Krüppel
A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and
where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory
information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a
few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple
transcription factors. Transcription factor binding sites have a tendency to cluster; the extent to which they do can be used
as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the
early Drosophila embryo, genomic regions containing unusually high concentrations of predicted binding sites were identified for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. One of the newly identified clusters, mapping upstream of the gap gene giant (gt) was tested; it acts as an enhancer that recapitulates the posterior expression pattern of gt (Berman, 2002).
The transcription factors Bicoid (Bcd), Caudal (Cad), Hunchback
(Hb), Krüppel (Kr), and Knirps (Kni) act at very early stages of
Drosophila development to define the anterior-posterior
axis of the embryo. Bcd and Cad are
maternal activators broadly distributed in the anterior and posterior
portions of the embryo, respectively. Hb, Kr, and Kni are zinc-finger
gap proteins that act primarily as repressors in specific embryonic
domains. Sequences of previously described binding sites were collected for these
five factors present in the cis-regulatory regions of known target
genes. The binding sequences for each factor were aligned by using the
motif-assembly program, and the
binding specificities of each factor were modeled with position weight matrices (PWMs). PWMs are a useful way
to represent binding specificities and provide a statistical framework
for searching for novel instances of the motif in genome sequences (Berman, 2002).
A freely available program PATSER was used to search
the genome for sequences that match these PWMs, and a
web-based visualization tool, CIS-ANALYST
(http://www.fruitfly.org/cis-analyst/) was devised to display the location
of predicted binding sites along with genome annotations in selected
genomic regions. PATSER assigns a score to each potential
site that reflects the agreement between the site and the corresponding
PWM. These scores approximate the free energy of binding between the
factor and site, and CIS-ANALYST uses a
user-defined cutoff parameter to eliminate predicted
low-affinity sites (Berman, 2002).
Using CIS-ANALYST, the distribution of Bcd,
Cad, Hb, Kr, and Kni binding sites were examined in a 1-Mb genomic region surrounding the well-characterized eve locus at a site_p value of
0.0003. At this relatively
high-stringency value, most experimentally verified binding sites are
retained; at more restrictive values, many of these sites would be
lost (Berman, 2002).
To investigate whether binding site clustering could help to explain
the specificity of these factors for eve, a
simple notion of binding site clustering was incorporated into
CIS-ANALYST, allowing searches for segments of a
specified length containing a minimum number of predicted binding
sites. When the 1-Mb region surrounding eve was searched for
dense clusters of predicted high-affinity sites (at least 13 Bcd, Cad,
Hb, Kr, or Kni sites in a 700-bp window), three discrete regions were
identified. Strikingly, these
three clusters are all adjacent to eve, and overlap the
previously characterized stripe 2, stripe 3 + 7, and stripe 4 + 6 enhancers (Berman, 2002).
To generalize and quantify these promising results, a
broader collection of 19 well-defined CRMs from 9 Drosophila genes known to be required for proper embryonic development was compiled. Each of these CRMs is sufficient to direct the expression of a
distinct anterior-posterior pattern in early embryos; genetic
evidence suggests that each CRM is regulated by at least one of the following: Bcd, Cad,
Hb, Kr, and Kni. Mutation and in vitro DNA binding studies
completed on a subset of the CRMs provide evidence for a direct
regulatory relationship. The same clustering criteria that were
successful for identifying CRMs in eve (700-bp regions with
at least 13 predicted binding sites) identified clusters overlapping 14 of these 19 known CRMs (Berman, 2002).
A search of the entire genome for 700-bp windows containing at
least 13 predicted binding sites identified 133 clusters in addition to
the 19 described above, or ~1 per 700 kb of noncoding sequence. As
expected, when more stringent clustering criteria are used, both the
number of known CRMs recovered and the number of novel clusters
identified decrease. The novel clusters identified with a density
of at least 15 binding sites per 700 bp, a level at which half of the
known CRMs are still recovered, were further examined. Binding site plots for the 22 novel clusters identified at this
high stringency condition, and 6 additional novel clusters
identified with an equally stringent search by using only Bcd,
Hb, Kr, and Kni have been published as supporting information on the PNAS
web site).
Twenty-three of these 28 clusters fall in regions between
genes, whereas the remaining 5 fall in introns. There are
therefore 49 genes that either contain a novel cluster of binding
sites or flank an intergenic region that does. The
expression patterns of these 49 genes in early embryos were examined by
whole-mount RNA in situ hybridization and DNA microarray
hybridization. At least 10 of the 28 clusters were adjacent to a gene that showed localized anterior-posterior
expression in the syncitial or cellular blastoderm stages, consistent with early regulation by
maternal effect or gap transcription factors. Although the numbers are small, this is significantly more than the 1 or 2 expected if the
positions of clusters had been chosen at random (Berman, 2002).
One of these clusters is located ~2 kb upstream of the gap gene
giant (gt). During cellularization,
gt is expressed in two broad domains, one in the anterior
and one in the posterior portion of the embryo.
The pattern of expression of the posterior expression domain is determined by the activities of Cad, Hb, and Kr. However, the cis-regulatory sequence controlling this posterior expression pattern has not been precisely identified. Whether this cluster of binding sites might be the gt posterior enhancer was evaluated. A 1.1-kb fragment containing this
cluster was placed in a reporter construct containing the eve minimal promoter fused to a lacZ reporter gene. The expression pattern of this construct largely recapitulates the early expression pattern of the gt posterior expression domain. In the absence of Kr function, the anterior border of the gt posterior domain shifts anteriorly, indicating repression by Kr. The construct containing the gt posterior enhancer exhibits a similar shift in the absence of Kr (Berman, 2002).
Posterior hairy stripe boundaries are established by gap protein repressors unique to each stripe. Krüppel limits the anterior
expression limits of both stripes 5 and 6 and is the only gap gene to do so, indicating that stripes 5 and 6
may be coordinately positioned by the KR repressor. Binding sites for the KR protein were located in both stripe enhancers. The stripe 6 enhancer contains
higher affinity KR-binding sites than the stripe 5 enhancer, which may allow for the two stripes to be
repressed at different KR protein concentration thresholds. The Knirps
activator binds to the stripe 6 enhancer and there appears to be a competitive mechanism of KR
with KNI for repression of stripe 6 (Langeland, 1994).
Gap genes Kruppel (Kr), knirps (kni), and tailless (tll) control the expression of the pair-rule gene hairy (h) by
activating or repressing independent cis-acting units that generate individual stripes. KR activates
stripe 5 and represses stripe 6, KNI activates stripe 6 and represses stripe 7, and TLL activates stripe
7. KR and KNI proteins bind strongly to h control units that generate stripes in areas of low
concentration of the respective gap gene products and weakly to those that generate stripes in
areas of high gap gene expression. These results indicate that KR and KNI proteins form overlapping
concentration gradients that generate the periodic pair-rule expression pattern (Pankratz, 1990).
