snail

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of sna at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

sna transcripts are first detected during late syncytial blastoderm (stage 4). By stage 5, SNA mRNA is detected along the ventral surface of the embryo, extending to the anterior and posterior poles. In the early blastoderm, the level of SNA mRNA transcripts in the posterior region begins to decrease. In early germ band elongation [Images], SNA mRNA is seen in the anterior midgut rudiment as well as on the dorsal surface of the invaginating amnioproctodeum [Images] (Alberga, 1991).

sna expression during neurogenesis evolves from segmentally repeated neuroectodermal domains to a pan-neural pattern (Ip, 1994b). Formation of SNA protein in the neurogenic region is predominantely cytoplasmic (Alberga, 1991).

Translation of SNA mRNA is apparently delayed as the SNA protein is not detected before the onset of gastrulation (Alberga, 1991).

Both escargot and snail are coexpressed in the wing imaginal disc beginning at stage 12 in embryonic development and in the genital disc (Fuse, 1996).

Analysis and reconstitution of the genetic cascade controlling early mesoderm morphogenesis in the Drosophila embryo

To understand how transcription factors direct developmental events, it is necessary to know their target or 'effector' genes whose products mediate the downstream cell biological events. Whereas loss of a single target may partially or fully recapitulate the phenotype of loss of the transcription factor, this does not mean that this target is the only direct mediator. For a complete understanding of the pathway it is necessary to identify the full set of targets that together are sufficient to carry out the programme initiated by the transcription factor, which has not yet been attempted for any pathway.In the case of the transcriptional activator Twist, which acts at the top of the mesodermal developmental cascade in Drosophila, two targets, Snail and Fog, are known to be necessary for the first morphogenetic event, the orderly invagination of the mesoderm. A system of reconstituting loss of Twist function by transgenes expressing Snail and Fog independently of Twist was used to analyse the sufficiency of these factors°Va loss of function assay for additional gene functions to assess what further functions might be needed downstream of Twist. Confirming and extending previous studies, Snail was shown to play an essential role, allowing basic cell shape changes to take place. Fog and at least two other genes are needed to accelerate and coordinate shape changes. Furthermore, this study represents the first step in the systematic reconstruction of the morphogenetic programme downstream of Twist (Seher, 2007).

In addition to Twist, Snail and Fog, there are genes in four regions of the autosomal genome which upon deletion lead to abnormalities during ventral furrow formation. Is it likely that all zygotically active genes that participate in normal mesoderm invagination have been detected? Although the assay proved to be sufficiently sensitive to identify a number of mutants, it is conceivable that further genes with even less pronounced mutant phenotypes were missed. Further, genes with completely redundant functions, for example, because duplicates exist in distinct regions of the genome, might not give a loss-of-function phenotype. Such genes might be identifiable only via sophisticated genetic screens (modifier screens) or appropriate molecular approaches (Seher, 2007).

The loss of the genes uncovered by the deficiencies results only in a delay of furrow formation, and not in the complete failure of invagination. If these genes are Twist targets, there are different possible explanations for the mutants showing such weak phenotypes. The genes might control an essential process parallel to that controlled by Snail, but act in a redundant manner in the pathway, such that disruption of only one of their functions does not lead to the disruption of furrow formation. Alternatively, only the pathway controlled by Snail may be essential, with other genes acting in parallel affecting only the speed and efficiency of furrow formation. The severe phenotype seen in the double mutants of Df(3R)Tl^P and fog as well as the enhancement of the fog phenotype by the loss of one copy of Snail argue for the latter scenario, i.e. two parallel pathways, both of which are essential (Seher, 2007).

Embryos were created in which the function of the mesodermal transcription factor twist was replaced by two of its downstream targets, snail and fog. The analysis of these embryos concentrated on the first phase of mesoderm morphogenesis, during which cell shape changes internalize the prospective mesoderm. The subsequent epithelial–mesenchymal transition, cell division and cell migration depend on other Twist targets, such as string, htl and dof. Since these are not expressed in the twist,PE::fog;2xPE::sna (twist driven fog and snail) embryos, this aspect of morphogenesis cannot occur in the reconstituted twist embryos (Seher, 2007).

