slit
A distal 2.2 kb region, part of the the 4.8 kb region contiguous with the start site, directs slit expression to midline glia, cardioblasts, ventral epidermis and ventral pharynx, antennomaxillary complex, midgut and Malpighian tubules. The proximal 2.6 kb of the larger 4.8 kb region directs expression in stage 10; activity is restricted to the brain. A 1.0 kb fragment, including the first intron of the structural gene (but not the proximal promoter region) directs expression to cardioblasts, a subset of brain, and midline glia. Thus two promoter regions can direct transcription to midline glia; a distal upstream fragment and an enhancer located in the first intron (Wharton, 1993).
A single DNA motif has been found in the intronic enhancer, regulated by single-minded. It consists of an E-box element ACGTG, also found in a Toll neural promoter. The same element is responsible for single-minded autoregulation in the midline (Wharton, 1994).
During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the
axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent
on Single-minded, a basic-helix-loop-helix (bHLH)-PAS transcription factor. Fish-hook/Dichaete, a Sox
HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene
expression. single-minded, Dichaete, and drifter are all expressed in developing midline cells, and both loss- and
gain-of-function assays reveal genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the
slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Dichaete directly associates with the PAS domain of Single-minded
and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Dichaete can form homodimers and also associates with
other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription
factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes (Ma, 2000).
To address whether the sim, Dichaete, and
dfr genes might functionally interact to regulate
development of the embryonic CNS midline, whether
they exhibit overlapping expression in developing midline cells was examined. This was accomplished using anti-Dichaete and anti-Dfr sera, as well as a
P[3.7sim-lacZ] marker that mimics sim midline expression. P[3.7sim-lacZ] embryos were immunostained using anti-ß-gal and either anti-Dichaete or anti-Dfr sera. Prominent overlapping expression was detected between Sim and Dichaete in developing CNS midline cells from stage 8 throughout the remainder of germ band extension. Overlap was also detected in a subset of prospective foregut cells. Similar overlapping expression was also detected between Sim and Dfr. Midline coexpression of Dichaete and Dfr was detected by immunostaining wild-type embryos with anti-Dichaete and anti-Dfr sera. Both genes are expressed together in the CNS midline throughout germ band extension. In germ band-retracted embryos, Dichaete exhibits overlapping expression with Sim and Dfr in the midline glia. Dichaete and Dfr are also detected together in lateral cells of the thoracic ganglia and a subset of ventral epidermal cells. These analyses indicate that sim, Dichaete, and dfr are coexpressed in developing CNS midline cells. The midline expression of these three genes also overlaps that of the slit gene, which is a downstream target of Sim (Ma, 2000).
Both loss-of-function and gain-of-function assays were used to
detect genetic interactions between sim, Dichaete,
and dfr. Mutants are known to show genetic interactions in CNS midline differentiation and in Slit protein expression. Potential cooperative interactions between sim, Dichaete, and dfr in
regulating slit gene transcription were examined through the use of a P[1.0slit-lacZ] marker. This reporter contains a portion
of a slit intron that drives lacZ expression mimicking that
of the native slit gene in developing midline glia;
P[1.0slit-lacZ] expression is first detected in germ
band-extended stage 11 embryos and is maintained throughout the
remainder of embryogenesis. Dichaete
null mutant embryos exhibit a misplacement and loss of midline glia,
as detected via anti-ß-gal immunostaining. P[1.0slit-lacZ] is expressed normally in stage 11 Dichaete mutant embryos, but during germ band retraction the number of midline glia becomes reduced from wild
type, and many cells are located at aberrant ventral positions within
the nerve cord. Similar, although less severe, defects are observed in
dfr mutant embryos, where some midline glia are displaced
from their normal positions. Notably, ß-gal-expressing midline glia are still detected in both Dichaete and dfr mutants, indicating that unlike Sim, Dichaete and Dfr are not absolutely required for
P[1.0slit-lacZ] expression or midline glial development (Ma, 2000).
A dfr-Dichaete double mutant strain was used to examine
whether Dichaete and dfr might act together to
regulate midline gene expression. Embryos mutant for both
Dichaete and dfr exhibit much more
severe defects in P[1.0slit-lacZ] expression than either Dichaete or dfr single mutants. Although P[1.0slit-lacZ] is activated normally in stage 11 dfr-Dichaete double mutant embryos, there is a striking loss of midline P[1.0slit-lacZ] expression during germ band
retraction. This synergistic effect strongly suggests that Dichaete and Dfr function together to regulate slit transcription. These functions may be mediated directly through Dichaete and Dfr binding sites present in the slit 1 kb regulatory region. Another, nonexclusive possibility is
that Dichaete and Dfr might indirectly control slit
transcription by regulating the expression of sim. To
address this possibility P[3.7sim-lacZ] expression was examined in wild-type and dfr-Dichaete embryos. Compared with
wild-type embryos, dfr-Dichaete double mutants exhibit a
severe decrease in P[3.7sim-lacZ] expression, a phenotype
that first becomes apparent during germ band retraction. Thus, Dichaete
and dfr also influence sim expression and hence may indirectly influence the expression of a wide array of midline genes (Ma, 2000).