The expression of the pair-rule gene hairy in seven evenly spaced stripes along the longitudinal axis of the Drosophila blastoderm embryo is mediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter gene
expression in place of the most posterior h stripe 7 (h7-element), contains a dense array of binding sites for factors providing the
trans-acting control of h stripe 7 expression as revealed by genetic analyses. The stripe seven enhancer is found in a minimal 932 bp region from a 1.5 kb DNA fragment of the h upstream region. The h7-element mediates position-dependent gene
expression by sensing region-specific combinations and concentrations of both the maternal homeodomain transcriptional activators,
Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Zygotic caudal expression is not required for activation. Caudal and Bicoid, which form
complementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that the
anterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the h
stripe-7 domain are brought about by the local activities of repressors that prevent activation. The spatial limit of h7 is significantly altered in the gap mutants tailless, knirps and kruppel, but not in embryos lacking either hunchback, giant or huckebein. There are seven binding sites for Bcd, twenty-three for caudal, five for Kruppel, fourteen for Knirps, eight for Hunchback and five for Tailless. In the absence of both cad and bcd, activation still occurs. Thus, a third activator, likely to be Kr, must function in such embryos. It is thought that Kr acts as both a repressor and an activator within the h7 element depending on its concentration. The posterior border is set in response to Tll activity under the control of the terminal maternal organizer system. The anterior border of the expression domain is due to repression in response to Kni. The results suggest that the gradients
of Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo (La Rosee, 1997).
Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the
anterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In
the anterior region, such elements mediate transcriptional activation in response to (1) the maternal
concentration gradient of the anterior determinant Bicoid and (2) repression by spatially distinct activities
of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on
the activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlled
by zygotic regulators. The zygotic activation of hairy stripe 6 expression is preceded
by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe
expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior
stripes. To establish the potential of Cad and Kni to interact with the cis-acting DNA that mediates hairy stripe 6-like expression in the embryo, in vitro footprinting experiments were performed with the 532 bp hairy stripe 6-element DNA. Cad and Kni bind to thirty six in vitro binding sites, some of which overlap, throughout the element. The sequence of the Cad and Kni binding sites matches the consensus described for each of the two proteins. Most of the potential Cad and Kni binding sites are close to or overlapped by binding sites for Kruppel (eight sites), Hunchback (eight sites), and Tailless (five sites). Tests using fragments of the 532 bp enhancer and of another element, 284-HT, show that sequences mediating activation of reporter expression are not maintained within a minimal activation element but instead are dispersed throughout the enhancer (Hader, 1998).
Krüppel represses giant in the central domain thus assuring separated anterior and posterior expression (Kraut, 1991).
knirps expression is repressed by tailless activity, whereas it is directly enhanced by Kr activity. Thus, Kr activity is present throughout the domain of kni expression and forms a long-range protein gradient, which in combination with kni activity is
required for abdominal segmentation of the embryo. A construct containing 4.4 Kb of kni upstream sequence located at -0.9 Kb from the start of transcriptiongives the correct spatial pattern of expression in the anterior and posterior
domains. Kr responsive elements in kni reside in this 4.4 kb fragment and
more precisely in 800 bp fragment located at the 3' end of the 4.4 Kb. Two adjacent Kr protein binding regions are present approximately in the middle of this 0.8 Kb fragment. More sites become protected in footprint experiments when using higher concentration levels of the Kr protein (Pankratz, 1989)
The Krüppel binds to the sequence AAGGGGTTAA. Binding sites are present for KR upstream of the two hunchback promoters. These could mediate the repression of hb by KR and perhaps allow hb to influence its own expression. A 10 Kb genomic DNA fragment contains the hb coding sequence and both promoters. The proximal promoter directs early zygotic expression of hb in
the anterior part of the embryo The distal hb promoter is transcribed maternally and also directs later zygotic expression . This latter fragment contains the KR binding sites. 300 bp upstream of the transcription
start of the 2.9 kb transcript are sufficient for normal regulation of the
expression of this transcript. The two KR binding sites are located at -676 and -359 bp from
the proximal hb promoter (Treisman, 1989).
Analysis of the initial paired expression suggests that the gap genes hunchback, Krüppel, knirps and giant activate paired expression in stripes. Specifically, in Krüppel mutants stripes 2 and 3 are replaced by a broader stripe posterior to the wild-type stripe 2. Stripes 5 and 6 are replaced by a stipe that is located posterior to wild type stripe 5 (Gutjahr, 1993).
A 480 bp region of the even-skipped promoter is both necessary and sufficient to direct a
stripe of LacZ expression within the limits of the endogenous eve stripe 2. The maternal morphogen
Bicoid and the gap proteins Hunchback, Krüppel and Giant all bind with high
affinity to closely linked sites within this small promoter element. Forming the posterior border of the stripe involves a delicate balance between limiting amounts of the BCD activator and the KR
repressor (Small, 1992).
The entire functional even-skipped locus of Drosophila
is contained within a 16 kilobase region. As a
transgene, this region is capable of rescuing even-skipped
mutant flies to fertile adulthood. Detailed analysis of the 7.7
kb of regulatory DNA 3' of the transcription unit reveals
ten novel, independently regulated patterns. Most of these
patterns are driven by non-overlapping regulatory
elements, including ones for syncytial blastoderm stage
stripes 1 and 5, while a single element specifies both stripes
4 and 6. Expression analysis in gap gene mutants shows
that stripe 5 is restricted anteriorly by Krüppel and
posteriorly by giant, the same repressors that regulate
stripe 2. Consistent with the coregulation of stripes 4 and
6 by a single cis-element, both the anterior border of stripe
4 and the posterior border of stripe 6 are set by zygotic
hunchback, and the region between the two stripes is
ëcarved outí by knirps. Thus the boundaries of stripes 4 and
6 are set through negative regulation by the same gap gene
domains that regulate stripes 3 and 7, but at
different concentrations (Fujioka, 1999).
Gap proteins Krüppel and Hunchback to function as
transcriptional regulators in cultured cells. Both proteins bind to specific sites in a 100-bp DNA
fragment located upstream of the segment polarity gene engrailed, which also contains functional
binding sites for a number of homeo box proteins. The Hunchback protein is a strikingly
concentration-dependent activator of transcription, capable of functioning both by itself and also
synergistically with the pair-rule proteins Fushi tarazu and Paired. In contrast, Krüppel is a
transcriptional repressor that can block transcription induced either by Hunchback or by several
different homeo box proteins (Zuo, 1991).
To understand the nature of the regulatory signals impinging on the second promoter of the
Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. Products of the zygotically-active segmentation genes ftz, hb, Kr, gt and kni then act as activators or repressors of
Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp (Riley, 1991).
Expression of the abdominal-A and Abdominal-B genes of the BX-C of Drosophila is controlled by a cis-regulatory promoter and by distal enhancers called infraabdominal regions. The activation of these regions along the anteroposterior axis of the embryo determines where abdominal-A and Abdominal-B are transcribed. There is spatially restricted transcription of the infraabdominal regions (infraabdominal transcripts) that may reflect this specific activation. These iab regions are named after the abdominal segments they control, iab-2 through iab-7 regulating abdominal segments 2 through 7 corresponding to parasegments 7 through 12 respectively.
