escargot
Gene expression is primarily ectodermal, occuring approximately at cell cycle 11 in the syncytial blastoderm stage. Transcripts are detected along the dorsal surface, just anterior to the region where the cephalic furrow will form. Prior to gastrulation, dorsal expression disappears and a grid-like pattern is found in the neurogenic region of the ventral ectoderm and in the head region. Later transcripts are found in the ventral midline. After germ band shortening, transcription is seen in leg, wing, haltere, and genital imaginal discs. Strong expression is also seen in the anterior region, including cells outlining the site of head involution [Images], and the dorsal ridge and frontal sac. histoblast cells of the abdominal segments also express escargot (Whiteley, 1992).
Expression of escargot in imaginal tissues is also found in third instar larvae (Hayashi, 1993).
Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).
At embryonic stage 11, the early expression of D11 expression marks the entire limb primordium that gives rise to both wing and leg discs. After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc. Double labeling of stage 15 leg discs reveals that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg, suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium (Kubota, 2003).
In the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage. Double labeling with antibodies against Esg and Hth reveal that the Esg expression overlaps with Hth expression. Esg is used as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region (Kubota, 2003).
The expression domain of wg and the position of wing and leg primordia were compared. Wg expression in the trunk ectoderm starts as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlap with wg stripes, as revealed by the wg-lacZ reporter. At late stage 11, wg-lacZ stripes break up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein becomes limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes. The ventromedial wg stripe also overlaps with proximal leg cells that are labeled with anti-Esg at stage 15. The ventral half of proximal leg cells is nearly completely included within the ventral wg stripes. The dorsal half of leg cells is also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia are first recognizable at stage 12 as cells expressing Vestigial (Vg), they do not overlap with the stripe of wg. Dorsal cell migration further separates wing primordia from the source of Wg at stage 15. The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development (Kubota, 2003).
To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).
Cell fate maintenance of proximal leg requires continuous signaling by Wg. The requirement for arm and wg is higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe, it is proposed that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal-proximal cells require a higher level of Wg to be produced to reach their position (Kubota, 2003).
Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp, is expressed in stripes abutting the Wg stripe; Dpp is known to be required for distal leg development (Kubota, 2003).
Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth, marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by a distal gene, as shown by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, Dll is known to repress proximal genes in larval leg discs. The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc have distinct cell-adhesive properties from those in the Dll-expressing distal domain, and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc (Kubota, 2003).
Although the analogous pattern of Wg and Dpp expression plays essential roles in PD patterning in embryonic and larval leg development, significant differences are noted. In embryonic leg discs, expression of both proximal and distal leg markers is lost in mutants of Wg signaling or Dpp signaling. Therefore, Wg and Dpp contribute to both proximal and distal leg development in the embryo. In the larvae, reduction of Wg and Dpp expression due to the loss of hh function causes a loss of the distal domain, but no effect on the proximal gene expression was observed, suggesting that Wg and Dpp play little or no role in the development of proximal domain. The inability of Wg or Dpp to participate in the proximal leg patterning in the larvae is due to, at least in part, the function of Hth to block activation of target genes for Wg and Dpp. In the embryo, however, Hth does not block expression of esg, a target gene for Wg, as demonstrated by coexpression of Esg and Hth. Therefore, proximal domains of embryonic and larval leg discs are different in the way Hth regulates target genes for Wg. This difference may reflect distinct stages of leg development in the embryo, where proximal leg and epidermal cells are continuous, as defined by Hth expression, and in the larvae, where they are separated by the peripodial membrane (Kubota, 2003).
The complementary pattern of Wg and Dpp expression in the larval leg disc is maintained by mutual repression. No evidence for mutual repression of Wg and Dpp was observed in embryonic leg discs. Perhaps the complementary expression pattern of Wg and Dpp in the embryonic leg disc is under the control of the mechanism regulating the global dorsoventral pattern of the embryo (Kubota, 2003).
