slouch
To determine the cis-acting regulatory elements controlling the cell-specific expression of NK-1, transiently expressed
chloramphencol acetyl transferase (CAT) reporter gene activities from transfected C2C12 myoblasts and NG108-15 neuroblastoma
cells were measured using various CAT constructs containing different 5' upstream regions of NK-1. From the initial analysis of 3.9 kb of the 5'
upstream region, it has been found that the regions from -1865 to -476 and from -476 to +100 contained strong negative and positive
regulatory elements, respectively. Within the positive cis-acting region an 86-bp DNA fragment (from -435 to -350) is sufficient to
activate the reporter gene in C2C12 cells, whereas additional regions (from -157 to -28 and from -510 to -425) are required for
optimal activity in NG108-15 cells. Gel shift and DNaseI footprinting assays have defined a plausible binding site for C/EBP,
5'-TTTCGCAAG-3' (-424 to -416), and a novel binding site for unknown factors, 5'-AATTACTCACATCC-3' (-370 to -357).
Further mutation analysis has revealed that the novel binding sequence for unknown factors is necessary and sufficient for
transcriptional activity for reporter gene expression in C2C12 myoblast cells in an orientation-independent manner (Kim, 1999).
Ectopic expression of muscle segment homeobox gene in the mesoderm results in altered
expression of the slouch/NK1/ S59 and nau/Dmyd genes, leading to a loss of some muscles and defects in the
patterning of others, suggesting that the muscle defects are at the level of recruitment and/or patterning of muscle precursor cells (Lord, 1995).
The mechanisms that underlie the segregation of muscle founder cells in the Drosophila embryo are
undefined. The proneural gene lethal of scute (l'sc) is expressed in clusters of cells in
the somatic mesoderm, from which individual muscle progenitors are singled out by progressive
restriction of l'sc expression. Coexpression of l'sc and slouch in a
subset of muscle progenitors shows that muscle founders are produced by division of muscle
progenitors. In neurogenic mutant embryos the restriction of l'sc expression fails and all cells in a
cluster coexpress l'sc and S59. Loss-of-function and overexpression phenotypes indicate a role for
l'sc in the segregation of muscle progenitors and the formation of the muscle pattern (Carmena, 1995).
Subsets of differentiating muscles in the Drosophila embryo express putative transcription factors,
such as slouch and vestigial. These genes are thought to control the development of specific muscle properties.
Myogenesis in embryos mutant for wingless is grossly deranged. Mesodermal expression of slouch is
lost, whereas some vestigial-expressing muscles develop. wingless dependence and independence
of specific muscle subsets correlates with an early derangement of twist expression in wingless
mutants. Ectoderm appears to have a possible role in the patterning of Drosophila mesoderm (Bate, 1993).
slouch expression in the midgut corresponds to a region where labial is expressed. Ubx, dpp and wg are required for labial expression and Slouch may be activated in parallel or downstream of labial (Dohrmann, 1990).
During Drosophila embryogenesis, mesodermal cells are recruited to form a complex pattern of larval muscles. The formation of the
pattern is initiated by the segregation of a special class of founder myoblasts. Single founders fuse with neighbouring nonfounder
myoblasts to form the precursors of individual muscles. Founders and the muscles that they give rise to have specific patterns of gene
expression, and it has been suggested that it is the expression of these founder cell genes that determines individual muscle attributes
such as size, shape, insertion sites and innervation. The segmentation gene Kruppel is expressed in a subset of founders
and muscles. Kruppel protein is expressed in a variety of muscles, including two dorsal muscles (the dorsal acute muscle 1 and the dorsal oblique muscle 1), three lateral muscles (including the lateral longitudinal muscle 1 and the lateral transverse muslces 2 and 4), and four ventral muscles (ventral longitudinal muscle 3, ventral acute muscle2 and ventral oblique muscles 2 and 5). Kruppel regulates specific patterns of gene expression in these cells, specifically the homeodomain gene slouch, also known as S59. Kruppel is required for the acquisition of proper muscle identity.
Gain and loss of Kruppel expression in sibling founder cells is sufficient to switch these cells (and the muscles to which they
give rise) between alternative cell fates. Thus Kr is not responsible for myogenic differentiation but for the specific characteristics of individual muscles. Ubiquitous expression of Kr does not alter the pattern of slouch expression in muscle progenitors and the onset of slouch expression is normal in the absence of Kr (Ruiz-Gomez, 1997).