The Abdominal-B (Abd-B) gene of the bithorax complex (BX-C) of Drosophila controls the identities of the fifth through seventh abdominal segments and segments in the genitalia (more precisely, parasegments 10-14). The gap genes hunchback,
Krüppel, tailless and knirps control abdominal-A and Abdominal-B expression early in development. The gradients of the Hunchback and Krüppel products seem to be key elements in this restricted activation (Casares, 1995).
Krüppel and Knirps act through the infraabdominal 5 fragment of Abd-B to limit anterior Abd-B expression and regulate the graded Abd-B domain respectively. Both hunchback and Polycomb are required for Abd-B silencing (Busturia, 1993).
The gap genes hunchback,
Krüppel, tailless and knirps control abdominal-A and Abdominal-B expression early in
development. The restriction of abdominal-A and Abdominal-B transcription is preceded by (and
requires) the spatially localized activation of regulatory regions, which can be detected by the
distribution of infraabdominal transcripts. The activation of these regions requires no specific gap gene. Instead, a general mechanism of
activation, combined with repression by gap genes in the anteroposterior axis, seems to be
responsible for delimiting infraabdominal active domains. The gradients of the hunchback and
Krüppel products seem to be key elements in this restricted activation (Casares, 1995).
The anterior Abdominal-B expression limit is apparently determined by Krüppel repression, whereas the Knirps repressor may be responsible for the graded Abd-B expression within the Abd-B domain. iab-5 and two other fragments called MCP and FAB show
region-specific silencing activity: they suppress at a distance beta-gal expression mediated by a
linked heterologous enhancer. Silencing requires hunchback as well as Polycomb function and evidently provides maintenance of Abd-B expression limits throughout embryogenesis (Busturia, 1993).
Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the
gap gene products and the Polycomb group proteins. DNA elements and
trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein have been investigated. Analysis of a 1.7-kb
enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that both Hb
and Kruppel (Kr) are required to set the Abd-A anterior boundary in parasegment 7. DNase I footprinting and site-directed
mutagenesis show that Hb and Kr are direct regulators of this iab-2 enhancer. The single Kr site can be moved to a new
location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results
suggest that Kr repression occurs through a local quenching mechanism. The gap repressor Giant (Gt)
initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. This iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (Pho). These iab-2
Pho sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the Pho
sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late
embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer (Shimell, 2000).
The gap gene product Kr
is required to set the iab-2(1.7) anterior expression border. However, since Kr is not expressed
anterior to PS5, some other factor must
also be required to repress the iab-2(1.7) enhancer in anterior
regions. A likely candidate is the Hb protein, which has
been shown to be important for repressing the bx and pbx
enhancers anterior to PS6. To examine whether Hb plays a role in setting the
iab-2(1.7) anterior expression boundary, this
construct was crossed into both hb and osk mutant backgrounds. Loss
of zygotic hb caused a slight broadening of the initial
expression band, indicating an anterior shift in the expression
pattern of this enhancer. The presence
of maternal Hb likely minimizes the anterior shift in these
zygotic hb mutant embryos. Consistent with this view, it has been found that, in an osk mutant background, in which the
maternal level of Hb is uniform throughout the embryo, expression from the iab-2(1.7) enhancer is
completely abolished. These findings
suggest that, as with the bx and pbx enhancers, Hb is
important for setting the initial anterior limit of iab-2
enhancer function (Shimell, 2000).
Hb, Kr, and Gt have been classified as short-range
repressors whose range of action is limited to approximately
50 to 150 bps. Two major mechanisms of
short-range repression are: competitive binding to an overlapping
activator binding site, and quenching, which entails
interference with function of locally bound activators. Since studies on Hb, Kr, and Gt action
have focused primarily on their control of pair-rule genes
such as eve, it was of interest to address mechanisms used by
these repressors in the alternative context of a homeotic
gene regulatory region.
The in vitro binding analysis identified five discrete Hb
sites on the iab-2(1.7) fragment. One of these sites,
Hb2, overlaps extensively with one of the Eve binding
sites. Since Eve acts as an activator of iab-2(1.7) expression, Hb may repress by competing with Eve for direct
binding to this site. Evidence for a direct competition
mechanism has been described for Hb repression through
the bx and bxd/pbx control regions of the Ubx homeotic gene. In these
cases, the anterior boundary is in PS6 rather than PS7, and
Hb competes with Ftz rather than Eve. However, mutational
analysis shows that Hb sites other than Hb2 also
contribute to iab-2 repression. These additional sites could
promote Hb competition with Eve by assisting Hb binding
at Hb2 through cooperative interactions. Similarly, the
single Gt binding site in iab-2(1.7) overlaps another Eve
binding site, suggesting that Gt may also repress by
direct competition with Eve in posterior parasegments.
In contrast, the single Kr binding site (Kr1) does not
overlap Eve sites. A distinct Kr mechanism is also supported
by the ability of Kr1 to repress even when relocated
100 bp away from its normal position in the iab-2(1.7)
fragment. This flexibility, together with failure of
Kr repression when Kr1 is further relocated by 300 bp, is
consistent with a short-range quenching mechanism. These
results argue against Kr repression by direct interference
with basal transcription factors, since
300 bp is small compared to the 20-kb distance between the
iab-2 enhancer and the abd-A promoter. Previous studies
using a synthetic regulatory region have shown that Kr can
repress by a quenching mechanism in vivo (Shimell, 2000).
Any proposed mechanism for Kr action through iab-2,
however, must account for the variability of Kr repression
within its own expression domain. Specifically, Kr represses
the iab-2 enhancer in PS3 and PS5 where Kr
concentrations are low, but it does not repress in PS7 where
Kr concentrations are high. This observation suggests that
simple occupancy of the Kr1 site is not sufficient for iab-2
repression and that another factor acts in concert with Kr.
The likely partner is Hb since Kr repression of iab-2 is
limited to parasegments that accumulate significant levels
of both Kr and Hb. In this view, repression just anterior to
PS7 requires both Kr and Hb, whereas repression in more
anterior parasegments, where Hb levels are highest, is
mediated by Hb alone. Kr-Hb synergy could involve direct
contact since the two proteins have been shown to interact
when bound to DNA. Whether Kr
synergizes with Hb by augmenting Hb binding to DNA in a
cooperative manner or by recruiting additional corepressors
is not clear. Kr, but not Hb, functions
together with the corepressor dCtBP (Shimell, 2000 and references therein).
After Hb and Kr decay during early gastrulation, the
repressed state is propagated through later stages of development
by the PcG proteins. How the transition from early
gap repressors to long-term PcG repressors occurs at the
molecular level is not known. Two basic models have been
proposed: (1) direct recruitment, and (2) chromatin recognition. Model (1): The gap gene products, especially Hb, have
been proposed to help recruit PcG proteins directly to
specific DNA sites. Based upon its
early time of action, a role for the PcG protein Extra sex
combs (Esc) as a molecular bridge between the two sets of
repressors has been suggested. However, direct interactions
between Esc and gap repressors have not been reported.
A better candidate for such a molecular link is
dMi-2, which binds directly to Hb and behaves genetically
as an enhancer of PcG repression. In its
simplest form the direct recruitment model is unlikely
because the iab-2, bx, and pbx enhancers all contain Hb
sites but do not effectively recruit PcG proteins. These
elements fail to maintain A-P boundaries of expression and
are unable to attract PcG proteins to sites on chromosomes. Furthermore,
the continuous requirement for PRE sequences during
development shows that DNA
site recognition by PcG proteins can occur long after Hb
and Kr have decayed. Model (2):The second model proposes that PcG
proteins recognize some feature of silenced chromatin,
rather than particular gap repressors.