Thus, in Drosophila, specific mechanisms are involved in embryonic development as opposed to larval leg development. This finding gives rise to the question as to which of the mechanisms is used in other primitive hemimetabolous insects, where the specification and growth of the leg occur simultaneously (Kubota, 2003).
Adult stem cells maintain organ systems throughout the course of life and
facilitate repair after injury or disease. A fundamental property of stem and
progenitor cell division is the capacity to retain a proliferative state or
generate differentiated daughter cells; however, little is currently known about
signals that regulate the balance between these processes. A proliferating
cellular compartment has been characterized in the adult Drosophila
midgut. Using genetic mosaic analysis it has been demonstrated that
differentiated cells in the epithelium arise from a common lineage. Furthermore,
reduction of Notch signalling leads to an increase in the number of midgut
progenitor cells, whereas activation of the Notch pathway leads to a decrease in
proliferation. Thus, the midgut progenitor's default state is proliferation,
which is inhibited through the Notch signalling pathway. The ability to
identify, manipulate and genetically trace cell lineages in the midgut should
lead to the discovery of additional genes that regulate stem and progenitor cell
biology in the gastrointestinal tract (Micchelli, 2006).
The adult Drosophila midgut can be identified on the basis of two
anatomical landmarks along the anterior-posterior axis of the gastrointestinal
tract: the cardia and pylorus. The inner surface of the midgut is lined with a
layer of cells that project into the gut lumen. These cells exhibit apical-basal
polarity; staining for F-actin reveals the presence of a distinct striated
border on their lumenal surface. This observation is consistent with the
suggestion that the midgut is lined by a cellular epithelium (Micchelli, 2006).
Wild-type midguts were stained with 4,6-diamidino-2-phenylindole (DAPI) to
reveal the distribution of cell nuclei within the tissue. Nuclei of the midgut
display a distinct distribution and fall into two main categories. The most
prominent cells lining the midgut contain large oval nuclei that stain strongly
with DAPI. These cells exhibit a region of the nucleus that does not stain with
DAPI, giving the nucleus a hollow appearance. This unstained region may
correspond to the large nucleolus characteristic of differentiated cells. A
second population of cells containing small nuclei can be detected at a basal
position within the tissue. The small nuclei are distant from the gut lumen and
often lie in close apposition to the two layers of overlying visceral muscle
that surround the gut. On the basis of nuclear size, position and morphology two
general populations of midgut cells can, therefore, be distinguished (Micchelli,
2006).
Previous studies in Drosophila have led to conflicting views over the
existence of cell proliferation in the adult gastrointestinal tract. Early
reports suggested that somatic stem cells were present in the adult because of
morphological similarity to certain larval cells and by analogy to different
insect species. In contrast, 3H-thymidine labelling experiments
detected DNA synthesis in the adult Drosophila midgut, but no mitotic
figures were observed in a large sample analysed. On the basis of these
observations, it was concluded that no somatic cell division occurs during the
lifetime of Drosophila. To distinguish between these possibilities, a
series of three independent assays was used to test whether cell proliferation
can be detected in the adult midgut. In the first assay genetically marked
wild-type cell lineages were used to identify dividing cells. The production of
marked clones after mitotic recombination depends upon subsequent cell division
and is, therefore, a direct means to assay proliferation. In these experiments,
wild-type lineages were positively marked in adult flies using the MARCM system.
Mitotic recombination was induced by heat shock and green fluorescent protein
(GFP)-marked clones could be detected in the midgut. Similar results were
obtained when adults were heat shocked up to 10 days after eclosion. This
suggests that the ability to generate clones is not transient, and probably
persists throughout the entire life of the animal (Micchelli, 2006).
Under the experimental conditions used, the MARCM system produced some
background GFP signal that could be detected in control animals. To quantify the
background signal, the number of GFP-labelled cells was compared in control and
experimental animals. A greater than sixfold increase in the number of
GFP-labelled cells was detected after heat shock. A second independent clone
marking method was used that did not rely on either Gal4 or Gal80. In these
experiments, clones were marked by the loss of a ubiquitously expressed GFP and
similar results were observed. It is concluded that a population of actively
dividing somatic cells is present in the adult Drosophila midgut
(Micchelli, 2006).