The fog and snail transgenes had distinguishable effects in twist embryos. The PE::fog transgene induces the earliest event of the typical cell shape changes, apical flattening, and enhanced apical constrictions of ventral cells. By contrast, nuclear movement away from the apical cell surface was not significantly improved, nor was cell shortening observed. While the 2xPE::snail transgene also led to some improvement in apical flattening, it had additional effects. A larger number of cells showed distinctive nuclear movement, and a higher frequency of deep invaginations were scored, suggesting a role for Snail in releasing nuclei. With the combination of both transgenes nearly normal cell shape changes occurred which resulted in the formation of proper furrows in a substantial number of embryos. Specifically, many cells assumed the typical wedge-shape of ventral furrow cells, showing that snail and fog are sufficient to induce this shape in the absence of further Twist targets (Seher, 2007).

However, it appears that snail and fog cannot be the only targets downstream of twist to control mesoderm invagination. If they were, they should be able to replace twist function completely and fully restore furrow formation. It is therefore concluded that besides snail and fog other twist target genes must exist which are necessary to orchestrate the formation of the ventral furrow in the accurate, fast and stable fashion in wildtype embryos (Seher, 2007).

The as yet unknown targets must be involved in those events which were not restored in twist,PE::fog;2xPE::sna embryos: the speed of the process, adhesion between apical surfaces, and cell shortening during invagination. The latter process occurs efficiently in snail mutants, confirming that it is not Snail-dependent. Since fog does not contribute to this process it is likely that one or more other twist targets are involved in cell shortening (Seher, 2007).

In summary, the zygotic control of ventral furrow formation branches into separable functions downstream of Twist, the induction of the basic cell shape changes of ventral cells, and the control of the speed, accuracy and coordination of the shape changes. One of the known targets of Twist, the repressor Snail, is necessary to allow the shape changes to occur, whereas Fog and probably other Twist targets are responsible for accuracy and efficiency. Together, they ensure the rapid and regular formation of the ventral furrow. Ventral furrow formation may be an adaptation to the rapid early embryogenesis of long germ insects, serving to position mesodermal cells at a site where they can efficiently begin their FGF-dependent spreading on the inner surface of the ectoderm. The experimental system used in this study may be extended to test the function of the whole set of Twist targets, once they have been identified, for their ability to re-establish mesoderm invagination in the absence of Twist, and thereby reconstruct fully the pathway from a 'selector' gene to the cell biological processes it controls (Seher, 2007).

Effects of Mutation or Deletion

Mitoses specific to the mesoderm are absent in the mutants twist and snail, that fail to differentiate the ventrally derived mesoderm. The lateral mesectodermal domain shows a partial ventral shift in twist mutants but a proportion of ventral cells do not behave characteristically, suggesting that twist has a positive role in the establishment of the mesoderm. In contrast, snail is required to repress mesectodermal fates in cells of the presumptive mesoderm. In the absence of both genes, the mesodermal and the mesectodermal anlage are deleted (Arora, 1992).

Aspects of midgut development, migration of anterior midgut (AMG) primordium and the posterior midgut (PMG) primordium, and transition to an epithelium are all necessary processes that depend on the mesoderm. The extension of the midgut primordia is achieved by cell migration along the visceral mesoderm that forms a continuous layer of cells within the germ band. In mutant embryos lacking the entire mesoderm or failing to differentiate the visceral mesoderm, AMG and PMG are formed but do not migrate properly. In addition, they fail to form an epithelium and instead either remain as compact cell masses anterior and posterior to the yolk (in twist and snail mutant embryos) or only occasionally wrap around the yolk before embryogenesis is completed (in tinman-deficient embryos). Thus the visceral mesoderm serves as a substratum for the migrating endodermal cells and that the contact between visceral mesoderm and endoderm is required for the latter to become an epithelium (Reuter, 1993).