This model is supported by patterns of PcG-dependent
silencing that reflect patterns of early gene activity rather
than the distributions of gap proteins. In
this view, PcG proteins sense the transcriptional off state
and then assemble locally to imprint this state through
later stages. These two models are not mutually exclusive. Both the
Hb-interacting protein dMi-2 and the
Kr-interacting protein dCtBP have
mammalian homologs that interact with histone deacetylases. Perhaps the gap repressors work by targeting these
deacetylases, whose action alters the local acetylation state
of the histone tails. This could provide a feature of silenced
chromatin that is recognized by PcG proteins and that
promotes their association at nearby PREs (Shimell, 2000 and references therein).
Kr activity is required for the establishment of Antennapedia third thoracic segment expression and is involved in restricting Abd-B products within the abdominal segments eight and nine (Harding, 1987).
Krüppel, caudal and cut are expressed in the Malpighian tubules before and during differentiation. Two of the genes,Krüppel and cut, are known to be required for development of the tubules. The absence of maternal and zygotic caudal function reduces their
normal growth and elongation. Normal Krüppel function, which is known to be required for caudal expression, is also required for cut expression, while cut and caudal are expressed independently of
each other. Cell type transformations of Malpighian tubules were studied by examining the effects
of mutations on the expression of markers specific to Malpighian tubules, hindgut, or midgut of
normal embryos. Loss of Krüppel activity confers hindgut characteristics on those cells that
normally form the Malpighian tubules with all markers tested. Loss of cut function alters the
expression of some markers but not others. The pathway of tissue specific gene regulation,
apparently, branches beyond Krüppel to form at least a cut and a caudal branch (Liu, 1992).
A target gene of Kr, termed knockout (ko), was isolated by virtue of Kruppel in vitro binding sites in the ko promoter. Loss and gain of function experiments show that Kr activity maintains ko expression in a subset of muscles. ko encodes a novel protein expressed in several embryonic tissues, including Kr-expressing muscles. knockout is initially expressed in stage 10 embryos in the pharynx and the esophageal region, in groups of cells in the developing CNS, in the distal part of the Malpighian tubule and in the dorsal vessel. It is expressed in specific muscle precursor cells, both transiently and persistently depending on the location, until mature muscle fibers have been formed. Cis-acting elements controlling muscle expression of ko are spread over 15 kb of DNA. While Kr is required for a distinct subset of muscles, it is also necessary in others where its role is not decisive. That is, ko expression is variably affected in certain muscle precursors of Kr mutants. ko is not ectopically expressed in Kr gain of function experiments. Movements of embryos deficient for ko activity are uncoordinated. Their muscle pattern is normal, but the patterns of neuromuscular innervation are specifically disarranged. The results suggest that the Kr target gene ko is required for proper innervation of specific muscles by RP motoneurons (Hartmann, 1997a).
After blastoderm formation, Kruppel is expressed in various spatially and temporally restricted patterns in the developing embryo, including a subset of muscle precursors. By virtue of Kruppel in vitro binding sites, a putative Kr target gene, termed KrT95D, has been identifed. It encodes a novel protein that contains evolutionarily conserved regions. KrT95D is expressed in spatially restricted patterns throughout embryogenesis. Kr and KrT95D expression overlap in several locations, including muscle precursor cells, the tip cell of the Malpighian tubules and the ventral midline cells of the central nervous system. Results from the analysis of the KrT95D expression pattern in Kr loss-of-fuction and Kr gain-of-function embryos suggest that Kr activity is not essential for KrT95D expression in most locations of the embryo, except in the muscle precursors VO5 (Hartmann, 1997b).
By examining expression of arc in different mutant embryos,
it was determined that transcription factors known to
be required for patterning and maintenance of various
developing epithelia control arc expression in those domains. tll and hkb, which
are required to pattern the posterior 15% of the embryo, control arc expression
in the posterior midgut primordium. fkh, which appears to
act as a maintenance, or permissive, transcription factor, is
required for expression of arc throughout the gut. byn,
which is required for hindgut development and specifies its
central domain (the large intestine),
controls expression of arc in the elongating hindgut. Kr and
cut, required for evagination and extension of the Malpighian
tubule buds
control expression of arc in the tubule primordia (Liu, 2000).
Krüppel (Kr), a member of the gap class of Drosophila segmentation
genes, encodes a DNA binding zinc finger-type transcription factor. In addition
to its segmentation function at the blastoderm stage, Krüppel also plays a
critical role in organ formation during later stages of embryogenesis. To
systematically identify in vivo target genes of Krüppel, DNA fragments were
isolated from the Krüppel-associated portion of chromatin and they were
used to find and map Krüppel-dependent cis-acting regulatory sites in the
Drosophila genome. Krüppel binding sites are not enriched in
Krüppel-associated chromatin and the clustering of Krüppel binding
sites, as found in the cis-acting elements of Krüppel-dependent
segmentation genes used for in silico searches of Krüppel target genes, is
not a prerequisite for the in vivo binding of Krüppel to its regulatory
elements. Results obtained with the newly identified target gene(s) ken and
barbie, together referred to as ken indicate that Krüppel
represses transcription and thereby restricts the spatial expression pattern of
ken during blastoderm and gastrulation (Matyash, 2004).
To establish whether the newly identified candidate genes are indeed
regulated in a Krüppel-dependent fashion, focus was placed on ken.
The reason for this choice was that ken, which encodes a DNA binding zinc
finger-type transcription factor, appears at a first glance unlikely to be a
Kr target gene. This is because (1) Kr activity is not required for
male genitalia formation and adult eye development, the two processes in which
ken is involved. (2) ken is expressed early in two stripes
that do not overlap with the Kr expression domain during blastoderm stage
and gastrulation. In contrast, it was found that the isolated 749-bp DNA fragment is highly enriched in the DNA of
Krüppel-associated chromatin and that
it contains five Krüppel binding sites confirmed by gel mobility shift assays (Matyash, 2004).
To solve this apparent dilemma and to thereby demonstrate that the screen has
indeed led to Krüppel target genes, it was asked whether Krüppel does
regulate ken expression in vivo by performing in situ
hybridizations of ken probes to whole mount preparations of wild type and
homozygous Kr1 lack-of-function mutant embryos.
In wild type, Krüppel is
initially expressed in a broad band in the central region of the blastoderm.
In contrast, ken is expressed in two distinct
stripes that are anteriorly adjacent and posterior to the Kr central
domain. In Kr mutant embryos, the two stripes
of ken expression are not altered, but an additional expression domain was observed
where Kr is normally expressed
at syncytial blastoderm stage. This expression domain appears earlier than the normal stripes of
ken expression, and it subsequently fades in a posterior to anterior
direction, resulting in a third narrow stripe that remains separated from the
anterior ken stripe.