Because homozygous sim mutants exhibit severe CNS midline defects, it is not informative to analyze the phenotypes of Dichaete-sim or dfr-sim double mutants. Instead, potential interactions between Dichaete and sim were examined via a gain-of-function approach using the Gal4/UAS targeted gene expression system. A P[GMR-Gal4] strain that drives Gal4 expression in and behind the morphogenetic furrow in the developing eye imaginal disc was crossed to P[UAS-Dichaete] and P[UAS-sim] strains. P[GMR-Gal4]/+;P[UAS-Dichaete]/+ animals exhibit a moderate eye roughening with disruption of ommatidia organization and loss of mechanosensory bristles. In contrast, ectopic sim expression results in essentially normal eye morphology. The effects of Dichaete and sim coexpression reveal a nonadditive phenotype; there is a stronger disorganization of ommatidia and mechanosensory bristles than seen in flies expressing Dichaete or sim alone, and there is also a dramatic loss of eye pigmentation. These results indicated that ectopic expression of Dichaete and sim synergistically alters normal eye development, and supports the hypothesis that these genes can interact functionally (Ma, 2000).
Analysis of a 380 bp slit midline
regulatory fragment has indicated the presence of a single CNS midline element (CME), through which Sim::Tgo heterodimers act. The CME is located within 300 bp of the distal end (farther from the promoter in the native slit gene) of this fragment. An inverted TTCAAT repeat (TTCAATTTCATTGAA) is located 20 bp proximal to the CME. This sequence resembles a (A/T)(A/T)CAAT consensus binding site for Sox proteins, although binding of Sox proteins to a TTCAAT sequence has not been reported.
Because sequences present in an extended 1 kb slit DNA
fragment are required for normal levels of slit expression
in vivo, additional DNA sequences have been obtained. This analysis indicated that no other CMEs are present in the 1 kb slit DNA fragment. However, two perfect Dfr consensus binding sites, ATGCAAAT and
CATAAAT, located within 500 bp of DNA proximal to the CME were identified. These two Dfr binding sites are separated by ~150 bp and flank a consensus Dichaete binding site, TACAAT. These data suggest
that Dichaete, Sim, and Dfr may all bind to sites present in the 1 kb
slit regulatory DNA fragment. To test this possibility, DNA
gel mobility shift assays were performed using the Dichaete HMG domain and
full-length Dfr protein on double-stranded oligonucleotide probes
corresponding to sequences from the slit 1 kb fragment. The
Dichaete HMG domain binds strongly to a 26 mer probe containing the TACAAT
site. In contrast, Dichaete does not bind consistently to a 26 mer probe containing both TTCAAT sites, suggesting that Dichaete can distinguish between closely related DNA sequences. Dfr protein binds very strongly to a 33 mer probe that contains the ATGCAAAT site, and less strongly to a 32 mer probe containing the CATAAAT site. Dfr binds the ATGCAAAT site
both as an apparent monomer and a dimer, because two distinct bands
with reduced mobilities are detected. The 1 kb slit
fragment thus may integrate the actions of at least three different
types of regulatory proteins, represented by Sim, Dichaete, and Dfr (Ma, 2000).
The ability of Dichaete, Dfr, Sim, and Tgo to directly
control slit transcription was examined using transient transcription assays in cultured Drosophila S2 cells. The P[1.0slit-lacZ] construct was used as a reporter with various combinations of plasmids
that express Dichaete, Dfr, Sim, or Tgo. Dichaete modestly activates P[1.0slit-lacZ] transcription, indicating that in both yeast and fly
cells, Dichaete can function as a direct transcriptional activator. Dfr
results in little if any activation of P[1.0slit-lacZ], and Dfr and Dichaete together do not exhibit any increased activation over the levels observed for Dichaete alone. Neither Sim nor Tgo alone is able to activate the P[1.0slit-lacZ] reporter, because only background levels of expression are detected. Furthermore, Sim and Tgo together yield only minimal activation. These results imply that although Sim::Tgo heterodimers strongly activate
expression of a P[6XCME-lacZ] reporter (>150 units) that contains
six multimerized CMEs, additional factors are
required to achieve high levels of reporter expression. Significantly, the combination of either Dichaete and Sim::Tgo or Dfr and Sim::Tgo both result in relatively high levels of activation. Thus, both Dichaete and Dfr strongly enhanced the ability of Sim::Tgo heterodimers to activate slit
transcription. Comparable levels of activation are observed when all four proteins are expressed together. Taken together, the DNA binding and transcriptional activation assays provide additional evidence that regulation of
slit expression in the midline glia requires functional
interactions between Dichaete, Dfr, Sim, and Tgo (Ma, 2000).
Functional interactions between Sim, Dichaete, and Dfr may also regulate
the midline expression of other genes, including sim and
breathless (btl). Thus, sim has
autoregulatory functions, and the combined functions of dfr and Dichaete are also required for sustained midline sim expression. In addition, a 2.8 kb interval in the P[3.7sim-lacZ] transgene used in this study contains six evolutionarily conserved CMEs as well as several consensus Dichaete and Dfr binding sites. btl encodes an FGF receptor homolog whose expression in the CNS midline and tracheal cells has been shown to depend, respectively, on Dfr as well as Sim and Tgo, or Trh and Tgo. A 200 bp btl midline/tracheal regulatory region contains three evolutionarily conserved CMEs. Inspection of this region also revealed the presence of a conserved consensus ATCAAT Dichaete binding site located in a 40 bp interval between CME2 and CME3, as well as a conserved consensus
GATAAAT Dfr binding site located 40 bp
downstream of CME3. Thus, functional interactions between Sim, Dichaete,
and Dfr could be a general mechanism to regulate gene transcription
during CNS midline development (Ma, 2000).
The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis. In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).