Hemocytes derive exclusively from the mesoderm of the head and disperse along several invariant migratory paths throughout the embryo. The origin of hemocytes from the head mesoderm is further supported by the finding that hemocytes do not form in Bicaudal D, a mutation that lacks all head structures, or in twist snail double mutants, where no mesoderm develops. All embryonic hemocytes behave like a homogenous population with respect to their potential for phagocytosis. Thus, in the wild type, about 80-90% of hemocytes become macrophages during late development. In the Drosophila embryo, apoptosis can occur independently of macrophages, since mutations lacking macrophages show abundant cell death (Tepass, 1994).

Scutoid is a classical dominant gain-of-function mutation of Drosophila, causing a loss of bristles and roughening of the compound eye. Previous genetic and molecular analyses have shown that Scutoid is associated with a chromosomal transposition resulting in a fusion of no ocelli (located at 35A4), a Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures, and snail (located at 35D2) genes. How this gene fusion event leads to the defects in neurogenesis has not been known until now. snail has been found to be ectopically expressed in the eye-antennal and wing imaginal discs in Scutoid larvae, and this expression is reduced in Scutoid revertants. The expressivity of Scutoid is enhanced by zeste mutations. snail and escargot encode evolutionarily conserved zinc-finger proteins involved in the development of mesoderm and limbs. Snail and Escargot proteins share a common target DNA sequence with the basic helix-loop-helix (bHLH) type proneural gene products. When expressed in the developing external sense organ precursors of the thorax and the eye, these proteins cause a loss of mechanosensory bristles in the thorax and perturbed the development of the compound eye. Such phenotypes resemble those associated with Scutoid. Furthermore, the effect of ectopic Escargot on bristle development is antagonized by coexpression of the bHLH gene asense. Thus, these results suggest that the Scutoid phenotype is due to an ectopic snail expression under the control of no ocelii enhancer, antagonizing neurogenesis through its inhibitory interaction with bHLH proteins (Fuse, 1999).

Prior studies have shown that the Sco phenotype is caused by the fusion of a chromosome fragment containing a part of noc and Adh genes placed with the region approx. 16 kb upstream of sna. Analyses of Sco revertants have demonstrated that both sna and noc portions of the fusion are necessary to cause the phenotype. It has been proposed that Sco is an ìantimorphicî allele of noc, based on the sensitivity of the Sco phenotype to the copy number of the noc gene. One model suggests that the noc-sna fusion on the Sco chromosome produces a fusion gene product that antagonizes the wild-type noc gene product. However, molecular analyses of the noc locus have led to questions about the model. noc is divided into physically separable units [l(2)35Ba, nocA, and nocB] based on the mapping of aberrations affecting noc functions. Mutations in l(2)35Ba lead to embryonic- larval lethality with defects in the optic lobe. nocA and nocB mutations cause a loss of ocelli and their associated bristles, with mutations in the former region having a stronger effect. l(2)35Ba encodes the No ocelli protein that contains a zinc-finger motif and requires nocA function for its expression. Since no transcript derived from the nocA region was identified, it is very likely that the l(2)35Ba-encoded protein carries out noc function, and that nocA is a cis-regulatory region of l(2)35Ba. Similar phenotypes of nocA and nocB suggest that nocB is also another regulatory region of l(2)35Ba, although more molecular analysis is required before a final conclusion can be drawn. The Sco transposition breaks between nocA and nocB, and places nocB next to sna, leaving l(2)35Ba and nocA in their original position far apart from the noc-sna fusion point. This makes the presence of an antimorphic sna-noc fusion gene product impossible. Given the current results showing that Sco is associated with ectopic sna expression, and that misexpression of wild-type sna mimics Sco phenotype, it is proposed that Sco is a gain-of-function, neomorphic mutation of sna (Fuse, 1999).

The Sco chromosome pairs tightly with the wild-type homolog in the polytene chromosome, suggesting that Sco and noc are placed in physical proximity. zeste is a mediator of transvection, a proximity-dependent, sometimes interchromosomal, interaction between enhancers. In this scenario sna expression from the Sco chromosome is normally down-regulated by the wild-type copy of the noc enhancer, and zeste mutations disrupt this trans-repression to enhance the phenotype. A mutation in noc would also interfere with this trans-repression. Such a copy number dependent repression of transcription has been shown for pairing dependent repression, in which the reporter gene white linked to a fragment containing a polycomb response element is repressed when two copies of the gene are placed in proximity (Fuse, 1999).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

