-hb stripe changes the profile of the anterior Hb gradient by generating a localized peak of protein that persists until after the broad domain has started to decline. This peak is required specifically for the formation of the mesothoracic (T2) segment. At the molecular level, the PS4-hb stripe is critical for activation of the homeotic gene Antennapedia, but does not affect a gradient of Hb repressive activity formed by the combination of maternal and Bcd-dependent Hb. The repressive gradient is critical for establishing the positions of several target genes, including the gap genes Kruppel, knirps, and giant, and the homeotic gene Ultrabithorax. Different Hb concentrations are sufficient for repression of gt, kni, and Ubx, but a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr. These results suggest that the individual phases of hb transcription, which overlap temporally and spatially, contribute specific patterning functions in early embryogenesis (Wu, 2001).
Primary zygotic expression of HB mRNA (P2-hb) covers
much of the anterior half of wild-type embryos early in
nuclear cleavage cycle 14. This pattern is soon transformed into the secondary pattern, which includes the PS4-hb stripe. This stripe
appears just before midcycle 14, and persists until the onset
of gastrulation. The distribution of Hb protein is similar to
the mRNA profile, but the protein seems to degrade more slowly. Thus, in midcycle-14 embryos, the Hb pattern consists of a broad anterior domain, with a peak at the position of PS4. Later, when cellularization is complete, Hb in anterior regions degrades further, leaving a clear stripe at PS4 (Wu, 2001).
To specifically remove this stripe, a misexpression
transgene (st2-kni) was used that directs barely detectable levels
of the gap gene kni at the position of PS3. Ectopic kni expression completely abolishes the PS4-hb stripe, and the
peak of Hb protein observed in wild-type embryos. By the end of cellularization, no protein is detectable at the PS4 position of st2-kni embryos. The relative levels of Hb were further quantified at
midcycle 14 in wild-type and st2-kni embryos. The PS4
position was determined by simultaneous fluorescence in
situ hybridization with an RNA probe directed against fushi tarazu, a pair-rule gene expressed in stripes that correspond to the even-numbered parasegments (ftz stripe 2 corresponds to PS4). In st2-kni embryos, nuclei at the center of ftz stripe 2 contain only about 50% of the Hb normally present at this position. Thus, in wild-type embryos, the PS4-hb stripe alters the profile of the anterior Hb gradient by creating a peak of protein concentration at
midcycle 14, and also ensures the perdurance of high Hb
levels at this position until the end of cellularization (Wu, 2001).
In contrast to previous hb rescue experiments, the st2-kni
transgene removes the PS4-hb stripe without changing the
normal maternal and P2-hb gradients. Embryos carrying this transgene were examined for morphological defects later in development. More than 75% of these embryos die by the end of embryogenesis and show deletions of
the T2 denticle band, which is derived from cells in PS4. While approximately 50% show a complete T2 deletion in dorsal and ventral regions, the phenotype is
more severe on the ventral side, with ~90% completely
lacking the T2 ventral denticle band. The cause of the phenotypic difference between dorsal and ventral regions is not clear. These results indicate that high levels of Hb at the position of PS4 are critical for T2 development (Wu, 2001).
To investigate the role of PS4-hb in T2 development, the expression patterns of segmentation and homeotic genes was examined in st2-kni embryos, and those fully rescued by the st2DeltaK-hb-1 transgene (a transgenic line that directs high levels of hb in a wide stripe that overlaps the PS4 position). No changes in the
expression patterns of the gap genes Kr, kni, or gt were detected in st2-kni embryos. This suggests that Hb-mediated repression of
these genes is not dependent on the PS4-hb stripe. However,
several genes normally expressed in PS4 were significantly altered. One such gene is ftz. In st2-kni embryos, activation of ftz stripe 2 is delayed, and reduced in intensity. To test whether this reduction affects ftz function, expression of the ftz target gene engrailed was examined. en stripe 4, which is normally activated by ftz stripe 2, is also significantly reduced in st2-kni embryos (Wu, 2001).
The observed reductions in ftz stripe 2 and en stripe 4 could
lead to the T2 deletion caused by the removal of the PS4-hb
stripe. Surprisingly, however, addition of the st2DeltaK-hb-1
transgene, which mediates complete rescue of morphological
defects in st2-kni embryos, does not detectably alter the
level of ftz stripe 2 or en stripe 4. The
failure to rescue these stripes may be due to the ectopic Kni
in this region. However, since these embryos are rescued to
adulthood, the reduced ftz and en stripes must be capable of
supporting the establishment of the segment (Wu, 2001).