The pattern and level of expression of axon guidance proteins must be choreographed with exquisite precision for the
nervous system to develop its proper connectivity. Previous work has shown that the transcription factor Lola is
required for central nervous system (CNS) axons of Drosophila to extend longitudinally. Lola is
simultaneously required to repel these same longitudinal axons away from the midline, and it acts, in part, by
augmenting the expression both of the midline repellant, Slit, and of its axonal receptor, Robo. Lola is thus the
examplar of a class of axon guidance molecules that control axon patterning by coordinating the regulation of multiple, independent guidance genes,
ensuring that they are co-expressed at the correct time, place and relative level (Crowner, 2002).
The reduction of Robo expression seen in lola mutants is relatively modest (~40%). It is known, however, that a 50% diminution in Robo is sufficient by itself to cause some inappropriate midline crossing, and this effect is strongly enhanced by a simultaneous 50% reduction in Slit. Loss of lola causes a greater reduction than this in Slit levels. Thus, it is plausible that the change in Slit and Robo levels could account for much of the midline phenotype observed in embryos that bear strong lola mutations. But why are weaker lola alleles like lola1A4 able to cause extra midline crossing when their effect on target gene expression is presumably proportionately less? It is likely that regulation of Slit and Robo expression is only one part of the control of midline crossing by lola, and that a significant contribution to the phenotype is made by changes in the expression of other, interacting guidance genes that are also controlled by lola. For example, aspects of the lola midline phenotype resemble details of the axon pattern observed upon mutation of genes encoding receptor tyrosine phosphatases, suggesting that these are good candidates for potential lola effectors. Moreover, it is known that the Notch-dependent mechanism that promotes the alternative (longitudinal) trajectory of CNS axons also requires lola. The multiplicity of genes contributing to the midline/longitudinal axon growth decision underscores the need for a gene, like lola, to coordinate the expression of all these cooperating guidance factors. It is suggested that it is the combination of many quantitative effects, each individually modest, which together produce the profound effects of lola on axon patterning (Crowner, 2002).
Many questions remain from these studies. (1) Though Lola itself is a transcriptional regulator, it is not known whether robo and slit are direct Lola targets or whether Lola initiates a longer chain of events leading only indirectly to robo and slit. For example, Lola could regulate other genes that themselves control the stability of robo or slit RNA or protein, or the splicing or translation efficiency of these genes. Analysis of this issue will require unambiguous identification of the exact lola isoforms required for expression of robo and slit, and characterization of their DNA-binding specificities in combination with their appropriate dimerization partner(s). (2) Only the accumulation of Robo and Slit protein has been characterized in lola mutants, and not transcript levels. The inherent variability of whole-mount RNA in situ hybridization has prevented sufficiently precise quantification of robo and slit RNA levels for this purpose. Nonetheless, the observation that ectopic expression of lola 4.7 leads to ectopic expression of slit RNA strongly argues that lola is upstream of slit transcription, though it remains possible that Robo and Slit expression are also subject to lola-dependent regulation at some post-transcriptional level (Crowner, 2002).
Formation of the neural network requires concerted action of multiple axon guidance systems. How neurons orchestrate expression of multiple guidance genes is poorly understood. This study shows that Drosophila T-box protein Midline controls expression of genes encoding components of two major guidance systems: Frazzled, ROBO, and Slit. In midline mutant, expression of all these molecules are reduced, resulting in severe axon guidance defects, whereas misexpression of Midline induces their expression. Midline is present on the promoter regions of these genes, indicating that Midline controls transcription directly. It is proposed that Midline controls axon pathfinding through coordinating the two guidance systems (Liu, 2009).
To address how Mid activates expression of the three axon guidance genes, the binding sequence of Mid was determined using an in vitro binding site selection method. Mid-binding sequence was selected from a pool of random oligonucleotides using Mid protein affinity-purified from an embryonic extract. The consensus sequence deduced from the selected oligonucleotides was (G/A/T)NA(A/T)N(T/G)(A/G)GGTCAAG. This sequence was found in the upstream regions or an intron of slit, frazzled, and robo, and all of these sites were conserved among several Drosophila species. To determine whether Mid binds to these regions in vivo, chromatin immunoprecipitation (ChIP) was performed using anti-Mid antibody. In all three genes, Mid was present around the Mid-binding sites, but not on regions without the binding site. In contrast, a potential Mid-binding site 32-kb upstream of the commisureless gene, whose expression is not affected in mid mutants, was not occupied by Mid (Liu, 2009).
The importance of the Mid-binding sites in frazzled and slit was assessed by transgenic reporter assays. To test the role of the Mid site in frazzled, reporter genes were constructed that contain the transcription start site of frazzled and an upstream region including a wild-type Mid-binding site (fraPlacZ) or a mutated site (fraMPlacZ). Compared with the wild-type reporter gene, the reporter with a mutated binding site showed reduced expression levels (33% reduction). Thus Mid-binding site is indeed required for the proper expression of frazzled. Mutating the Mid-binding site in slit also caused a severe effect on slit expression. The lacZ expression in sliPlacZ is driven by the slit regulatory element and the endogenous promoter. While sliPlacZ with the wild-type binding site recapitulated the slit expression in the midline glia and lateral cells, base substitutions in the Mid-binding site in sliMPlacZ abolished the lacZ expression. It is possible that the Mid-binding site resides in an essential promoter element of slit, and hence, the base substitutions abolished slit transcription in all cells. However, the same results were obtained using sli4.5HHlacZ and sliM4.5HhlacZ in which the slit regulatory element is fused to a heterologous hsp70 promoter. Since mid was expressed in the lateral cells but not in midline glia, these results suggest that Mid-binding sites in slit control slit transcription via binding to multiple factors: Mid in lateral cells and unknown factor(s) in midline glia. Taken together, these results demonstrate a direct role for Mid in the regulation of frazzled and slit, and suggest that Mid governs the expression of multiple axon guidance genes through directly binding of the Mid sites in their regulatory regions (Liu, 2009).