To extend these findings, 5-bromodeoxyuridine (BrdU) incorporation studies
were constructed. Both large and small BrdU-labelled midgut cells were detected.
Large nuclei adjacent to each other can be differentially labelled, suggesting
asynchrony in the timing or extent of DNA synthesis over the course of the
labelling period. This is consistent with the notion that the large nuclei are
endoreplicating. However, both endoreplication and the canonical cell cycle
require new DNA synthesis. To distinguish endoreplicating from dividing cells in
the midgut the tissue was stained with an antibody raised against
phospho-histone H3. Careful examination revealed that very low levels of
phospho-histone H3 staining could be detected in all cells. However, double
staining with DAPI revealed that elevated levels of phospho-histone H3
indicative of mitosis could be detected only among the population of cells with
small nuclei. Thus, cells in the midgut seem to have two distinct cell cycles;
whereas both large and small nuclei undergo DNA synthesis, only the cells with
small nuclei undergo cell division (Micchelli, 2006).
In order to characterize further the small cell population, an expression
screen was conducted to identify cell-specific molecular markers. Three markers
expressed in small cells were identified: escargot (esg), a
transcription factor that belongs to the conserved Snail/Slug family;
prospero (pros), a conserved homodomain transcription factor, and
Su(H)GBE-lacZ, a transcriptional reporter of the Notch signalling.
Simultaneous detection of esg expression (esg-Gal4,
UAS-GFP), anti-Pros, Su(H)GBE-lacZ expression and DAPI
has demonstrated that small cells can be subdivided into the following classes on
the basis of differential gene expression: esg-positive
(esg+), pros-positive (pros+),
esg-negative pros-negative
(esg- pros-), esg-positive
Su(H)GBE-lacZ-positive
[esg+ Su(H)GBE-lacZ+] and
esg-positive Su(H)GBE-lacZ-negative
[esg+ Su(H)GBE-lacZ-].
esg+ and pros+ expression define distinct
cell populations, whereas Su(H)GBE-lacZ expression subdivides the
esg+ class into
esg+ Su(H)GBE-lacZ+ and
esg+ Su(H)GBE-lacZ- subpopulations.
Quantification reveals that each cell type is present in the midgut in different
proportions. The ability to distinguish different cell types using molecular
markers enabled determination of the cell lineage relationships in this tissue.
If the large and small nuclei are lineally distinct then marked clones should be
restricted to one or the other cell type. However, if a common stem cell
progenitor exists in the adult midgut, then marked lineages should contain both
large and small nuclei within a clone. To distinguish between these
possibilities positively marked MARCM clones were generated and nuclei were
labeled using DAPI. Lineage analysis shows that marked clones generated in the
adult contain both large and small nuclei. In addition, both esg
expression and anti-Pros-labelled cells could be detected within the clones.
These lineage-tracing experiments suggest that a stem cell progenitor exists and
is sufficient to generate the distinct cell types of the adult midgut. This cell
is referred to as the adult intestinal stem cell (ISC) (Micchelli, 2006).
esg expression in diploid cells has been shown to be necessary for the
maintenance of diploidy. In addition, the distribution of esg messenger
RNA has been used as a marker for male germline stem cells. Together, these
observations raise the hypothesis that esg expression may also mark a
population of progenitors in the midgut. It was therefore asked whether
esg expression correlates with markers of cell proliferation.
Simultaneous staining with anti-BrdU and DAPI reveals that esg-expressing
cells are among the population of cells that are also positively labelled by
BrdU. To ask whether esg-expressing cells also undergo cell division, the
midgut was double stained to detect both esg expression and
phospho-histone H3. High levels of phospho-histone H3 can be detected
specifically in esg-expressing cells. These results demonstrate that
esg expression marks a population of proliferating progenitor cells in
the midgut (Micchelli, 2006).