twi and sna, were found to be necessary for development of the tubules beyond evagination. These genes could function downstream of Kruppel and parallel to cut to implement the Malpighian tubule specific pattern of gene expression. Both twi and sna function in determining mesodermal fate. The effect of sna mutations on the morphogenesis of the tubules, which are of ectodermal origin, can be explained by the fact that sna is also expressed in the Malpighian tubules. twi expression has not been reported in the Malpighian tubules although the distribution of the protein throughout embryogenesis has been described. One possibility is that twi is expressed in the Malpighian tubules at a level that was undetected or at a time before the budding of the tubules. twi is active in the development of the embryonic termini. Lack of twi activity could lead to the failure of the tubules to develop by virtue of a defect in terminal specification that occurs prior to budding of the tubules. Alternatively, the effect of twi may be indirect, possibly occurring through an inductive interaction of another tissue on the Malpighian tubules (Jack, 1999).

Three snail family genes -- snail, escargot and worniu -- encode related zinc finger transcription factors that mediate Drosophila central nervous system (CNS) development. Simultaneous removal of all three genes causes defective neuroblast asymmetric divisions; inscuteable transcription/translation is delayed/suppressed in the segmented CNS. Furthermore, defects in localization of cell fate determinants and orientation of the mitotic spindle in dividing neuroblasts are much stronger than those associated with inscuteable loss of function. In inscuteable neuroblasts, cell fate determinants are mislocalized during prophase and metaphase, yet during anaphase and telophase the great majority of mutant neuroblasts localize these determinants as cortical crescents overlying one of the spindle poles. This phenomenon, known as 'telophase rescue', does not occur in the absence of the snail family genes; moreover, in contrast to inscuteable mutants, mitotic spindle orientation is completely randomized. These data provide further evidence for the existence of two distinct asymmetry-controlling mechanisms in neuroblasts both of which require snail family gene function: an inscuteable-dependent mechanism that functions throughout mitosis and an inscuteable-independent mechanism that acts during anaphase/telophase (Cai, 2001).

CNS development is abnormal in Df(2L)osp29 embryos due to deletion of Sna family proteins. Both Sna and Wor are expressed strongly in all NBs, including those in the procephalic region, during early neurogenesis. The expression of Esg is also seen in NBs and other tissues, as visualized with anti-Esg immunostaining. Expression of Esg can be detected in the midline cells as well as GMCs during embryonic development. The functions of these three genes are overlapping; the early CNS defects are detected only when all three genes are removed simultaneously. In order to test whether the defects of localization of Mir/Pros and Pon/Numb seen in Df(2L)TE35BC-3 embryos are due to the absence of the three sna family genes, the localization of Mir/Pros and Pon/Numb was examined in embryos single mutant for sna, esg or wor, a double mutant for sna/esg and deletions that removed sna/wor or esg/wor, as well as embryos double mutant for sna/esg and further subjected to wor double-stranded RNA (RNAi) treatment. In single and double mutant embryos, both Mir/Pros and Pon/Numb form normal basal crescents in mitotic NBs. Only the sna/esg double mutant embryos that have been injected with wor RNAi reproduce the phenotype found in Df(2L)TE35BC-3 embryos (Cai, 2001).

In wild-type embryos, NBs are located between the ectoderm and mesoderm. The Df(2L)TE35BC-3 embryos lack mesoderm. Therefore, it is possible that correct NB asymmetry requires signal(s) from the mesoderm, and the asymmetry defects seen in Df(2L)TE35BC-3 could be due simply to the absence of mesoderm in these embryos. This is unlikely since NB asymmetry is intact in sna embryos, which lack mesoderm and share the abnormal morphology of Df(2L)TE35BC-3 embryos. Furthermore, the partial rescue of mesoderm in Df(2L)TE35BC-3 embryos by ectopic expression of the Sna protein driven by twist-gal4 does not reverse the asymmetry defects. Thus, it is concluded that mislocalization of Mir/Pros and Pon/Numb in Df(2L)TE35BC-3 embryos is due to the absence of all three sna family genes. Based on this conclusion, Df(2L)TE35BC-3 is referred to as sna/esg/wor deficient and was used in subsequent studies (Cai, 2001).