This study has shown that Mid directly controls transcription of key components of the two major axon guidance systems: the Netrin/Frazzled system and the Slit/ROBO system. Because these two systems are considered to have opposing outputs, it is interesting that the expression of both systems are induced by the same transcription factor, Mid. Dynamic expression of Frazzled and ROBO is required for growth cones to simultaneously respond to both attractants and repellents, integrate these signals, and then respond to the relative balance of forces. These molecules also provide nonautonomous functions required for cell motility, such as mediating cell adhesion and promoting axon elongation. The coordination of axon guidance systems by Mid may thus ensure cooperative actions of multiple guidance molecules in growth cone dynamics, axonal adhesion, and elongation. The role of Mid in the transcriptional regulation of axon guidance
might be a conserved function, because its orthologs of human, mouse, and zebrafish Tbx20 are also expressed in motor neurons (Liu, 2009).
Drosophila single-minded (sim) encodes a nuclear protein that plays a critical role in
the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the
embryonic CNS. In sim mutant embryos the
midline cells fail to differentiate properly into their mature CNS cell types and do not take their
appropriate positions within the developing CNS. sim is required
for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at
91F. The sim mutant CNS defect may be largely due to loss of midline slit expression. The snail gene is required to repress sim and other midline genes in the presumptive mesoderm (Nambu, 1990).
The availability of expressed Slit proteins enabled an examination of their interactions with Roundabout proteins.
The interaction between Slit and Robo proteins was further demonstrated using soluble forms of the
ectodomains of rRobo1, rRobo2, and dRobo1 fused to either the constant region (Fc) of the human
immunoglobulin molecule or to alkaline phosphatase. rRobo1-Fc and rRobo2-Fc bind transfected
cells expressing hSlit2, and Drosophila Robo bound cells expressing Drosophila Slit.
These results indicate a high degree of specificity in the interaction between Slit and Robo proteins. In
addition, in cross-species experiments it was found that the binding interactions are evolutionarily
conserved. Thus, Drosophila Slit binds cells expressing either rRobo1 or rRobo2, and hSlit2 binds cells
expressing dRobo1, although these interactions appeared weaker than those observed within species. Similarly, Robo1-Fc and Robo2-Fc bind cells expressing Drosophila Slit,
and Drosophila Robo binds hSlit2-expressing cells. The amino-terminal LRRs of Drosophila and mammalian Slit proteins have homology to a number of
ECM molecules, including the laminin-binding molecule biglycan. This
prompted an examination of whether Slit proteins can also bind laminins. hSlit2, applied in
conditioned medium from transfected cells, binds to a substrate with laminin 1, but not a substrate
coated with fibronectin. Since Netrin proteins show homology to a portion of the
laminin molecule, whether Netrin 1 and Slit2 can bind one
another was examined. Netrin 1 binds to COS cells expressing hSlit2 in a pattern that was indistinguishable from binding observed with
Robo1-Fc and Robo2-Fc (Brose, 1999).
Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands, Netrin and Slit,
are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a
modular function encoded in the cytoplasmic domain of these receptors, chimeras were created carrying the ectodomain of one receptor and the cytoplasmic domain
of the other and their function in transgenic Drosophila was tested. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin
receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an
attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline (Bashaw, 1999).
In Drosophila, the same midline cells normally secrete both Netrins and Slit. Growth cones can simultaneously respond to both ligands in a cell-specific fashion.
Some growth cones express high levels of Fra and low levels of Robo, and they extend toward and across the midline. Other growth cones appear to express high
levels of both receptors, and they can extend toward the midline, but they do not cross it. Growth cones can dramatically change their
levels of Robo expression; once they cross the midline, growth cones increase their level of Robo, a change that prevents them from crossing the midline again. Such complex and dynamic behavior requires growth cones to be able to simultaneously respond to both attractants and repellents and to integrate these signals and
respond to the relative balance of forces. Introducing a chimeric receptor into this finely tuned system leads to dramatic phenotypes. Adding a receptor that responds
to Netrin as a repellent leads to a comm-like phenotype in which too few axons cross the midline. Adding a receptor that responds to Slit as an attractant leads to
the opposite robo- or slit-like phenotypes, in which too many axons cross the midline or remain at the midline, respectively. These phenotypes are dose dependent,
suggesting that by adding more chimeric receptor, the relative balance can be tipped and in tis way the growth cone's response is selectively controlled. This striking dosage
sensitivity raises the possibility of using these phenotypes as the basis for genetic suppressor screens to identify signaling components that function downstream of
attractive and repulsive guidance receptors (Bashaw, 1999).
Another finding of this study is that the signal transduction machinery for attraction and repulsion downstream of these receptors appears to be present in all neurons,
and probably in all migrating muscle precursors as well. All neurons expressing either Fra-Robo or Robo-Fra appear to behave the same, regardless of their environment:
if they express Fra-Robo, they stay away from the midline; if they express Robo-Fra, they extend toward the midline. No other factor appears to intrinsically commit
one growth cone or another to only one kind of response. The same is true for migrating muscle precursors. Normally, many of them express Robo and migrate away from the Slit-expressing midline. However, given the opportunity
(by transgenic expression of Robo-Fra), they clearly contain the full machinery for the opposite response. In all these transgenic experiments, the growth cone or
muscle response always correlated with the level of receptor (Brashaw, 1999 and references).