Mutations in wingless leads
to the complete loss of a subset of muscle founder cells characterised by the expression of slouch/S59.
Wingless acts directly on the mesoderm to ensure the formation of
slouch-expressing founder cells. Wg can signal across germ
layers: in the wild-type embryo, Wg from the ectoderm constitutes an inductive signal
for the initiation of the development of a subset of somatic muscles (Baylies, 1995).
Muscle founder cells arise from the asymmetric division of muscle progenitor cells, each of which develops from a group of cells in the somatic mesoderm that express lethal of scute. All the cells in a cluster can potentially form muscle progenitors, but owing to lateral inhibition, only one or two develop as such. Muscle progenitors, and the subsequent founder cells, then express transcription factors such as Krüppel, S59 and Even-skipped, all of which confer identity on the muscle. Definition of some muscle progenitors, including three groups that express S59, depends on Wingless signaling. Lateral inhibition requires Delta signaling through Notch and the transcription factor Suppressor of Hairless. Since the Wingless and lateral-inhibition signals are sequential, one might expect that muscle progenitors would fail to develop in the absence of Wingless signaling, regardless of the presence or absence of lateral-inhibition signaling. The development of the S59-expressing muscle progenitor cells has been examined in mutant backgrounds in which both Wingless signaling and lateral inhibition are disrupted. Progenitor cells fail to develop when both these processes are disrupted. This analysis also reveals a repressive function of Notch, required before or concurrent with Wingless signaling that is unrelated to its role in lateral inhibition (Brennan, 2000).
During wild-type development, expression of S59 is first seen during stage 10 in a single muscle progenitor cell either side of the midline in every segment. By stage 11, this pattern has evolved in abdominal segments such that S59 expression is seen both in the nervous system and in two groups of muscle progenitor cells. During stage 12, a third muscle progenitor cell starts to express S59. These muscle progenitor cells give rise to three muscle founder cells that maintain the expression of S59. Fusion of these founder cells with myoblasts results in the S59-expressing muscles seen in late stages of embryogenesis (Brennan, 2000).
Disruption of lateral-inhibition signaling, in either Notch (N) germline-clone, suppressor of Hairless germline-clone or Delta zygotic mutant embryos, increases the number of cells expressing S59 compared with wild type at stage 11. Because of general degeneration of these embryos during germ-band retraction, however, it is difficult to examine the expression of S59 after stage 11, but the mesoderm clusters that can be identified are expanded (Brennan, 2000).
Unlike the disruption of lateral-inhibition signaling, attenuation of Wingless signaling, by removing either wingless (wg) or dishevelled function, blocks the expression of S59 in the mesoderm. In contrast, increasing Wingless signaling, either by overexpressing the Wingless protein in the mesoderm using the GAL4/UAS system (twist-GAL4>UASwg embryos), or by removing shaggy function (sggm11 germline-clone embryos), leads to enlarged groups of S59-expressing muscle progenitor cells during stage 11. However, during germ-band retraction, the groups are reduced in size. In the twist-GAL4>UASwg embryos the reduction in cluster size leads to a largely normal set of three muscles, whereas in the sggm11 embryos the reduction is more extreme and leads to the loss of S59-expressing muscles (Brennan, 2000).
Since Wingless signaling is required for the initiation of S59 expression in the mesoderm and lateral-inhibition signaling is required for the subsequent restriction of S59 expression to one or two cells within each cluster, it is expected that in the absence of Wingless signaling S59 will not be expressed, even if lateral-inhibition signaling is also blocked. This appears to be the case in wgS107.5;DlFX3 zygotic and wgS107.5,Su(H)SF8 germline-clone embryos. In contrast, mesodermal S59 expression is observed in Df(1)N81k1,dshv26 and Df(1)N81k1;wgCX4 germline-clone embryos, in which Wingless signaling is blocked and Notch function is removed. Finally, as with the single-mutant embryos, the double-mutant embryos degenerate during germ-band retraction, making it difficult to examine S59 expression after stage 11 (Brennan, 2000).