In wild-type embryos, Baz, Insc and Pins form a complex that is localized to the apical cortex of the dividing NBs. The apical complex is required for the asymmetric distribution of cell fate determinants such as Pros and Numb to the basal cortex of NBs and coordinates the orientation of the mitotic spindle along the apical-basal axis of the NB. In embryos deficient for the sna family genes, Mir/Pros and Pon/Numb are no longer concentrated to the basal cortex of mitotic NBs, indicating defects in NB asymmetry. It is possible that the asymmetry defects seen in sna/esg/wor-deficient NBs are due to the alteration of Insc expression. Anti-Insc staining indicates that Insc protein is indeed undetectable in the segmented CNS of sna/esg/wor-deficient embryos. Although the signal intensity in the procephalic region is comparable to that in the wild-type controls, the number of cells with anti-Insc staining appears to be decreased. This altered expression of Insc in the mutant embryos suggests that the mislocalization of Mir/Pros and Pon/Numb in sna/esg/wor-deficient embryos is, at least in part, due to a lack of Insc protein expression in dividing NBs. As expected, Baz protein levels are low and undetectable in the great majority of mutant NBs. The lack of easily detectable Baz in NBs is probably due to the instability of the protein when Insc is absent since the baz mRNA levels remain unchanged in sna/esg/wor NBs. Pins protein localization is also affected in sna/esg/wor-deficient embryos (Cai, 2001).

The down-regulation of Insc protein in NBs is also dependent on the simultaneous loss of sna, esg and wor functions. Insc expression in double mutant embryos of sna/esg was similar to that of wild-type embryos. In sna/esg double mutant embryos, further removal of the third member of sna gene family, wor, with RNAi leads to the total loss of Insc protein expression. Moreover, ectopic expression of any one of the sna family genes under the control of an early neural driver sca-gal4 in sna family gene mutant embryos largely restores the Insc expression in NBs (sna 79%; esg 64% and wor 44%), further indicating that Insc expression is indeed regulated by the Sna family proteins (Cai, 2001).

insc transcript levels were examined in the sna/esg/wor-deficient embryos. In wild-type stage 9-10 embryos, insc RNA is expressed prominently in NBs of the segmented CNS and in the procephalic region. The transcript level is maintained in the segmented CNS and procephalic NBs throughout embryogenesis. In sna/esg/wor-deficient embryos, RNA in situ hybridization data indicate that the insc RNA is absent in the segmented CNS at stages 9-10 but is detectable in the procephalic NBs. This suppression of insc RNA transcription in the segmented CNS of sna/esg/wor-deficient embryos provides evidence that the Sna family proteins are essential for insc mRNA transcription during early neurogenesis. The suppression of insc transcription in the segmented CNS is transient and insc RNA can be detected, at a lower level, in late stage 11 embryos. However, Insc protein in the segmented CNS of sna/esg/wor-deficient embryos remains undetectable at late stage 11 when the insc RNA levels partially recover by an unknown mechanism. It is obvious that translation of insc RNA in late stage 11 embryos is inhibited in the segmented CNS of embryos deficient for sna/esg/wor. Although the inhibition mechanism is unknown, it is believed that the insc 5'- and/or 3'-untranslated regions (UTRs) are involved since Insc protein can be ectopically expressed in sna/esg/wor-deficient embryos from a uas-insc transgene in which the 5'- and 3'-UTRs have been partially removed. Considering that the Sna family proteins are localized to nuclei, it is unlikely that they interact directly with 5'- and/or 3'-UTRs of insc RNA. Presumably other genes regulated by the Sna family proteins mediate the observed translational effect (Cai, 2001).

The observation of delayed and decreased insc mRNA transcription and the inhibition of Insc protein synthesis in the segmented CNS of sna/esg/wor-deficient embryos suggests the dual regulation of insc expression by the Sna family proteins at both transcriptional (stage 9-10) and translational (stage 11 onwards) levels. This dual regulation mechanism is prominent in the segmented CNS but insc RNA and protein expression in the procephalic region is only partially affected in sna/esg/wor-deficient embryos. The mechanism that enables the partial restoration of insc transcription in NBs of the segmented CNS at late stage 11 in the absence of sna family gene function remains to be identified (Cai, 2001).