The finding that the cytoplasmic sequence determines the response of a guidance receptor raises a number of interesting questions. Attraction might lead to a local
change favoring actin polymerization over depolymerization, while repulsion might lead to the opposite change. But is guidance that simple? The
cytoplasmic sequences of five different families of repulsive guidance receptors are now known: UNC-5s, Eph
receptors, Neuropilins, Plexins, and Robos. Interestingly, they
appear to share little if any sequence similarity to one another in their cytoplasmic domains. It is possible, of course, that they bind different adapter proteins that
converge on the same repulsive motility machinery. But it is equally likely that not all repulsion is the same and that different classes of repulsive receptors mediate
different types of responses in the growth cone. It could be that what is lumped together under the term 'repulsion' actually represents several molecularly distinct mechanisms
that negatively influence local growth cone behavior. Just what these different cytoplasmic domains do, and how many different types of repulsion exist, awaits future
investigation (Bashaw, 1999 and references).
Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo
mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must
have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline,
even though they cross and recross it. The Drosophila genome is shown to encode three Robo receptors and Robo and Robo2 have distinct functions,
which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit (Simpson, 2000).
Robo and Robo2 also cooperate in other developmental processes. Slit, Robo, and Robo2 function during mesoderm migration. After gastrulation in Drosophila, many myoblasts migrate laterally away from the ventral midline. In slit mutant embryos, some mesoderm cells do not migrate away from the midline and, instead, form muscles abnormally near the midline that often stretch across the midline. A weak version of this phenotype is observed in the robo mutant, suggesting that it alone cannot control mesoderm migration away from Slit. A similarly weak phenotype is observed in the robo2 mutant. However, a strong phenotype is observed in the robo,robo2 double mutant. This phenotype is very similar to the slit phenotype; many mesodermal cells do not migrate away from the midline, and, instead, some developing muscles are found ectopically crossing the midline. Thus, Robo and Robo2 appear to cooperate in controlling mesoderm migrations away from the midline. Robo and Robo2 also appear to cooperate in governing proper cell migrations and alignment of cardioblasts in the embryonic heart and in the further development of muscle, including the identification of proper insertion sites (Simpson, 2000).
The commissureless phenotype observed at the higher levels of Robo2 overexpression can be partially genetically suppressed by heterozygosity (i.e., removing one copy) of robo, slit, or enabled. Although the number of commissures that form in these backgrounds is increased, the phenotype is more complex than simple suppression because in many cases the crossovers that now occur are inappropriate. Adding a robo dominant-negative transgene (truncated just after the transmembrane domain) changes the phenotype at all levels of Robo2. The Robo dominant negative (roboDN) increases the ectopic crossing seen at low levels of Robo2 overexpression, and it causes ectopic crossing at higher levels of Robo2 overexpression as well. It is unclear whether this is suppression by interference with Robo2 repulsion directly or, alternatively, whether it results from cumulative loss of repulsion by reducing the efficacy of the Robo pathway. However, increasing levels of RoboDN in a wild-type background only look like a robo loss of function, no matter how much RoboDN is added, and not like a robo,robo2 double mutant or slit mutant. This suggests that the RoboDN affects Robo output and not Robo2 output, making the second alternative above seem more likely (Simpson, 2000).
Ectopic expression of low levels of Robo2 by all neurons causes ectopic crossing of axons reminiscent of a robo mutant. A possible explanation is that small amounts of Robo2 can interfere with repulsion by Robo. Perhaps Robo2, which lacks some of the conserved motifs found in the Robo cytoplasmic domain, has a less robust repulsive output than Robo. Extra Robo2 could interfere with Robo by dimerizing with it and creating a weaker receptor. Alternatively, Robo2 might interfere by competing for Slit binding or by sequestering downstream signaling components needed by Robo. In vitro analysis shows that the cytoplasmic domains of Robo2 and Robo can bind to one another (and homodimerize), suggesting that the interference might be direct (Simpson, 2000).
The CNS of bilateral symmetric organisms is characterized by intensive
contralateral axonal connections. Genetic screens in Drosophila have identified only a few genes required for guiding commissural growth cones toward and across the midline. Two evolutionarily conserved signaling molecules, Netrin and Slit, are expressed in the CNS midline cells. Netrin acts primarily as an attractive signaling cue, whereas Slit mediates repulsive functions. A detailed analysis is provided of the Drosophila gene schizo, which is required for commissure formation. schizo leads to a commissural phenotype reminiscent of
netrin mutant embryos. Double-mutant analyses indicate that Netrin
and Schizo act independently. The schizo mutant phenotype can be
suppressed by either expressing netrin in the CNS midline cells or by a reduction of the slit gene dose, indicating that the balance of attractive and repulsive signaling is impaired in schizo mutants. Overexpression of the schizo RNA in the CNS midline using the GAL4/UAS system leads to a slit phenocopy, suggesting that
schizo primarily antagonizes Slit signaling. This is further
supported by cell type-specific rescue experiments. The schizo gene
generates at least two proteins containing a conserved Sec7 and a pleckstrin homology domain (PH) characteristic for guanine nucleotide exchange factors (GEF) acting on ARF GTPases, which are known to regulate endocytosis, In support of the notion that schizo regulates Slit expression via endocytosis, it was found that blocking endocytosis leads to a schizo-like phenotype. It is thus proposed that the balance of the two signaling cues Netrin and Slit can be regulated, controlling membrane dynamics (Önel, 2004).