Previous experiments have implicated Hb as a repressor of the gap genes Kr, kni, and gt, but the expression domains of these genes are not affected by the removal of the PS4-hb stripe. Another gene controlled by Hb-mediated repression is the homeotic gene Ultrabithorax, which is strongly expressed as a stripe at the position of PS6 in late-blastoderm embryos. Since the PS4-hb stripe is also expressed at this stage, whether it is required for setting the anterior Ubx expression border was tested. Like the gap gene targets (Kr, kni, and gt), Ubx expression is undisturbed in embryos specifically lacking the PS4-hb stripe. Thus, the peak of protein provided by this stripe is not required for repression of any of these four genes (Wu, 2001).
In wild-type embryos, the anterior borders of Kr, kni, gt, and Ubx are located at different positions along the
anterior-posterior axis, suggesting that they respond
to different thresholds of Hb concentration. The
position of the gt border, which lies most posteriorly, is
established by the maternal Hb gradient. There is very
little change in the position of this border in zygotic hb
mutants, suggesting that the maternal gradient is sufficient for gt
repression. The anterior kni border, which lies six to
eight nuclear diameters from nuclei that produce zygotic
HB mRNA, is also initially established by the maternal
gradient. In mutants lacking zygotic hb, this border is
correctly positioned early in cycle 14, but there is an
anterior expansion at midcycle 14, possibly due to degradation of the maternal
gradient. Whether this expansion is sensitive to
ectopic Hb driven by the st2DeltaK-hb construct was tested. These
experiments show a dose-dependent repression of kni
back to its original position. Since only
one copy of the transgene causes a strong repression, kni
is sensitive to very low levels of Hb. The homeotic gene
Ubx, which is normally expressed about four nuclear
diameters from the posterior edge of the hb domain, also expands anteriorly in zygotic hb mutants. Addition of the st2DeltaK-hb transgene causes a dose-dependent repression of this gene as well. In this case, two copies of the transgene are significantly more effective than one, suggesting that Ubx repression requires a higher level of Hb than kni (Wu, 2001).
Finally, the gap gene Kr, whose anterior border
overlaps the hb expression domain in wild-type embryos, was tested.
This gene expands anteriorly in zygotic hb mutants, but cannot be repressed even by two copies of the st2DeltaK-hb transgene.
This suggests that a very high level of Hb, or a combinatorial
mechanism, is required for repression of Kr (Wu, 2001).
In presumptive cephalic regions, zygotic hb mutants lack
elements of the labial segment, including the H-piece and
labial sense organs, and other head structures are severely
disorganized, probably due to a disruption of head involution. Introduction
of the st2DeltaK-hb-1 transgene causes a very weak
rescue of these defects, compared to the mostly
wild-type head structures found in hb mutants rescued by
P2-hb transgenes. Since the st2DeltaK-hb transgene is activated at a later stage than P2-hb, perhaps high levels of Hb present earlier
in anterior regions are required for the correct formation of
these structures. However, the expression patterns of the
head gap genes orthodenticle, button-head, and
empty-spiracles are not affected in zygotic hb mutants, raising the possibility that Hb may function indirectly by preventing the expression of repressors of head development. A candidate for such a repressor
is Kr, which cannot be repressed by even two copies of the
st2DeltaK-hb-1 transgene. Perhaps the expanded Kr domain
causes the remaining head defects in hb mutants that are
partially rescued by the st2DeltaK-hb transgene (Wu, 2001).
To test this, one copy of the Kr
gene was genetically removed from these embryos. This manipulation causes a
dramatic rescue of most head structures, and a few
embryos (~10%) display anterior phenotypes that are indistinguishable
from wild type. This suggests that the expansion
of Kr into presumptive head regions prevents normal
head development, and that an important function of P2-hb
is to restrict Kr to more posterior regions. However, the
larval heads of Kr/+;hb/hb embryos still exhibit many
morphological defects, suggesting that Hb also patterns the head by mechanisms that are independent of its role in Kr repression (Wu, 2001).
To understand how loss of Grunge activity affects segmentation, the expression of hunchback (hb), Krüppel (Kr), knirps (kni) and fushi tarazu (ftz) was analyzed in embryos derived from Gug35 germline clones fertilized by Gug35 sperm. In wild-type embryos, the expression of these segmentation gene products localizes to discrete domains in the early embryo. In almost all of the expression domains, loss of Gug activity increases the number of cells expressing these segmentation genes, suggesting that Gug plays a role in their repression. Later the expression of ftz displays a more complex defective pattern with some stripes being broader, and others narrower, than wild type (Erkner, 2002).