However, the esg+ cell population can be divided on the
basis of Su(H)GBE-lacZ expression. To distinguish functionally the two
esg+ populations, the consequences of altering Notch
signalling in the adult midgut were examined. The effect of globally reducing
Notch signalling was tested using the conditional Notch
temperature-sensitive (Nts) mutant.
Nts flies were first crossed to an allelic series that
included N55e11, N264.47,
Nts1 and Nnd.1. The strongest
loss of function combinations
(Nts/N55e11 and
Nts/N264.47) failed to
generate viable adult flies even at the permissive temperature, often dying as
pharate adults. Nts/Nts flies
produced viable adults at the permissive temperature with midguts similar to
wild type. Nts/Nts flies
shifted to the non-permissive temperature led to a mild increase in the number
of small cells. The weakest allelic combination,
Nts/Nnd.1, also produced
viable adults at the permissive temperature but showed no detectable phenotype
when shifted to the non-permissive temperature (Micchelli, 2006).
The requirement of N only in esg+ progenitor cells
was tested. To obtain both spatial and temporal control over transgene
expression in esg-expressing cells, the temperature-sensitive Gal80
inhibitor, Gal80ts was combined with the
esg-Gal4 transcriptional activator. To verify that the
Gal80ts transgene functions in the midgut, the temporal
and spatial induction of a UAS-GFP transgene was characterized. Adult
esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the
permissive temperature showed no detectable GFP expression in their midguts In
contrast, when these flies were shifted to the non-permissive temperature they
showed high levels of GFP expression that were detectable after 1 day and
maximal by 2 days (Micchelli, 2006).
The requirement of Notch was then tested in esg+ cells
using a UAS-NRNAi transgene, to reduce Notch
signalling. In control experiments, UAS-NRNAi;
esg-Gal4,UAS-GFP, tub-Gal80ts flies grown at the
permissive temperature appear to have wild-type midguts and show no detectable
GFP expression, suggesting that under these conditions UAS transgenes are
efficiently suppressed. In contrast, UAS-NRNAi;
esg-Gal4,UAS-GFP, tub-Gal80ts flies shifted to
the non-permissive temperature show an increase in the number of small cells (19
out of 20 midguts). Notably, the presence of esg-Gal4,
UAS-GFP in this experiment enabled a determination that the
increased number of small cells were also esg+. When these
guts were co-stained with anti-Pros antibody ectopic small cells were observed
that also expressed pros, and these cells were often associated with
lower levels of esg expression. Taken together these experiments suggest
that Notch signalling in esg+ cells is necessary to restrict
proliferation (Micchelli, 2006).
The effect of Notch activation was tested in esg+ cells
using Nintra, a constitutively active form of Notch. In
control experiments, esg-Gal4,UAS-GFP,
tub-Gal80ts; UAS-Nintra flies
grown at the permissive temperature appear to have wild-type midguts and show no detectable
GFP expression. In contrast, esg-Gal4,UAS-GFP,
tub-Gal80ts; UAS-Nintra flies
shifted to the non-permissive temperature showed a decrease in phospho-histone
H3 staining compared to controls that were not shifted. In addition, although
some esg+ cells appear to be wild type, a region-specific
decrease was observed in the levels of esg expression and a concomitant
increase in nuclear size similar to that of midgut epithelial cells. These
observations demonstrate that Notch activation is sufficient to limit
proliferation of esg+ cells and suggests that Notch may also
be sufficient to promote early steps of epithelial cell differentiation
(Micchelli, 2006).