Only four zygotically active genes were found in a screen for mutations affecting commissure formation (frazzled, weniger, schizo and the
netrin gene complex). Two EMS-induced schizo mutants
(schizoC1-28 and schizoU112) were
initially identified. Subsequently two P-element induced schizo alleles (schizol(3)3 and schizoP244) were identified. All these alleles led to a reduction in the number of commissural fibers crossing the CNS midline. Interestingly, the anterior commissures were affected more prominently. Not all neuromeres were equally affected and the strongest defects were generally observed in abdominal segments A1-A4. All CNS midline cells formed in normal number in the absence of schizo function. However, as generally observed in mutants affecting formation of commissures, the midline glial cells migrated out laterally along the few remaining commissural fibers. In addition to the commissural phenotype, defects in the longitudinal connectives were noted (Önel, 2004).
The most prominent function of schizo is its role in commissure
development. Two major signaling cascades are known to control axonal growth
across the midline. They are initiated by the signaling molecules Netrin and
Slit, which are both secreted by the CNS midline glial cells in the
Drosophila embryo. First genetic interaction studies of schizo and frazzled or schizo and netrin function demonstrate a much stronger commissural phenotype in double mutants than embryos mutant only for schizo, frazzled or netrin. The commissural
phenotypes of the double-mutant embryos suggest that schizo is not
acting within the Netrin signaling pathway but may be required for a
Netrin-independent attractive pathway. Alternatively, schizo may be
necessary for suppressing the perception or the generation of a repulsive
signal normally generated by the CNS midline cells (Önel, 2004).
The main axonal repulsive signal is encoded by
slit. Slit is an LLR protein secreted by the CNS midline glial cells. schizo function appears to be required to downregulate repulsive signaling, either by affecting the generation of active Slit protein or by preventing signaling in the commissural growth cones; the mutant schizo phenotype could be explained by an upregulated Slit signaling. Thus, one might expect that the schizo commissural phenotype could be suppressed by a concomitant reduction in the dose of slit function. slit-/+;
schizo–/– as well as
robo-/+; schizo–/–
embryos were generated, and in both cases a suppression of the schizo CNS
phenotype was observed. Thus schizo might be required to negatively regulate Slit signaling (Önel, 2004).
If schizo is indeed a negative regulator of slit
function, an increase of schizo gene dose should
result in a decrease of active Slit signaling. One might thus be able to
enhance the mutant slit phenotype by using a schizo gene
duplication. Following mapping of schizo to the base of the left arm of chromosome 3 a chromosomal translocation of the corresponding
part of the third chromosome to the Y chromosome (Tp(3;Y)A81) was used to
generate embryos with three copies of schizo. In an otherwise
wild-type background, this triplication of the schizo region did not
result in an abnormal CNS phenotype. However, when
the schizo translocation was placed in a heterozygous
slit–/+ background, a slit-like
phenotype was observed that was never detected in heterozygous slit embryos (Önel, 2004).
schizo maps to chromosome region 78A/B between the genes poils aux pattes and knockout. To identify the schizo gene in this chromosomal interval P-element-induced schizo
alleles were used. The chromosomal insertions of the P-elements in l(3)3 and P224 were determined by inverse PCR
and Southern analyses and the results suggested that schizo corresponds to CG32434. The
lethality associated with the P-element-induced l(3)3 schizo mutation
could be reverted by precise excision of the P-element. Mutant
l(3)3 embryos displayed a schizo phenotype with reduced
commissures and defective fasciculation in the longitudinal connectives. Subsequent sequencing of cDNA clones LP01489, RE44556 and GH10594
isolated by the BDGP showed that the schizo locus encompasses 41 kb of genomic DNA. At least two different promoters direct the expression of two isoforms of 1325 amino acids (SchizoP1) and 1313 amino acids (SchizoP2) in length. Verification of the cloning of schizo was obtained by genetic rescue experiments. These deduced schizo proteins correspond to the Iso1 and Iso2 variants of the loner gene, which was recently identified in a screen for mutations affecting mesoderm development. By contrast Chen (2003) GH10594 was found to be entirely contained within the LP01489 sequence and no evidence was found for a third schizo protein isoform (Önel, 2004).
The deduced Schizo proteins share three conserved sequence modules. In the
N-terminal region there is a so-called IQ domain, which is predicted to
interact with calmodulin. Within the C-terminal third of the protein a Sec7 domain is directly adjacent to a PH-domain. Proteins
characterized by such a domain signature are generally acting as guanine
nucleotide exchange factors (GEFs). The Anopheles homolog is about
90% identical. The closest human homologs are EFA6, being
32% identical to Schizo, lacking the IQ domain, and ARF-GEP100 showing a 40% identity to Schizo. Both human proteins were shown to act as ADP ribosylation factor 6 (ARF6)-GEFs suggesting that schizo might have a similar function (Önel, 2004).
The molecular identification of schizo allowed for the determination of the expression pattern throughout development. schizo expression is already detected in the unfertilized egg, indicating a prominent maternal contribution.
schizo expression stays almost uniform until the end of stage 10.
Within the developing nervous system, expression can be noted in the CNS
midline cells. In addition, schizo expression can be detected in the epidermis and the visceral mesoderm (Önel, 2004).