Loss of Gug activity severely affects the process of segmentation and the expression of segmentation genes when missing from the female germ line. At the blastoderm stage, most of the expression domains of hb, Kr, kni and ftz genes are expanded compared with wild type. These observations indicate that maternal production of Gug is crucial for the repression of these genes to precise domains in the early embryo. Gap proteins, including Hb, Kr and Kni are known to be required to restrict each others domains of expression. It will be interesting to test if Gug acts with these proteins for these repression activities (Erkner, 2002).
Cooperative interactions by DNA-binding proteins have been implicated in
cell-fate decisions in a variety of organisms. To date, however, there are few
examples in which the importance of such interactions has been explicitly tested
in vivo. This study tests the importance of cooperative DNA binding by the Bicoid
protein in establishing a pattern along the anterior-posterior axis of the early
Drosophila embryo. bicoid mutants specifically defective in
cooperative DNA binding fail to direct proper development of the head and
thorax, leading to embryonic lethality. The mutants do not faithfully stimulate
transcription of downstream target genes such as hunchback (hb), giant, and
Krüppel. Quantitative analysis of gene expression in vivo indicates that bcd cooperativity mutants are unable to accurately direct the extent to which hb is expressed along the anterior-posterior axis; they display a reduced ability to generate sharp on/off transitions for hb gene expression. These failures in precise transcriptional control demonstrate the importance of cooperative DNA binding for embryonic patterning in vivo (Lebrecht, 2005).
Irregular facets (If) is a dominant mutation of
Drosophila that results in small eyes with fused ommatidia. Previous
results showed that the gene Krüppel (Kr), which is
best known for its early segmentation function, is expressed ectopically in
If mutant eye discs. However, it was not known whether ectopic Kr
activity is either the cause or the result of the If mutation. This study
shows that If is a gain-of-function allele of Kr. The If
mutation was used in a genetic screen to identify dominant enhancers and
suppressors of Kr activity on the third chromosome. Of 30 identified
Kr-interacting loci, two were cloned, and whether they also represent
components of a natural Kr-dependent developmental pathway of the embryo
was tested. The two genes, eyelid (eld) and
extramacrochaetae (emc), which encode a Bright family-type DNA
binding protein and a helix-loop-helix factor, respectively, are necessary to
achieve the singling-out of a unique Kr-expressing cell during the
development of the Malpighian tubules, the excretory organs of the fly. The
results indicate that the Kr gain-of-function mutation If provides
a tool to identify genes that are active during eye development and that a
number of them function also in the control of Kr-dependent developmental
processes (Carrera, 1998).
Kr expression defines the Malpighian tubule anlage at late blastoderm
stage and becomes restricted to a ring of cells at the midgut/hindgut boundary
from where Kr-expressing Malpighian tubule precursors evert. Previous
studies have shown that the specification of Malpighian tubule fate and the
segregation of the cells depend on Kr expression in the Malpighian tubule
anlage. In Kr-deficient embryos, the respective cells become part of the
hindgut epithelium (Carrera, 1998).
Once the tubules evert, Kr expression becomes restricted to a single
cell, termed the "tip mother cell". The singling-out process of this cell from
an equivalence group of Malpighian tubule precursors involves the activated
Notch pathway, which restricts the proneural bHLH proteins encoded by the
achaete-scute-complex (ASC) genes to the tip mother cell. In this
cell, the ASC proteins act in concert with bHLH protein encoded by
daughterless (da) to maintain Kr expression. The tip mother
cell divides once, and the daughters give rise to the tip cell, which controls
proliferation during tubule elongation and differentiates neuronal
characteristics, and an excretory cell, termed "satellite cell". The satellite
cell loses Kr expression in a Notch-dependent manner, whereas
Kr expression is maintained in the tip cell until the end of
embryogenesis (Carrera, 1998).
emc expression accompanies Malpighian tubule development in a manner
similar to Kr expression. However, once the tip cell is formed, the
patterns of expression become complementary, meaning that emc expression
continues in all cells of the elongating Malpighian tubules except in the tip
cell. To test whether the complementary patterns of Kr and emc
expression reflect a regulatory effect of emc on Kr, as indicated
during eye development in the If mutant, Kr expression was
examined in the Malpighian tubules of emc mutant embryos. Multiple
Kr-expressing cells are seen in emc mutant Malpighian tubules.