This characterization of the adult Drosophila midgut suggests that a
population of adult stem cells resides within this tissue. This analysis of the
Notch signalling pathway in esg+ cells suggests that
esg+ Su(H)GBE-lacZ- cells mark a
population of dividing progenitors and that Notch is necessary and sufficient to
regulate proliferation. A model is proposed in which
esg+ Su(H)GBE-lacZ- progenitors
generate at least two different types of daughter cells depending on the level
of Notch activation. Under conditions of reduced Notch function an expansion of
both esg+ progenitor cells and pros+ cells
is observed. These observations suggest that esg+ cells give
rise to pros+ cells in a Notch-independent manner. Under
conditions of Notch activation a decrease is observed in the proliferation and
promotion of epithelial cell fate differentiation, while the number of
pros+ cells remains unaffected (Micchelli, 2006).
Several lines of evidence suggest that pros+ cells
correspond to gut enteroendocrine cells. Previous studies show that
prox1, the vertebrate pros homologue, is associated with
post-mitotic cells and early steps of differentiation in the central nervous
system. Furthermore, in Drosophila, pros is thought to be a
pan-neural selector gene that is both necessary and sufficient to terminate cell
proliferation. Finally, although vertebrate enteroendocrine cells arise from
endodermal origins they are known to express neural-specific markers. Therefore,
pros+ cells probably define a population of enteroendocrine
cells in the midgut (Micchelli, 2006).
Studies of stem cell compartments in Drosophila have led to the
characterization of two types of progenitor cells in the germ line. The first is
referred to as the germline stem cell and is sufficient to give rise to the
respective cells of either the male or female germ line. The second type of
progenitor cell described is called the cystoblast in female germ line and
gonialblast in the male germ line. Although the cystoblast and gonialblast both
have the capacity to generate the differentiated cells of their respective
tissues, they are thought to be more restricted in their fate than the germline
stem cells. On this basis, it is suggested that an analogous progenitor may also
exist in the adult Drosophila midgut; this cell is referred to as the
enteroblast (EB). The population of
esg+ Su(H)GBE-lacZ- progenitor cells,
which has been described, displays characteristics of both the ISC and the EB;
therefore, additional experiments will be necessary to distinguish unambiguously
these alternatives (Micchelli, 2006).
Escargot regulates tracheal branch fusion in Drosophila. During development of tubular networks such as the mammalian vascular system, the kidney
and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous
network. Little is known of the cellular mechanisms or molecular control of epithelial tube
fusion. A tracheal cell located at the
developing fusion point expresses a sequence of specific markers as it grows out and contacts a
similar cell from another tube; the two cells adhere and form an intercellular junction, and they
become doughnut-shaped cells with the lumen passing through them. The early fusion marker
Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy,
activating the expression of later fusion markers and repressing genes that promote branching.
Ectopic expression of escargot activates the fusion process and suppresses branching throughout
the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous
malformations in humans. This establishes a simple genetic system to study fusion of epithelial
tubes (Samakovlis, 1996).
The mutant phenotypes of cdc2 are similar to
those of escargot: many diploid cells in imaginal discs, salivary glands and the central
nervous system enter an endocycle, characterized by DNA replication without a subsequent mitotic phase. Such endocycling cells are often polytene, possessing thick chromosomes with DNA replicated many times over. When escargot function is eliminated, diploid imaginal
cells that were arrested in G2 lose Cyclin A, a regulatory subunit of G2/M cdk, and
entered endocycle. escargot genetically interacts with cdc2, suggesting an intimate biological interaction.
Since mitotically quiescent abdominal histoblasts still require cdc2 to remain
diploid, the inhibitory activity of Cdc2 on DNA replication appears to be
separable from its activity as the mitosis promoting factor. These results suggest that
in G2, escargot is required to maintain a high level of G2/M cdk, which actively inhibits
the entry into S phase. Expression of Cyclin A is lost in escargot mutants, suggesting that Cdc2 activity (dependent on its regulatory subunit Cyclin A), indirectly depends on escargot (Hayashi, 1996).
In some escargot mutants, abdominal histoblasts become polypoid. It has been suggested that one role of esg is to maintain diploidy of imaginal cells (Fuse, 1994 and Hayashi, 1993).