These observations establish that in the absence of Kr activity,
ken is activated in the central region of the embryo and that this aspect
of ken activity is normally repressed in a Krüppel-dependent manner (Matyash, 2004).
Previous results have shown that the expression of the anterior stripe of
ken is activated in response to the transcription factors encoded by
bicoid and hunchback, whereas the posterior stripe is activated by
the transcription factor of tailless, and its shape and size are due to
repression by Huckebein. To establish whether ectopic
expression of Krüppel also causes the repression of ken, a
heat shock-driven Kr transgene was used to misexpress
Kr uniformly in the blastoderm embryo.
The posterior stripe of ken expression is
not affected by ectopic Kr activity, whereas the anterior ken
stripe is lacking. Collectively, the results demonstrate that Krüppel
participates in early ken regulation by acting as a local repressor of
the gene in wild type embryos (Matyash, 2004).
This study was directed at the identification of Krüppel-dependent
genes involved in neurogenesis, muscle, and Bolwig organ development. Genes identified that
are involved in neurogenesis include tup, cut and short stop. In fact, 55 of the 82 isolated
genes are known to participate in these developmental processes. Thus, it is expected that
Krüppel regulates possibly several hundreds of genes during the entire
life cycle of the fly (Matyash, 2004).
Two of the Kr target genes (emc and osa) have been
identified in a genetic modifier
screen for gene products that mediate Kr activity. In addition, a DNA fragment
corresponded to the intron of the gene CG7097, a
putative regulatory target of segmentation genes expressed during blastoderm
formation. Microarray-based expression data and whole
mount in situ hybridization of early embryos
shows that this gene as well as additional 29 of the 43 candidate
genes are expressed during the first 14 h
of embryonic development. These observations and the results of the genetic
studies with ken indicate that the DNA isolated from
Krüppel-associated chromatin revealed in vivo target sites of the
transcription factor (Matyash, 2004).
Previous analysis has shown that during segmentation Krüppel controls the
activity of other transcription factors that are part of a cell fate-determining
gene network. The results
suggest that this earlier finding is not restricted to Kr segmentation
function since the majority of the Krüppel target genes identified in this
study (18% of the total isolates) encode transcription factors as well. The more
important notion is, however, that Krüppel not only participates in the
regulation of transcription factor networks at the different levels of the
segmentation gene cascade but also assists signaling
events by regulating various pathway components, as exemplified by target genes
coding for components of the JAK/STAT-signaling pathway. Krüppel target DNA
includes portions of the genes ken, STAT92E, and stc, which code
for JAK/STAT-mediating transcription factors as well as factors known to participate in
signaling by the epidermal growth factor receptor (Asteroid)
and Rho GTPases (Gef64C).
Moreover, the isolation of genes encoding lipid metabolism-related enzymes and
the lipid carrier Neural Lazarillo (NLaz) suggests
that Krüppel not only takes part in embryonic fat body development
but also participates in metabolic functions (fat storage
or fat consumption) of the organ (Matyash, 2004).
The majority of the newly isolated Krüppel target sites lack Krüppel
binding site clusters as revealed in cis-acting elements of the
Krüppel-dependent segmentation genes.
However, the isolated and subsequently tested set of DNA
fragments is enriched in Krüppel-associated chromatin, as has been found
with the eve stripe 2 element, which contains clustered Krüppel
target sites. This finding suggests that the
clustering of binding sites is not the sole biologically relevant marker for
Krüppel-dependent cis-acting control elements. Furthermore, the algorithm
applied to detect Krüppel binding sites only counted matches of sequences
to a weighted matrix that were arbitrarily set above a certain threshold. In
consequence, functional low affinity binding sites or Krüppel-dependent DNA
segments that contain only few and unclustered high affinity binding sites were
left undetected (Matyash, 2004).
Interestingly, more than half of the Krüppel target DNA fragments (68%)
were located in introns and exon/intron overlap sequences or in exons
and not at the canonical 5' termini of
protein-coding genes. The location of these fragments downstream of the
transcription start sites suggests that they may represent distal regulatory
elements (e.g., enhancers or silencers) or promoters for non-coding RNAs,
as implied by a most recent study on transcription factor binding along human
chromosome 21 and 22. Because noncoding transcripts
within the Drosophila genome are not systematically annotated, it cannot be
decide whether Krüppel participates in the transcription of such
transcripts (Matyash, 2004).
A surprising result of this study was that ken, which is not expressed in
the Krüppel domain of wild type blastoderm embryos, is in fact a target of Krüppel. In the absence of
Kr activity, ken is activated in the central region of the
blastoderm. Thus, in addition to the regulation of ken expression in the
anterior and posterior stripe domains, which involves the activities of
bicoid in cooperation with the gap genes hunchback, tailless, and
huckebein, Krüppel is needed to prevent
ectopic ken activation in the blastoderm embryo. This finding and the
notion that ubiquitous Krüppel expression abolishes ken activity in
the anterior but not in the posterior stripe domain suggest that the two stripes
of ken expression are under the control of separate cis-acting elements,
of which only one mediates repression by Krüppel (Matyash, 2004).
Genetic studies have revealed that segment determination in Drosophila
melanogaster is based on hierarchical regulatory interactions among maternal
coordinate and zygotic segmentation genes. The gap gene system constitutes the
most upstream zygotic layer of this regulatory hierarchy, responsible for the
initial interpretation of positional information encoded by maternal gradients.
A detailed analysis of regulatory interactions involved in gap gene
regulation is presented based on gap gene circuits, which are mathematical gene network
models used to infer regulatory interactions from quantitative gene expression
data. The models reproduce gap gene expression at high accuracy and temporal
resolution. Regulatory interactions found in gap gene circuits provide
consistent and sufficient mechanisms for gap gene expression, which largely
agree with mechanisms previously inferred from qualitative studies of mutant
gene expression patterns. These models predict activation of Kr by Cad and
clarify several other regulatory interactions. This analysis suggests a central
role for repressive feedback loops between complementary gap genes.
Repressive interactions among overlapping gap genes show anteroposterior
asymmetry with posterior dominance. Finally, these models suggest a correlation
between timing of gap domain boundary formation and regulatory contributions
from the terminal maternal system (Jaeger, 2004b).
Although activating contributions from Bcd and Cad show some degree of
localization, positioning of gap gene boundaries
during cycle 14A is largely under the control of repressive gap-gap
cross-regulatory interactions. Thereby, activation is a prerequisite for
repressive boundary control, which counteracts broad activation of gap genes in
a spatially specific manner.
In addition, gap genes show a tendency toward
autoactivation, which increasingly potentiates
activation by Bcd and Cad during cycle 14A.
Autoactivation is involved in maintenance of gap gene expression within given
domains and sharpening of gap domain boundaries during cycle 14A (Jaeger, 2004b).
Regulatory loops of mutual repression create positive regulatory feedback
between complementary gap genes, providing a straightforward mechanism for their
mutually exclusive expression patterns. Such a mechanism of 'alternating
cushions' of gap domains has been proposed previously. The results
suggest that this mechanism is complemented by repression among overlapping gap
genes. Overlap in expression patterns of two repressors imposes a limit on the
strength of repressive interactions between them. Accordingly, repression
between neighboring gap genes is generally weaker than that between
complementary ones. Moreover, repression among
overlapping gap genes is asymmetric, centered on the Kr domain.