The commissural schizo phenotype does not allow the deduction of
cell type in which Schizo normally acts. To test the cell-type requirement the GAL4 system was used and UAS-schizoP1 and
UAS-schizoP2 transgenic flies were established. Expression of the different schizo proteins was directed in the CNS midline cells of mutant
schizo embryos using the sim-GAL4 or sli-GAL4
driver strains. In both cases expression could rescue the schizo
mutant CNS phenotype indicating that Schizo acts in the midline glial cells,
which express both Slit and Netrin (Önel, 2004).
Genetic data indicate that schizo impairs Slit signaling in the CNS. This was further supported by overexpression of schizo. Whereas expression of schizo (P1 or P2) in all CNS midline cells of wild-type embryos did not evoke an abnormal phenotype, the same expression of schizo in heterozygous slit mutant embryos was able to induce a mild slit phenocopy. These results were similar to the ones obtained using chromosomal translocations, supporting the notion that Schizo acts in the CNS midline by regulating the level of Slit expression (Önel, 2004).
To reduce Slit activity, schizo might suppress exocytosis of
Slit-containing vesicles, or it might promote endocytosis of Slit-containing
vesicles from the membrane. Work from vertebrate tissue culture models has
shown that Arf-GEFs such as Schizo can activate endocytosis. To test
whether endocytosis might be relevant for commissure formation a
dominant negative Shibire protein was expressed; this efficiently blocks endocytosis specifically in the CNS midline cells using the sim-GAL driver. In
about 60% of such embryos a schizo phenocopy was observed. When higher levels
of the dominant negative Shibire protein were expressed using the
rho-GAL driver all embryos developed a schizo phenocopy,
suggesting that endocytosis participates in the regulation of Slit function in
the CNS midline cells. To further support the notion that Schizo induces
endocytosis of Slit, the negative Shibire protein was expressed in a
heterozygous slit mutant background. This indeed led to clear
suppression of the Shibire-induced phenotype. Schizo and its
vertebrate homologs exert at least part of the function through the small
GTPase Arf6. Arf6 mRNA is supplied maternally and is expressed
ubiquitously during embryonic development. To determine
whether Schizo acts via Arf6 to control endocytosis of Slit by the midline glial cells, a dominant negative Arf6 construct
(Chen, 2003) was expressed. Following
expression in the midline cells using the sim-GAL driver
no mutant phenotype was observed. Following expression of higher levels of Arf6DN using the rho-GAL driver, about 15% of the embryos
developed a schizo phenocopy, supporting the notion that Schizo acts via Arf6-regulated
endocytosis to control the level of Slit expression on the midline glial
cells (Önel, 2004).
Thus genetic and molecular data support a model in which schizo
negatively regulates the expression of Slit in the CNS midline cells. This study shows that schizo acts in a rather similar way to
commissureless; however, rather than affecting the Roundabout
receptor, schizo appears to act on the expression of the Slit ligand. First it was found that the triplication of the schizo gene interfers
with slit function and that reduction of slit expression in
schizo mutant embryos rescues the schizo mutant phenotype.
Finally, expression of a schizo transgene in the Slit-expressing CNS
midline cells (1) was able to rescue the schizo mutant phenotype and
(2) could induce a slit phenocopy when expressed in wild-type
embryos. The deduced nature of the Schizo protein suggests that it affects
Slit expression by post-transcriptional mechanisms (Önel, 2004).
Guanine-nucleotide exchange factors (GEFs) help to convert the inactive GDP-bound form of small GTPases into a GTP-bound active form. Schizo is a new Sec7 domain containing GEF, which shows 40% homology to human Arf-GEP100. Arf-GEP100 localizes to endosomal membranes and promotes GDP/GTP exchange on ARF6. The small GTPase ARF6 is a plasma membrane-localized protein and functions in the regulation of membrane ruffling, cell motility, aspects of endocytosis and exocytosis, membrane recycling, reorganization of the cortical actin cytoskeleton and activation of phospholipase D. In Drosophila, Arf6 is remarkably well conserved, being more than 96% identical to the human counterpart (Önel, 2004).
One aspect that might hint at how Schizo regulates Slit expression is the role of ARF6 in endocytosis and exocytosis. The function of ARF6 in
endocytosis is twofold. It either regulates clathrin-mediated endocytosis at the apical surface of polarized epithelial cells or it
is able to regulate non-clathrin-mediated endocytosis and the recycling
pathway in non-polarized cells. ARF6 has also been postulated to play a role in Ca2+-activated dense core vesicle (DCV) exocytosis by regulating phosphatidylinositol(4,5) biphosphate (PIP2).
Overexpression of a UAS-ARF6 construct in midline glia cells does not result in a schizo-like phenotype, whereas expression of a
dominant negative form of Arf6 results in a phenocopy of several phenotypes associated with the schizo mutant
(Chen, 2003). This suggests that Arf6 might also be involved in the regulation of Slit expression (Önel, 2004).
In-vivo Slit and Netrin are both expressed by the same CNS midline cells and their expression needs to be in an intricate balance. The importance of this balance and not the individual expression levels is highlighted by the fact that it is possible to rescue the schizo mutant phenotype by both
increased Netrin expression or reduced Slit expression. Within the midline glia, however, Schizo appears to primarily affect Slit expression either by inducing its endocytosis and subsequent degradation or by blocking exocytosis and thus release of Slit (Önel, 2004).