These results first confirm that Wingless signaling is required for the initiation of S59 expression and that a Delta-initiated lateral-inhibition signal is required for the restriction of S59 expression to one or two cells of each initial cluster. They also confirm the prediction that, in the absence of a Wingless signal, S59 is not expressed, regardless of whether lateral-inhibition signaling is occurring. Also, even though hyperactivating Wingless signaling leads to initially enlarged groups of S59-expressing muscle progenitor cells, a reasonably normal muscle pattern is obtained (Brennan, 2000).
The observed S59 expression in Df(1)N81k1, dshv26 and Df(1)N81k1; wgCX4 embryos can be explained if it is assumed that Notch has a repressive function that precedes Wingless signaling. In this situation, removal of Notch function will lead to the derepression of S59 expression before Wingless signaling. Consequently, it does not matter whether or not Wingless signaling occurs. This repressive function cannot be related to Delta signaling, however, because the removal of Delta or Su(H) function in embryos where Wingless signaling is not occurring does not result in S59 expression. The repressive function of Notch uncovered in these experiments must therefore be distinct from its repressive role during lateral inhibition (Brennan, 2000).
The second observation suggests that in response to increased Wingless signaling there is a linked increase in lateral-inhibition signaling. This would mean that increased Wingless signaling will only lead to a significant increase in the number of muscle progenitors if lateral inhibition cannot occur. The observed difference in the final muscle pattern between twist–GAL4>UASwg and sggm11 embryos is probably due to the difference in how Wingless signaling is activated in the different embryos. In the twist–GAL4>UASwg embryos, Wingless signaling is activated only transiently and is restricted to the mesoderm. In contrast, Wingless signaling is activated globally and throughout embryogenesis in sggm11 germlineclone embryos. This difference, along with the proposed linkage between Wingless signaling and lateral inhibition would mean that lateral inhibition is much greater in the sggm11 embryos. This situation would explain the greater reduction in the size of the groups of S59-expressing muscle progenitor cells observed in the sggm11 embryos and the loss of muscles if the restriction is too great (Brennan, 2000).
The link between Wingless signaling and lateral inhibition could occur in a number of ways. For example, Wingless signaling may directly alter a component of the Delta signaling pathway that would then increase the ability of this pathway to transduce the Delta signal. Alternatively, Wingless signaling could affect Delta signaling by altering the transcription of one of the components of the pathway. Either of these mechanisms would allow the organism to generate a lateral-inhibition signal appropriate to the input signal: a strong Wingless signal would lead to a strong lateral-inhibition signal and prevent unnecessary and unwanted development, whereas a weak Wingless signal would lead to a weak lateral-inhibition signal that allows development to proceed even though the input signal is weak. This would allow normal development to occur even if there are fluctuations in the input signal (Brennan, 2000).
It is thought that the muscle progenitor cells develop from a large pool of developmentally equivalent cells that is refined through two steps to produce one muscle progenitor cell. A very large group of cells is initially defined that have the potential to become muscle progenitor cells but are prevented from doing so by the novel function of Notch identified here. Wingless signaling then alleviates this repressive function of Notch within a few cells of the cluster to establish an equivalence group. This triggers the process of lateral inhibition, which subsequently selects a single cell to become a muscle progenitor. In this situation, overexpressing Wingless or constitutively activating Wingless signaling will alleviate the initial repressive function of Notch in all the cells is observed, revealing the larger extent of the initial cluster. The linked increase in lateral-inhibition signaling, however, ensures that the normal number of muscle progenitor cells develop (Brennan, 2000).
This model contrasts with others in which Wingless signaling is instructive and defines the position at which muscle progenitor cells will develop, but can explain why overexpressing Wingless leads to the development of S59-expressing muscles in their normal position. In this model the Wingless signal is permissive and not instructive: it does not define where S59 will be expressed but merely reveals places defined by earlier mechanisms. Finally, these data suggest that the loss of S59 expression in the absence of a Wingless signal is due to the early repression mediated by Notch (Brennan, 2000).
The patterning of the Drosophila mesoderm requires Wingless.
Little is known about how Wg provides patterning information to the mesoderm,
which is neither an epithelium nor contains the site of Wg production. By
studying specification of muscle founder cells as marked by the lineage-specific
transcription factor Slouch, it was asked how mesodermal cells interpret the steady
flow of Wg. Through the manipulation of place, time and amount of Wg signaling,
it has been observed that Slouch founder cell cluster II is more sensitive to Wg
levels than the other Slouch-positive founder cell clusters. To specify Slouch
cluster I, Wg signaling is required to maintain high levels of the myogenic
transcriptional regulator Twist. However, to specify cluster II, Wg not only
maintains high Twist levels, but also provides a second contribution to activate
Slouch expression. This dual requirement for Wg provides a paradigm for
understanding how one signaling pathway can act over time to create a diverse
array of patterning outcomes (Cox, 2005).