Posterior to this domain, only posterior neighbors
contribute functional repressive inputs to gap gene expression, while anterior
neighbors do not. This asymmetry is responsible for
anterior shifts of posterior gap gene domains during cycle 14A (Jaeger, 2004b).
Repression by Tll mediates regulatory input to gap gene expression by the
terminal maternal system. Tll provides the main repressive
input to early regulation of the posterior boundary of posterior gt,
and activation by Tll is required for posterior
hb expression. Note that these two features
form only during cycle 13 and early cycle 14A,
while other gap domain boundaries are already present at the transcript level
during cycles 10-12 and largely
depend on the anterior and posterior maternal systems for their initial
establishment. The delayed formation of posterior patterning features and
their distinct mode of regulation are reminiscent of segment determination in
primitive dipterans and intermediate germ-band insects, supporting a conserved
dynamical mechanism across different insect taxa (Jaeger, 2004b).
The set of regulatory interactions presented here provides a consistent and
sufficient dynamical mechanism for gap gene expression. In
summary, this set of interactions consists of the following five basic
regulatory mechanisms: (1) broad activation by
Bcd and/or Cad, (2) autoactivation, (3) strong repressive feedback between
mutually exclusive gap genes, (4) asymmetric repression between overlapping gap
genes, and (5) feed-forward repression of posterior domain boundaries by the
terminal gap gene tll. In the following subsections, evidence is discussed
concerning specific regulatory interactions involved in each of these basic
mechanisms in some detail (Jaeger, 2004b).
Activation by Bcd and Cad:
Activation of gap gene expression by Bcd and Cad is supported by the following.
Bcd binds to the regulatory regions of hb, Kr, and kni.
The kni regulatory region also contains binding sites
for Cad. The anterior domains of gt and hb are
absent in embryos from bcd mothers. The posterior domain of gt is
missing in embryos mutant for both maternal and zygotic cad, while the
posterior domain of kni is absent in embryos mutant for maternal
bcd plus maternal and zygotic cad. These
results suggest partial redundancy of activation of kni by Bcd,
consistent with evidence from zygotic cad embryos from bcd
mothers, where maternally provided Cad is sufficient to activate kni (Jaeger, 2004b).
Kr expression expands anteriorly in embryos from bcd mothers,
which is due to the absence of the anterior gt and hb
domains. Bcd has been shown to activate expression of
Kr reporter constructs. The fact that Kr is still expressed in
embryos from bcd mutant mothers has been attributed to activation by
general transcription factors or low levels of Hb. In contrast, the
models predict that this activation is provided by Cad. Although Kr
expression is normal in embryos overexpressing cad, repressive
control of Kr boundaries could account for the lack of expansion of the
Kr domain in such embryos (Jaeger, 2004b).
The activating effect of Cad on hb found in gap gene circuits is likely
to be spurious. The anterior hb domain is absent in embryos from
bcd mutant mothers,
which show uniformly high levels of Cad. Moreover, the
complete absence of the posterior hb domain in tll mutants
suggests activation of posterior
hb by Tll rather than by Cad. It is believed that this spurious activation of
hb by Cad is due to the absence of hkb in gap gene circuits. The
posterior hb domain fails to retract from the posterior pole in
hkb mutants, suggesting a repressive role of Hkb
in regulation of the posterior hb border. Consistent with this, the
posterior boundary of the posterior hb domain never fully forms in any of
the circuits. Moreover, Tll is constrained to a
very small or no interaction with hb due
to the absence of the posterior repressor Hkb, since activation of hb by
Tll would lead to increasing hb expression extending to the posterior
pole (Jaeger, 2004b).
Autoactivation:: A role for autoactivation
in the late phase of hb regulation
is supported by the fact that the posterior border of anterior
hb is shifted anteriorly in a concentration-dependent manner in embryos
with decreasing doses of zygotic Hb.
Weakened and narrowed expression of Kr in mutants encoding a functionally
defective Kr protein suggests Kr autoactivation. Similarly, a
delay in the expression of gt in mutants encoding a defective Gt protein
indicates gt autoactivation. However, the results suggest that
gt autoactivation is not essential. It is generally weaker than
autoactivation of other gap genes, and
circuits lacking gt autoactivation show no specific defects in gt
expression. Finally, in the case of kni, there is no experimental
evidence for autoactivation, while some authors have even suggested kni
autorepression. No such autorepression has been detected in any gap gene circuit (Jaeger, 2004b).
Repression between complementary gap genes:
Mutual repression of gt and Kr is supported by the following.
gt expression expands into the region of the central Kr domain in
Kr embryos. In contrast, Kr expression is not
altered in gt mutants before germ-band extension.
However, Gt binds to the Kr regulatory
region, and the central domain of Kr is absent in embryos overexpressing
gt. Moreover, Kr expression extends further
anterior in hb gt double mutants than in hb mutants alone.
The above is consistent with this analysis, which shows no
significant derepression of Kr in the absence of Gt even though
repression of Kr by Gt is quite strong (Jaeger, 2004b).
Hb binds to the kni regulatory region, and the posterior kni
domain expands anteriorly in hb mutants. Embryos overexpressing hb
show no kni expression at all, and embryos
misexpressing hb show spatially specific repression of kni
expression.There is no clear posterior expansion of kni in hb mutants.
This could be due to the relatively weak and late repressive contribution of Hb on the
posterior kni boundary or due to partial redundancy with repression by Gt
and Tll. The posterior hb domain
expands anteriorly in kni mutants, but anterior hb expression is
not altered in these embryos.
Nevertheless, a role of Kni in positioning the anterior hb
domain is suggested by the fact that misexpression of kni leads to
spatially specific repression of both anterior and posterior hb domains. Moreover, only
slight posterior expansion of anterior hb is observed in Kr
mutants, while hb is completely derepressed between its anterior and
posterior domains in Kr kni double mutants (Jaeger, 2004b).
Repression between overlapping gap
genes: gt, kni, and Kr show repression by their immediate
posterior neighbors hb, gt, and kni, respectively.
Retraction of posterior Gt from the posterior pole
during midcycle 14A fails to occur in hb mutants,
and no gt expression is observed in embryos overexpressing
hb. The posterior kni boundary is
shifted posteriorly in gt mutant embryos, and kni
expression is reduced in embryos overexpressing gt. Note that
these effects are very subtle and were not reported in similar studies by
different authors. A weak but functional interaction of Gt with kni
is consistent with these results. This interaction was found to be essential even
in a circuit where it was deemed below significance level. Finally, Kni has been
shown to bind to the Kr regulatory region, and the central Kr domain
expands posteriorly in kni mutants (Jaeger, 2004b).
In contrast, no effect of Kr on hb was detected.
However, hb expression expands
posteriorly in Kr mutants. This effect is
likely to involve repression of hb by Kni. Kni levels are reduced in
Kr embryos. hb is completely derepressed between its anterior and
posterior domains in Kr kni double mutants, whereas anterior hb
does not expand at all in kni mutants alone. Taken together these results
suggests that there is direct repression of hb by Kr in the embryo,
but it is at least partially redundant with repression of hb by Kni (Jaeger, 2004b).