The latter case would suggest that Slit and Netrin are brought to the
membrane of the midline glial cells in distinct vesicle populations, whereas the former case would require a specific membrane receptor for the Slit protein expressed by the CNS midline glia. Given the fact that the secreted Slit protein is found at very high levels at the midline glial cell membrane, this appears probable. Moreover, expression of a dominant negative Shibire protein in the midline glia leads to a schizo phenocopy. shibire encodes the Drosophila dynamin and is required for endocytosis and a block of shibire function leads to a block of endocytosis, which might result in higher levels of Slit expression. Thus, regulation of membrane dynamics appears crucial in controlling the function of the signaling molecule Slit (Önel, 2004).
Biochemical studies suggest that axon guidance activity requires cell-surface heparan sulfate to promote binding of mammalian Slit/Robo homologs. Drosophila Syndecan, a heparan sulfate proteoglycan (HSPG), is required for proper Slit signaling. Slit, the ligand for the Roundabout (Robo) receptors, is secreted from midline cells of the Drosophila central nervous system (CNS). It acts as a short-range repellent that controls midline crossing of axons and allows growth cones to select specific pathways along each side of the midline. In addition, Slit directs the migration of muscle precursors and ventral branches of the tracheal system, showing that it provides long-range activity beyond the limit of the developing CNS. Syndecan (Sdc) mutations have been generated; they affect all aspects of Slit activity and cause robo-like phenotypes. sdc interacts genetically with robo and slit, and double mutations cause a synergistic strengthening of the single-mutant phenotypes. The results suggest that Syndecan is a necessary component of Slit/Robo signaling and is required in the Slit target cells (Steigemann, 2003).
Genetic assays provide a sensitive means of detecting an in vivo interaction between different components in a pathway, but they do not show that the association is direct. Thus, a biochemical assay was developed to determine whether Sdc binds to Slit and/or Robo in cellular extracts in which all three proteins are endogenously expressed. Immunoprecipitation of either Slit or Robo and subsequent detection with anti-Sdc antibodies reveals that Sdc associates with both Slit and its receptor, suggesting the possibility of a ternary complex. This association is specific because no Sdc is trapped by nonspecific IgG or N-Cadherin antibodies that successfully IP other signaling molecules. Thus, Sdc participates directly in a complex with Slit and Robo (Johnson, 2004).
Homozygous sdc mutants are semilethal and show identical phenotypes in the CNS and in the muscle pattern. In order to unambiguously demonstrate that the lack of sdc activity is responsible for the mutant phenotype observed, Sdc-RA was panneurally expressed using the GAL4/UAS system in sdc mutant individuals. The neural phenotype of the mutants was rescued, indicating that the mutant phenotype was caused by the lack of Sdc and that the transgene provides functional Sdc activity (Steigemann, 2003).
In order to examine the possible defects in axonal guidance and muscle patterning, sdc mutant embryos were stained with both Fasciclin II (FasII; mAB 1D4) antibodies, which label three longitudinal axon tracts at each side of the midline, and anti-Mhc antibodies, which visualize the muscle pattern. The results show that the lack of sdc activity causes phenocopies of robo and robo2 mutants; i.e., it affects both midline guidance of axons and the establishment of the muscle pattern. The defects in CNS axon guidance were strikingly similar to robo2 mutants but less pronounced than in robo mutants. The muscle and CNS phenotypes were also weaker than in slit mutants, in which signaling through all Robo receptors is impaired (Steigemann, 2003).
In order to link embryonic Sdc requirement genetically to Slit/Robo signaling, it was next asked whether sdc mutations can enhance loss-of-function slit and robo phenotypes. It was found that the number of ventral muscles, which cross the midline dorsal of the CNS in homozygous sdc and robo2 single mutants, is significantly increased in double mutant combinations of sdc and robo2, resulting in a muscle phenotype indistinguishable from slit and homozygous robo, robo2 double mutants. In the CNS, the FasII-expressing longitudinal fascicles of robo2, sdc double mutants converged into a single thick axon bundle at the ventral midline, resembling the effects seen with slit mutants. Similar observations were obtained with the mAb BP102 against all CNS axons, showing strongly condensed fascicles in robo2, sdc double mutant embryos. The synergistic strengthening of both the muscle and the CNS phenotypes in robo2, sdc double mutants, which are similar to a weak slit mutant phenotype, indicates that only some Slit-derived repellent activity is received along the midline. In contrast to robo2, the robo mutant phenotype was not significantly enhanced by the simultaneous lack of sdc. The data suggest that Robo can, in part, compensate for the lack of Robo2 and vice versa and that Robo is more sensitive to reduced Sdc-dependent Slit activity than Robo2 (Steigemann, 2003).
The results imply that sdc, slit, and robo are components of the same genetic circuitry. This proposal was tested by genetic means, asking whether the gene activities interact in vivo. Loss of only one copy of sdc led to the development of a normal muscle pattern, whereas the simultaneous absence of one copy of both slit and sdc in slit/+, sdc/+ double heterozygous embryos caused an increase in the number of longitudinal transverse muscles. Furthermore, the number of FasII-expressing inner fascicles that cross the midline is increased (3.3%) as compared to slit heterozygous embryos (0.6%;). More clearly, homozygous sdc mutant embryos, which also lack one copy of either robo, robo2, or slit, show an enhanced axonal guidance defect with multiple midline crossings of the fascicles (72%, 47%, and 95%, respectively), a phenotype very similar to the robo mutant. These results establish that Sdc acts in the same genetic circuitry as Slit and the Robo receptor family and represents a critical component of the Slit/Robo signaling pathway (Steigemann, 2003).
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