In wg mutant embryos, the heart and approximately half the body
wall muscles are lost. One subset of these Wg-dependent body wall muscles can be
visualized using an antibody to the NK-homeodomain protein Slouch (S59). Slouch
expression arises in a precise, stereotypic pattern during embryonic
development. It is
first expressed in a single progenitor cell during early stage 11 of embryonic
development; this cell divides to give rise to two founder cells (Ia and Ib)
which together form cluster I (cI). During late stage 11, two additional
Slouch-positive progenitors appear at a different ventral location and divide
sequentially to form four founder cells that make up cluster II. Still
later, at stage 12, a single progenitor arises dorsally and divides to give
rise to cluster III. These muscle founder cells contain all the information
needed to create a particular subset of muscles and contribute to the
stereotypic set of larval muscles in each abdominal segment. After stage 12,
Slouch expression is maintained in a subset of these founder cells
that give rise, in the final muscle pattern, to muscle VT1 (from cI), VA2
(from cII) and DT1 (from cluster III).
Maintenance of Slouch expression in these founder cells is crucial to the
development of these muscles; removal of slouch leads to complete
(VT1) and partial muscle transformations (VA2; DT1). In this
study, focus was placed on the role of Wg in patterning the Slouch muscle founder
cells. For simplicity, focus was placed solely on two ventral Slouch clusters (I and
II), which develop independently and arise in a similar position along the dorsoventral axis but
have different anterior-posterior positions within each abdominal
hemisegment (Cox, 2005).
Through the manipulation of the amount and time of exposure to Wg signaling
in the Drosophila mesoderm, it is shown that Slouch founder cell
cII requires more Wg signaling than its neighbor, cI. Because cII arises in
the mesoderm beneath the source of Wg signal, it was initially thought that the
sensitivity that was detected would be due to Wg acting as a classic morphogen.
Specifically, during stage 11, Wg would directly elicit
concentration-dependent responses, leading to Slouch cI specification at low
levels and cII at higher levels. Instead, the data suggest an alternative
mechanism underlying this sensitivity. For Slouch cI, Wg signaling through
Twist is sufficient for fate specification. However, for Slouch cII, a second,
Twist-independent Wg signal is also necessary (Cox, 2005).
It has been shown that wg mutants fail to maintain high
levels of Twist.
Overexpression of Twist leads to expanded somatic mesodermal fates at the
expense of other mesodermal fates, such as heart and gut muscle. Conversely,
decreasing Twist levels leads to a reduction in somatic mesodermal fate, while
heart and gut muscle remain largely unaffected. These
findings underscore the importance of high Twist levels for the proper
implementation of somatic muscle fate. Because loss of high Twist levels leads
to loss of muscle founder cells, including all Slouch-positive clusters of
founder cells, it has always appeared that each Slouch cluster requires the
same amount of Wg signal (relayed through Twist) to assume its particular
fate. In this study, the requirement for Wg in maintaining high
Twist levels was uncoupled from the later role of Wg in specifying cII fate. The fact that
Twist specifically rescues Slouch cI in a wg mutant background
suggests that Slouch cII requires an additional, Twist-independent
contribution from Wg for proper patterning. Consistent with these results, wg
hypomorphs were found that provided sufficient signaling to maintain
high Twist levels during early mesoderm development and therefore pattern cI,
but that do not pattern cII. Temperature-shift experiments using wg
temperature-sensitive alleles have shown that Slouch cII specification and
engrailed expression in the ectoderm require Wg expression at later
stages of embryonic development. Thus, the absence of Slouch cII in the
different wg alleles, in hh mutant embryos and in a Twist
rescued wg mutant embryo, all suggest that proper patterning requires
not only an earlier Wg-dependent regulation of Twist, but also an additional
Wg contribution to specify its identity (Cox, 2005).
Manipulations of Wg signaling also revealed two additional aspects of
Wg signaling to the mesoderm. (1) It was found that the mesoderm, in general,
has a different threshold for Wg signaling when compared with the ectoderm.