Unlike repression by posterior neighbors, no or only weak
repression was found of posterior kni, gt, and hb by their anterior
neighbors Kr, kni, and gt, respectively.
Most gap gene circuits show weak activation of
hb by Gt. Graphical analysis
failed to reveal any functional role for such activation. Moreover, no functional
interaction was found between
gt and Kni. Although relatively
weak repression of kni by Kr was found in 6 out of 10 circuits,
no specific patterning defects could be
detected in the other 4. Consistent with the above, expression of posterior
hb is normal in gt mutants, and both the anterior boundaries of
posterior gt and kni are positioned correctly in kni and
Kr mutant embryos, respectively (Jaeger, 2004b).
Note that activation of kni by Kr, which has been proposed to explain
decreased expression levels of kni in Kr mutants, was never found.
The results strongly support the view that this interaction is indirect through Gt, which is
further corroborated by the fact that kni expression is completely
restored in Kr gt double mutants compared to that in Kr mutants
alone (Jaeger, 2004b).
A significant repressive effect of Hb on Kr was found.
Consistent with this, Hb has been shown to
bind to the Kr regulatory region, and the central Kr domain
expands anteriorly in hb mutants. However,
partial redundancy of this interaction is suggested by correct positioning and
shape of the anterior Kr domain in a circuit that does not show
repression of Kr by Hb (Jaeger, 2004b).
It has been proposed that Hb plays a dual role as both activator and repressor
of Kr. In the framework of the gene circuit model,
concentration-dependent switching of regulative action could be implemented by
allowing genetic interconnection parameters to switch sign at certain regulator
concentration thresholds. The current model explicitly does not include such a
possibility. Nevertheless, circuits have been obtained that reproduce
Kr expression faithfully, suggesting that
a dual role of Hb is not required for proper Kr expression. Moreover,
activation of Kr by Hb was ever observed in any of the circuits.
Therefore, the results support a mechanism
in which the activation of Kr by Hb is indirect through derepression of kni (Jaeger, 2004b).
Repression by Tll: Only a few earlier
theoretical approaches have considered terminal gap genes. Gap gene
circuits accurately reproduce tll expression. However,
in gene circuits, tll is subject to regulation by other gap genes, which
is inconsistent with experimental evidence. In contrast, the correct expression pattern
of tll in gap gene circuits allows its effect on other gap genes to be studied
in great detail. Strong repressive effects of Tll on Kr,
kni, and gt have been found. Tll binding sites have
been found in the regulatory regions of Kr and kni. In
tll mutants, Kr expression is normal,
whereas expression of kni expands posteriorly, and the
posterior gt domain fails to retract from the posterior pole.
No expression of Kr, kni, or
gt can be detected in embryos overexpressing tll under a
heat-shock promoter (Jaeger, 2004b).
A fundamental problem in functional genomics is to determine the structure and dynamics of genetic networks based on expression data. A new strategy is described for solving this problem and it is applied to recently published data on early Drosophila development. The method is orders of magnitude faster than current fitting methods and allows fitting of different types of rules for expressing regulatory relationships. Specifically, this approach is sused to fit models using a smooth nonlinear formalism for modeling gene regulation (gene circuits) as well as models using logical rules based on activation and repression thresholds for transcription factors. The technique also allows inference of regulatory relationships de novo or testing network structures suggested by the literature. A series of models is fitted to test several outstanding questions about gap gene regulation, including regulation of and by hunchback and the role of autoactivation. Based on the modeling results and validation against the experimental literature, a revised network structure is proposed for the gap gene system. Interestingly, some relationships in standard textbook models of gap gene regulation appear to be unnecessary for, or even inconsistent with, the details of gap gene expression during wild-type development (Perkins, 2006).
The regulatory structure of the Combined model is itself sufficient to reproduce all six gap gene domains using either the gene circuit or logical formalisms for production rate functions. Support is cited for the Combined model, and then consider the results of the individual models in light of several outstanding questions about gap gene regulation are discussed (Perkins, 2006).
The maternal proteins Bcd and Cad are largely responsible for activating the trunk gap genes, with Bcd being more important for the anterior domains and Cad more important for the posterior domains. Bcd is a primary activator of the anterior hb domain, the anterior gt domain, and the Kr domain. Cad activates posterior gt. The kni domain is present in bcd mutants and in cad mutants, but not in bcd;cad double mutants. This suggests redundant activation by the two maternal factors. Such redundant activation of kni is present in the Unc-GC model. For the other models, the optimization selected one or the other as activators, but not both. Tll is crucial for activating the posterior hb domain, while it represses Kr, kni, and gt, preventing their expression in the extreme posterior. All the regulatory relationships between the gap genes in the Combined model are repressive. The complementary gap gene pairs, hb–kni and Kr–gt are known to be strongly mutually repressive, as was found in nearly all the models. [Repression of hb by Kni is not part of the Rivera-Pomar and Jäckle (RPJ) regulatory relationships (Rivera-Pomar, 1996), but the unconstrained gene circuit (Unc-GC) model and Unc-Logic model (that employs the regulatory structure discovered by the unconstrained gene circuit fit, except that Gt activation of hb and Kni activation of gt were removed) included the link.] The models also suggest that mutual repression between hb and Kr helps to set the boundary between those two domains. A chain of repressive relationships, hb-gt-kni-Kr, causes the shifts in the Kr, kni, and posterior gt domains. Autoactivation by hb is well-established, and there is also some evidence for autoactivation by Kr and gt (Perkins, 2006).
Does Hb have a dual regulatory effect on Kr? There is a long-running debate about whether or not low levels of Hb activate Kr. In hb mutants, the Kr domain expands anteriorly, suggesting that Hb represses Kr. However, Kr expression in these mutants is lower than in wild-type and expands posteriorly in embryos overexpressing Hb. Further, in embryos lacking Bcd and Hb, the Kr domain is absent, but can be restored in a dosage-dependent manner by reintroducing Hb. These observations suggest that Hb activates Kr. It has been suggested, therefore, that low levels of Hb activate Kr while high levels repress it. An alternative explanation, however, is that the apparently activating effects of Hb are indirect, via Hb's repression of kni and Kni's repression of Kr. Optimization of the Unc-GC model, which could have resulted in activation or repression of Kr by Hb, but not both, resulted in repression. The RPJ models allow for a dual effect, but activation by Hb was eliminated during optimization of the RPJ-Logic model. The RPJ-GC model retained functional activation and repression of Kr by Hb. However, Kr expression in this model is defective. Kr is not properly repressed in the anterior. Further, Kr is ectopically expressed in a small domain in the posterior of the embryo. Thus, the current models provide no support for activation of Kr by Hb. The only support found, which is crucial in all models except Unc-Logic and also consistent with the mutant and overexpression studies, is for repression of Kr by Hb (Perkins, 2006).