This finding is consistent with the previous finding that emc mutant
embryos develop multiple tip cells and that each of them continues to express
achaete. Virtually the same observations have been made with Notch
mutants, and Notch acts toward restricting the activity of the proneural
bHLH proteins, which are required to maintain Kr expression first in the
tip mother cell and subsequently in the tip cell. However, although the
activated Notch pathway acts through transcriptional repression of the
ASC genes, emc protein antagonizes proneural bHLH activities by
sequestering the proteins as heterodimers that are incapable of binding to DNA.
The results are therefore consistent with the proposal that emc functions
in the control of Kr expression by antagonizing proneural bHLH activities
that are required to maintain Kr expression in the tip mother cell
(Carrera, 1998).
The Eld protein shows a nuclear localization, consistent with its suspected
function as a transcription factor. It appears to act in multiple signaling
pathways because it antagonizes wingless activity, suppresses Ras1
activity in the eye, and blocks Notch-dependent neuronal differentiation.
During Malpighian tubule development, eld is expressed in a restriced
area of the everting precursors that corresponds to the equivalence group of
cells expressing the proneural genes (Carrera, 1998).
eld mutant embryos exert a distinct phenotype during Malpighian tubule
development that is linked to Kr activity. Whereas the anlage and the
four tubules evert normally, each tubule develops two instead of the normal one
tip cell. Tip cell development is under the control of Kr activity, so it
was next asked whether and when Kr expression is altered in eld
mutant embryos. In correspondence with the mutant phenotype, the initial
expression of Kr, including its restriction to the tip mother cell,
appears to be normal. However, once the tip mother cell has undergone division,
two instead of only one of the daughter cells maintain Kr expression.
This indicates that eld activity is necessary to prevent Kr
expression in the sibling of the tip cell and allows for its differentiation
into a satellite cell. Thus, although emc is necessary for the
restriction of Kr to the tip mother cell, eld functions
specifically at the subsequent step during Malpighian tubule development where
an alternative and Kr-dependent cell fate decision is taken between the
daughters of the tip mother cell (Carrera, 1998).
Notch signaling is required first for the selection of the tip mother
cell and subsequently for the distinction between its daughters to either
develop a tip cell or a satellite cell. Consistently, in Notch mutant
embryos, all cells of the proneural equivalence group develop first into tip
mother cells; these cells divide and subsequently develop into the multiple tip
cells that continue Kr expression. In contrast, only two tip cells were
found in eld mutants. This finding implies that, if eld acts in a
Notch-dependent manner and/or mediates Notch signaling, its
activity is required only for the second of the two Notch-dependent
differentiation steps during Malpighian tubule development. Thus, eld
participates as an optional component in the Notch-signaling pathway and
is needed to prevent, directly or indirectly, the maintenance of Kr
expression in the satellite cell that would otherwise develop into a second tip
cell (Carrera, 1998).
The results of this study demonstrate that gene activities that were
identified via an artificial experimental situation, namely the ectopic
expression of Kr in the developing eye disc, can lead to the
identification of integral components of a Kr-dependent developmental
pathway during embryogenesis. In the eye imaginal disc, emc suppresses
Kr activity whereas eld has an opposite effect, but both act
during embryonic Malpighian tubule development as negative regulators of
Kr. No explanation is available for this phenomenon. It could mean, in
negative terms, that the Kr misexpression screen turned up
dosage-sensitive genes affecting cell fate that were several steps downstream
from Kr activity and thus have no direct interaction with Kr.
Thus, each gene identified in the modifier screen represents a candidate gene
that needs to be evaluated critically through additional criteria as outlined
here for eld and emc. The additional screening is essential to
distinguish between direct Kr interactors and genes that mediate
different read-outs of the Kr pathway in cells that have a different
organ or tissue competence. However, in view of the fragmentary information
concerning the spatial and temporal control of postblastodermal Kr
expression and in view of the fact that the few Kr target genes of
Kr were identified by molecular approaches, this experimental strategy to
assess components of a Kr-dependent regulatory circuitry seems a valid
one (Carrera, 1998).
Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).
To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).
Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).
Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).
Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).
A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).
The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).
Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).
In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).
The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.
The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).
Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).
To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).
To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).
In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).
Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).
The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).
The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).
Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).
S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).
To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).
Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).
Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).
Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).
Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).
Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).
The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).
It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).
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 them those expressed in somatic muscle precursors including the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).
Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).
The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).
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