By the time of neuroblast delamination, Sna is present in most of the neuroblasts that have segregated from the ectoderm. Despite the extensive expression in the neuroblasts, prior to this study, Sna had no known function in the developing nervous system. The neuroblast pattern of sna resembles that of a group of genes called pan-neural genes. One of these genes, scratch (scrt), encodes a protein that has sequence similarity to Sna in
the zinc-finger domain. Mutations of scrt have no obvious phenotype except that viable escapers have morphological defects in the eyes. Furthermore, no nervous system defect can be seen in sna scrt double
mutants. However, the scrt dpn double mutants exhibit some defects in nervous system development. deadpan (dpn) is another
pan-neural gene that encodes a basic helix-loop-helix protein. Therefore, scrt does have a function in the central nervous system
(CNS), but the function of sna, if any, in the nervous system does not overlap with that of scrt (Ashraf, 1999 and references therein).
Escargot (Esg) is another protein that contains five zinc fingers with sequences highly homologous to those of Sna. The expression of esg is rather dynamic during embryonic development. The gene is
expressed in the epidermis, neuroectoderm and imaginal precursor cells. The Esg protein probably acts through the cdc2 kinase to
maintain the proper cell cycle in larval imaginal disc cells; in esg mutant larvae the imaginal disc cells lose their diploidy as
they re-enter the S phase without going through mitosis. Moreover, esg and sna are both expressed in the embryonic
wing imaginal disc primodia and the two genes have redundant functions in this tissue; the vestigial marker gene
expression in the disc is lost in esg sna double mutants. Despite a clear demonstration of the redundant requirements of
sna and esg in the wing disc, the double mutant has been reported to have no significant embryonic CNS phenotype. Thus, the function of sna in nervous system development has remained a mystery (Ashraf, 1999 and references therein).
Evidence is provided that CNS expression of Snail is required for nervous system development. The neural function of snail is masked by two closely linked genes,
escargot and worniu. worniu (pronounced war-niu, Chinese for 'snail') encodes a protein with a
zinc-finger domain highly homologous to those of Sna and Esg; it has been identified from the Berkeley Drosophila Genome Project database. RNA in situ
hybridization reveals extensive expression of worniu in the developing nervous system. wor is located between
esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Although not affecting expression of early neuroblast markers, the deletion of the region
containing all three genes correlates with loss of expression of CNS determinants including fushi tarazu, pdm-2 and
even-skipped. Transgenic expression of each of the three Snail family proteins efficiently rescues the fushi tarazu
defects, and partially rescues the pdm-2 and even-skipped CNS patterns. These results demonstrate that the Snail family
proteins have essential functions during embryonic CNS development, around the time of ganglion mother cell
formation (Ashraf, 1999).
The putative Wor protein sequence contains a C-terminal domain with six zinc fingers that are very similar to those of Sna and Esg, even though those proteins contain only five fingers. The N-terminal halves of these proteins have rather divergent sequences, except that they all contain a conserved basic motif very close to the N-termini. The function of this motif is not known. Moreover, the proteins contain two P-DLS-K motifs. The P-DLS-K domains in Sna have been shown to interact with the Drosophila C-terminal binding protein (dCtBP) and to play important roles in transcriptional repression. Since all three Sna family proteins contain highly homologous corepressor-interacting and DNA-binding domains, and can bind to similar DNA sequences, it is possible that they bind to promoters of overlapping sets of target genes and repress transcription (Ashraf, 1999 and references therein).
While there is no maternal RNA deposition of wor, zygotic expression can be detected first at the onset of neurogenesis. At a late stage 8, WOR transcript can be observed in two small patches of cells in the dorsal head region anterior to the cephalic furrow, representing precursor cells of the developing brain. At stage 9 wor expresses in the first wave of delaminating neuroblasts along either side of the midline, as well as in cells in the head region. Later in the germ band-extended embryo, most of the neuroblasts contain WOR mRNA. This pattern greatly resembles that of sna at this stage of development, except that sna expression in some of the centrally located neuroblasts in each hemisegment is at lower levels. In later stages, wor continues to express in the brain and part of the ventral nerve cord. No expression of wor is detected in any other embryonic tissue (Ashraf, 1999).