In insc²² mutant NBs, in which the apical complex required for correct asymmetric division is abolished, basal components such as Mir/Pros and Pon/Numb often form random crescents, sometimes broad and loose, from prophase to metaphase; however, Pros/Mir and Pon/Numb can eventually be redistributed to the 'budding site' of the future GMCs, although sometimes not as exclusively as seen in wild-type embryos, at anaphase and telophase even when the spindle is misorientated. Consequently, the great majority of all GMCs inherit, at least in part, cell fate determinants such as Pros and adopt correct GMC fate. This phenomenon, referred to as 'telophase rescue', does not occur in NBs lacking the three sna family genes. For example, in sna/esg/wor-deficient NBs, basal proteins Mir/Pros and Pon/Numb form a randomly localized crescent in dividing NBs but, unlike in insc embryos, these proteins are not redistributed at anaphase/telophase to the region of the cortex that gives rise to the GMC. Consequently, the great majority of the GMCs do not inherit the basal proteins Mir/Pros and Pon/Numb and thus lose their GMC identities. This finding explains why GMCs are not specified correctly in Df(2L)osp29 embryos (Cai, 2001).

Furthermore, it is known that the mitotic spindle in NBs rotates 90° during metaphase so that it is realigned along the apical-basal (A/B) axis of the embryos; in insc mutants, this spindle rotation during metaphase occurs only in a small proportion (~20%) of NBs; nevertheless, even some of these NBs are able to reorient spindles late in mitosis. The NB spindle orientation during anaphase or telophase was measured in wild-type and mutant embryos and they were catagorized into four equal quadrants depending on the angle that the spindle forms with the A/B axis. Based on the spindle orientation in wild-type embryos, all spindles with an angle >45° relative to the A/B axis during late mitosis are considered to be misoriented. The misoriented spindles in insc²² mutant embryos are limited; the great majority of NBs (90%) have their spindles oriented within 45° of the A/B axis, compared with 100% in wild-type NBs. In contrast to wild-type and insc NBs, in sna/esg/wor-deficient NBs, spindle orientation is completely randomized with almost equal distribution for each of the four quadrants. Moreover, a small number of NBs (10%) completely reverse their polarity, giving rise to a small apical GMC, which has never been reported in any known asymmetry mutant (Cai, 2001).

These observations indicate that removal of Insc alone has only a limited effect on NB asymmetric divisions in terms of basal protein localization and spindle orientation late in mitosis, suggesting that the Insc-dependent mechanism is not the only apparatus that controls the asymmetric divisions in NBs. It appears that an Insc-independent mechanism exists that functions in parallel to coordinate the asymmetry events at later stages (anaphase onwards) of mitosis. This Insc-independent asymmetry-controlling mechanism, which is responsible for the 'telophase rescue' phenomenon and for prevention of random spindle orientation in insc²² embryos, is destroyed upon removal of the three sna family genes. However, one might argue that the severe asymmetry defects seen in the absence of the sna family genes might be artifactual, caused by the combination of loss of insc expression and the absence of the mesoderm. This possibility is suggested because in insc/sna double mutant embryos, which lack both insc and the mesoderm, NBs exhibit phenotypes that are indistinguishable from those seen in the insc single mutant. It has therefore been concluded that in the absence of the sna family genes, both the Insc-dependent and -independent asymmetry-controlling mechanisms are destroyed, leading to asymmetry defects that are more severe than those seen in insc single mutants (Cai, 2001).