What represses hb between the anterior and posterior domains? Another point of disagreement in the literature is what prevents the expression of hb between its two domains. In the model of Rivera-Pomar and Jäckle (1996), repression by Kr is the explanation. The RPJ models confirm that this mechanism is sufficient. Specifically, in these models Kr repression prevents hb expression just to the posterior of the anterior hb domain. Between the Kr and posterior hb domains, there is no explicit repression of hb. Rather, Hb is not produced simply because of a lack of activating factors. In contrast, the models of Jaeger (2004a and b) detected no effect of Kr and attributed repression solely to Kni. The Unc-GC and Unc-Logic models found repression by Kni, but in addition to repression by Kr, not instead of it. Kr is more responsible for repression near the anterior hb domain and Kni is more responsible for repression near the posterior hb domain. This is consistent with observations of expression in mutant embryos. Embryos mutant for Kr show slight expansion of the anterior hb domain, while kni embryos show expansion of the posterior hb domain. In Kr;kni double mutants, hb is completely derepressed between its two usual domains. This suggests, as seen in the Unc-GC and Unc-Logic models, that Kr and Kni are both repressors of hb, that their activity is redundant in the center of the trunk, and that Kr and Kni are the dominant repressors for setting the boundaries of the anterior and posterior domains, respectively. This interpretation was also favored by Jaeger (2004a and b), on the basis of the mutant data, even though Jaeger's models did not find repression by Kr (Perkins, 2006).
The posterior hb domain. In all of the current models, the posterior hb domain is activated by Tll and sustained by Tll and hb autoactivation. Rivera-Pomar (1996) did not consider the posterior hb domain, and did not include activation by Tll in his model. That link was added to the RPJ network structure because otherwise it was not possible to capture the posterior hb domain. The model of Jaeger (2004a and b) captured the domain without Tll activation by substituting activation from cad. However, there is no confirming evidence for such an interaction. The absence of posterior hb in tll mutants and the inability of the models to explain posterior hb by other means, leads to the straightforward hypothesis that Tll activates posterior hb. Posterior hb is unique in that the domain begins to form later than the other five domains modeled. In the RPJ models, this happens simply because high levels of Tll are needed to activate hb -- levels that are reached only at about t = 30 min. The Unc-GC and Unc-Logic models also employ repression by Cad to slightly delay Hb production in the posterior. However, there is no confirming evidence for such repression, and it is omitted from the Combined model (Perkins, 2006).
Shifting of the Kr, kni, and posterior gt domains. Domain shifting was first observed by Jaeger (2004a and b) and attributed to a chain of repressive regulatory relationships, hb-gt-kni-Kr. The current models largely support the importance of this regulatory chain, particularly the final two links. Repression of Kr by Kni was significant in all of the current models. Repression of kni by Gt was present in all models except RPJ-Logic, where it would be of little impact anyway, since RPJ-Logic has a defective posterior gt domain. Consistent with these findings, Kni binds to the regulatory region of Kr, and the Kr domain expands towards the posterior in kni mutants. Similarly, the kni domain expands posteriorly in gt mutants, while embryos overexpressing gt show reduced kni expression (Perkins, 2006).
Repression of gt by Hb is not as well supported by the current models. The Unc-GC model included the link, though the regulatory weight was the smallest of all those in the model. The link was eliminated from Unc-Logic and, of course, not present in the RPJ network structure. Instead, the models utilized decreasing activation by Cad (Unc-GC, Unc-Logic) and repression by Tll (Unc-GC, RPJ-GC) to shift the posterior gt domain. Even with these links, however, shifting of the domain is not well-captured. RPJ-GC appears to capture the posterior gt shift best ( Activating or repressing links that oppose the direction of the repressive chain were eliminated by optimization of the Unc-Logic, RPJ-GC, and RPJ-Logic models. In agreement with this result, the boundaries of the kni and posterior gt domains are correctly positioned in Kr and kni mutants, respectively. Thus, the simplest picture supported by the current models and consistent with the mutant studies is that there is no regulation from Kr, kni, or posterior gt to any of their immediate posterior neighbors, and that the repressive chain highlighted by Jaeger (2004a and b) is indeed responsible for domain shifting (Perkins, 2006).
Do gap genes autoregulate? All four of the current models include autoactivation by hb. This is supported by the observation that late anterior hb expression is absent in embryos lacking maternal and early zygotic Hb
. The models suggest hb autoactivation also plays a crucial role in sustaining the posterior domain, once it has been initiated by Tll, a role not previously emphasized. Autoactivation for the other genes was found by the Unc-GC model, but is not part of the RPJ network structure. It included autoactivation only for Kr and gt in the Combined model, on the basis of a weakened and narrowed Kr domain in embryos producing defective Kr protein and a delay in gt expression in embryos producing defective gt protein. Interestingly, the gene circuit models of Jaeger (2004a and b) also found autoactivation for all four gap genes, but they considered autoactivation by gt to be the weakest and least certain. In contrast, the Unc-Logic model retained gt autoactivation while eliminating autoactivation for Kr and kni. The RPJ-Logic model was unable to reproduce the posterior gt domain. However, it was found that by adding gt autoactivation to the model, it was able to create and sustain posterior gt correctly, bringing the error of the model down to 15.34. This suggests that, after hb, gt is the most likely candidate for autoactivation. However, even this is not strictly necessary. The RPJ-GC model is able to reproduce and sustain the posterior gt domain without autoactivation by relying on cooperative activation from Bcd and Cad (Perkins, 2006).
Comparison of regulatory architectures. The regulatory relationships proposed by Rivera-Pomar and Jäckle (1996) are not fully consistent with the data and require amending. Repression of gt by Kni, which contradicts the mechanism of domain shifts described by Jaeger (2004a and b), was eliminated by the optimization in both of the current models based on the RPJ regulators. Activation of kni by Kr was never observed. No support was found for a dual regulatory effect of Hb on Kr. Activation of Kr at low levels of Hb was eliminated in the RPJ-Logic model. It was retained in the RPJ-GC model, but resulted in serious patterning defects. Inclusion of Tll as an activator of hb was sufficient to produce the posterior hb domain. Based on the current fits and the primary experimental literature, there are likely other regulatory links missing from the model of Rivera-Pomar and Jäckle, though they are not strictly required to reproduce the wild-type gap gene patterns. Foremost is repression of hb by Kni, which appears important for eliminating hb expression anterior of the posterior domain. Fits based on the Sanchez and Thieffry (2001) regulatory relationships also support these conclusions (Perkins, 2006).
In contrast, the regulatory relationships in the Combined model and both the Unc-GC and Unc-Logic models are able to capture the wild-type gap patterns without gross defects. The relationships in the Unc-GC model are very similar to those obtained by Jaeger (2004a and b). For example, the regulation of Kr and kni is qualitatively equivalent in both models, and there is a single minor difference in the regulation of gt. The optimizations correctly identified activation of hb by Tll, which was missed by Jaeger (2004a and b), though the current models did less well at capturing shifting of the posterior gt domain. These regulatory relationships are also similar to those found by Gursky (2004), though that study was based on gap gene expression data with much lower accuracy and temporal resolution than the data used in this study. These similarities show that differences in the mathematical formulations of these models—as ordinary versus partial differential equations, how diffusion and nuclei doubling are modeled, and choice of boundary conditions and other simulation parameters—are not important for the reproduction of the gap gene patterns nor for the inference of regulatory relationships from the data (Perkins, 2006).
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