Conditions that completely rescue the ventral ectoderm and epidermis
(wgPE6 at the permissive temperature) fail to completely
rescue the mesoderm. (2) It was found that different mesodermal targets respond
differently to Wg signaling. For example, expression of the
DeltaNTcf (dominant negative form of Pangolin) has mild effects
on Twist but significant effects on Slouch cII.
Although it is predicted that TCF binds slouch regulatory regions
directly, it was found that Wg regulates Twist both directly through TCF and
indirectly through the pair-rule gene sloppy-paired. Whether or not
the difference in Wg regulation of
twist and slouch is due to the structure of the regulatory
regions, additional factors that integrate on these promoters in these
contexts and the activity of the Arm/dTCF complex remains to be uncovered (Cox, 2005).
This study also underscores the contribution that other factors make to
position the Slouch clusters: ectopic Wg expression in the mesoderm does not
produce uniform Slouch expression. This aspect of Wg signaling is reflected in other tissues
such as the epidermis. The
size of Slouch cII could not be further enlarged beyond that seen when Wg signaling was
initially increased. This suggests a
prepatterning mechanism, perhaps involving the activity of the pair-rule genes
that have been shown to be responsible for segmentation of the mesoderm, as well as
the integration of other signal transduction pathways, such as EGF/FGF and
Notch signaling. The data suggest that Wg signaling then works on this
prepattern to regulate the domain of Slouch expression (Cox, 2005).
The effect of Wg on muscle patterning is similar to
that described for even-skipped muscle progenitor specification; that
is, Wg signaling (in collaboration with such signals as Decapentaplegic) is
first required to set up a region of 'competence' through activation of
mesoderm-specific factors such as Twist and Tinman. Wg then later cooperates
with these intrinsic factors to induce the expression of even-skipped
in dorsal muscle progenitors, much as would be suggested for Slouch cII. However, the
observations suggest an important variation of Wg signaling in mesodermal
patterning. In the case of Slouch patterning, Wg creates temporal as well as
spatial diversity, while in patterning eve it only acts temporally.
Wg signaling contributes to the expression of Slouch in its two discrete
ventral patches by two distinctive mechanisms: through the regulation of an
upstream transcription regulator (Twist), which is sufficient for one domain
of expression; and through the cooperation of this factor with a second,
temporally distinct Wg input for the second domain of expression. The
expression of the same gene but at two different times and places, through two
Wg-dependent means, gives insight into how an organism may generate diverse
tissues in response to the same signal (Cox, 2005).
Work carried out in the wing imaginal disc suggests that Wg acts as a
morphogen. In this tissue, Wg protein was visualized in a graded
distribution and it appears to activate multiple target genes directly, in a
concentration-dependent manner. Based on these criteria, Wg has been labeled as a classical
morphogen. However, careful inspection of the molecular mechanisms underlying
Wg activation of both short- and long-range targets in the wing have revealed
that the pattern of Wg expression changes during wing imaginal disc
development, and that Wg collaborates with other pathways to set up the
expression of these genes. These studies have cast doubt on whether Wg is a
true morphogen in this tissue (Cox, 2005).
Investigating the molecular mechanisms that govern patterning of
the embryonic mesoderm, similarly suggests that Wg does not act on Slouch
clusters I and II as a classical morphogen. Wg does not
activate cI directly, but instead maintains high levels of Twist,
which sets up a somatic mesodermal competency domain that is sufficient to
create cI. Additional Wg is then required later to pattern cII. It can be
argued that Wg acts as a morphogen to regulate Twist expression (at low
levels), and then to control Slouch expression (at high levels) within cells
of cII. However, the precise regulation and dependence of Slouch clusters I
and II on Wg within both the dorsoventral and anteroposterior axes suggest
that there must be additional patterning information available to properly
place these two cell types. As more putative morphogens are held up to the
lens of molecular biology, it will be interesting to see whether there are
unexpected, new twists in the molecular underpinnings of morphogens (Cox, 2005).
The PRD-repeat domains of Slouch and PRD protein are sufficient to
mediate protein-protein interaction, suggesting that the
PRD-domain functions as a protein-binding interface and
thereby may increase the DNA binding specificity of
homeodomain transcription factors (Kim, 1995).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
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