The existence of two distinct asymmetry-controlling mechanisms in wild-type NBs raises an interesting issue: how do these two mechanisms work in concert to mediate asymmetric divisions? Since embryos deficient for the sna family genes lack both mechanisms, it was reasoned that by restoring the Insc-dependent mechanism in these embryos the consequences of missing just the insc-independent mechanism could be assessed. Ectopic expression of full-length Insc protein with an early neural driver sca-gal4 in NBs of sna family gene mutant embryos shows complete rescue of the protein localization defects. The apical complex forms normally, as indicated by the formation of apical Insc as well as Pins and Baz crescents. The defects in basal protein localization are also completely rescued; Mir/Pros and Pon/Numb form tight basal crescents in mitotic NBs. These results suggest that, with respect to protein localization, Insc protein is the only component missing in the Insc-dependent asymmetry machinery, and replacement of Insc through ectopic expression is sufficient to restore wild-type localization of the apical and basal components. Furthermore, it indicates that the Insc-independent mechanism is cryptic with respect to protein localization since it is dispensable when the Insc-dependent mechanism is in place. Either mechanism alone is able to distribute basal proteins to the cortex of the future GMC 'budding site' with clear temporal and efficiency differences: the Insc-dependent mechanism localizes basal proteins starting in late prophase in the form of tight crescents, while the Insc-independent mechanism is only able to redistribute, sometimes partially, mislocalized basal proteins late in mitosis (telophase rescue) (Cai, 2001).

The spindle misorientation phenotype in sna family gene mutant embryos is also largely corrected by ectopic Insc expression. However, unlike protein localization, the rescue of mitotic spindle orientation is incomplete; the population of NBs with misoriented spindles drops from 45% to only 12%. These data suggest that both the Insc-dependent and -independent mechanisms are required for correct spindle orientation in wild-type embryos since ~10% of the mitotic spindles are misoriented in anaphase/telophase NBs defective for either mechanism. However, a complete randomization of spindle orientation is seen when both mechanisms are absent (Cai, 2001).

Thus, the underlying cause for the asymmetry defects associated with some deficiencies uncovering the 35B-D region of the genome, e.g. Df(2L)TE35BC-3, is the simultaneous loss of three members of the sna gene family: sna, esg and wor. All available lethal complementation groups uncovered by Df(2L)TE35BC-3, all deficiencies that remove only two out of the three sna family members and a sna/esg double mutant generated from recombination do not show any defects in any aspect of NB asymmetric division; only embryos double mutant for sna/esg, and further subjected to wor RNAi, reproduce the asymmetry defects seen in the deficiencies. These data indicate that the defects in sna/esg/wor-deficient embryos are caused by the simultaneous functional loss of all three sna family genes. The observation that the ectopic expression of sna, esg or wor reverses the asymmetry phenotypes in the segmented CNS of sna/esg/wor-deficient embryos further supports this conclusion. These conclusions are in agreement with an earlier study reporting that the sna family genes are required for CNS development (Cai, 2001).

It has been observed that in insc embryos, cell fate determinants such as Pros and Numb are mislocalized early during mitosis; however, in anaphase and telophase, the effect termed 'telophase rescue' causes the misplaced crescents to redistribute and overlie one spindle pole, enabling the basal cell fate determinants to segregate, exclusively or partially, to the GMCs. The insc loss-of-function alleles insc²², insc^P49 and insc^P72 all show telophase rescue. It has been found that essentially all NBs in insc embryos can redistribute Pros and Numb, at least partially, into GMCs. These observations suggest the existence of a second asymmetry-controlling mechanism that does not require insc functions, which operates late in mitosis to coordinate protein localization with spindle orientation. These observations explain why insc mutants have minimal effect on GMC cell fate. The Insc-independent mechanism corrects the earlier errors caused by absence of Insc during anaphase/telophase, thereby enabling cell fate determinants to be inherited by the GMC. This mechanism is apparently less efficient, as shown by the fact that in some insc NBs, normally basal components form a broad and loose crescent and are only partially sequestered into GMCs. Furthermore, the observation that mitotic spindle orientation is only mildly affected in insc NBs is also consistent with an Insc-independent compensatory mechanism (Cai, 2001).

Analysis of NB divisions in embryos deficient for the three sna family genes provides further support for the existence of an Insc-independent mechanism. In these embryos, the Insc-dependent mechanism is clearly abolished; both the transcription and the translation of insc are suppressed in the mutant NBs. In addition, telophase rescue no longer occurs; the normally basally localized components are misplaced in mitotic NBs and not redistributed to the future GMCs even at anaphase/telophase. Moreover, the spindle orientation in embryos deficient for the sna family genes becomes randomized; ~45% of NBs exhibit misoriented spindles with an angle >45° with respect to the A/B axis at anaphase/telophase, which is not seen in wild-type NBs and is at a much higher frequency than that seen in insc²² NBs. Thus, NBs deficient for the sna family genes show two defects that are not seen in insc NB: (1) the absence of telophase rescue, and (2) randomization of the spindle orientation late in mitosis. These observations indicate that both the Insc-dependent and -independent mechanisms require the sna family genes (Cai, 2001).

These two mechanisms can apparently function independently. In insc NBs, the Insc-independent mechanism functions in the absence of the Insc-dependent mechanism to correct the earlier (prophase to metaphase) asymmetry defects during anaphase/telophase. In sna/esg/wor-deficient NBs that have been forced to express Insc, the Insc-dependent mechanism can act in the absence of the Insc-independent mechanism to mediate the localization of the basal components from prophase to telophase, obviating the requirement for telophase rescue; however, although the Insc-dependent mechanism can reduce the extent of the mitotic spindle orientation defects seen in the sna/esg/wor NBs, it does not restore wild-type spindle orientation. Therefore, it appears that both mechanisms are required and act in concert to mediate mitotic spindle orientation. However, with respect to localization of the basal components, the effects of the Insc-independent mechanism are only visible when the Insc-dependent mechanism is absent (Cai, 2001).

For the Insc-dependent mechanism, three components have been identified: Baz, Insc and Pins are known to form an apically localized functional complex. The function of this complex requires the participation of all members. Insc appears to be the only component of the Insc-dependent mechanism missing in sna/esg/wor-deficient embryos since ectopic expression of Insc restores its function. Little information is available on the components of the Insc-independent mechanism. Other members of asymmetry machinery identified so far in NBs are the basal components such as Mir/Pros, Pon/Numb, Stau and pros RNA. These downstream components are controlled and coordinated by both Insc-dependent and -independent mechanisms (Cai, 2001).

In embryos deficient for the sna family genes, one of the major defects is the absence of Insc protein expression in the segmented CNS. RNA in situ hybridization indicates that the insc RNA transcripts are not detected in NBs of stage 9-10 embryos. Even in late stage 11 embryos when the insc RNA levels partially recover, Insc protein is never seen in the segmented CNS, indicating that the down-regulation of insc occurs at both the transcriptional and translational levels. In the procephalic region of these sna/esg/wor-deficient embryos, Insc expression is only partially affected. The 5'- and/or 3'-UTRs of the insc transcript appear to play an important role in the translational regulation of Insc expression. This is supported by two observations: (1) Insc protein can be detected in sna/esg/wor embryos following ectopic expression of a cDNA construct containing the complete insc coding region but with the 5'- and 3'-UTRs partially removed; (2) transcripts derived from lacZ driven by a 1.2 kb insc 5' CNS promoter sequence are not subjected to this translational repression in sna/esg/wor embryos, although their expression pattern is identical to that of Insc in the CNS. Given that the Sna family proteins are localized to nuclei, it is unlikely that they play a direct role in translational regulation. Other unknown intermediates must be involved (Cai, 2001).

Snail was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4²²¹) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4²²¹-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).

In addition to the effects on primary dendrite extension, RNAi of each of these 18 genes limits the number and length of lateral dendrite branches. RNAi of some genes such as snail or knirps almost completely blocked dendrite branching, whereas RNAi of other genes such l(3)mbt had more modest effects on dendrite branching. In addition, a significant reduction of branching was noticed at the distal tip of the dorsally projected primary dendrite. In control treated stage 17 embryos, branchpoints are distributed along the primary dendrite, with the most distal branchpoint usually located within a few microns of the distal tip of the dendrite. In contrast, branching is rarely observed within 10 microns of the distal dendritic tip following RNAi of these group B genes. In some cases, such as snail(RNAi), knirps(RNAi), or l(3)mbt(RNAi), the most distal branchpoint is located 25 microns or further from the distal tip of the primary dendrite. Therefore, these TFs inhibit primary branch extension but promote lateral branching and lateral branch extension (Parrish, 2006).

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date revised: 1 August 2008

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