There is no extensive expression of esg in the neuroblasts similar to that shown for wor or sna. However, it has been demonstrated that esg RNA is expressed in the ventral neuroectoderm. Careful examination of the expression reveals that esg transcript is probably present in the CNS, albeit at variable levels. Based on the expression analyses, it is hypothesized that the newly identified wor might serve a redundant function with that of sna or esg during neural development. This would explain why neither single nor double mutants of sna and esg show severe defects in the nervous system (Ashraf, 1999).
To test the hypothesis that the Sna family proteins function redundantly in the developing nervous system, the neural phenotype associated with a deletion that uncovers all three genes was examined. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Advantage of the close proximity of these genes and the phenotypes of a deficiency mutant are examined. Since high levels of Sna and Wor are present in the neuroblasts, the expression of the proneural gene achaete, which marks a subset of early delaminating neuroblasts was examined. This expression is not affected in the osp29 deficiency mutants. The expression patterns of additional neuroblast markers including hunchback, dpn, scrt and lethal of scute also are similar in wild-type and mutant embryos. Therefore, the early waves of neuroblast delamination are normal in the absence of the Snail family proteins (Ashraf, 1999).
The CNS patterns of GMC markers ftz, pdm-2 and eve were examined. ftz is expressed in a number of midline precursor cells and extensively in GMC. In contrast to the neuroblast markers, the ftz expression is almost abolished in the mutant embryo. The pdm-2 gene is also expressed in some neuroblasts and GMC. The early neuroblast expression of pdm-2 in the mutant is nearly normal, while the expression in later staged embryos is highly defective. eve gene products are present in a number of GMC and postmitotic neurons during normal development. All the eve CNS expression is absent in homozygous osp29 deletion mutant embryos. Taken together, the deletion mutant that uncovers the three sna family genes shows severe defects in CNS development (Ashraf, 1999).
To confirm the function of these three proteins in neural development, transgenic rescue plasmids were constructed in which individual genes (esg, wor or sna) were placed under the control of a sna promoter, containing an enhancer element that directs expression in the neuroblasts. The transgenic flies obtained were then crossed with the osp29 strain and analyzed for CNS development. In the presence of any one of the three constructs the ftz expression is restored significantly. Analysis of the rescued pattern under higher magnification reveals that part of the ftz staining is clearly absent. However, more detailed analysis is required to pinpoint the exact cell lineages that are missing. Nevertheless, the results demonstrate that each of the three sna family genes can perform essential functions in the CNS in the absence of the other two.
The rescue by the transgenes of the expression of pdm-2 and eve, both of which are defective in the osp29 mutant, was also examined. While all three sna family genes clearly can rescue the expression of pdm-2 , the effect is not as extensive compared with that of ftz. For eve RNA, the transgenes rescue the expression in a significant number of cells when compared with the total loss of expression in the parental osp29 mutant. The rescue of eve, again, is not as extensive as that of ftz. Later stage CNS morphology in the rescued embryos was also monitored by BP102 staining. The embryos carrying the transgenes have slightly better overall CNS axonal morphology, but they are still highly abnormal when compared with the wild type (Ashraf, 1999).
Pairwise recombination of the transgenes were constructed and a test was performed to see whether they could achieve better rescue. By staining embryos obtained from stable lines that are homozygous for two transgenes, the constructs were found to give slightly direct the expression of ftz slightly better. Meanwhile, the eve and BP102 antigen expression in the presence of two transgenes reveals only minor improvement of the axonal morphology. These results suggest that the three proteins may have some collaborative function. It is also possible that the promoter used has some limitation in driving the rescue transgenes or that there are additional genes involved for the severe CNS phenotype (Ashraf, 1999).
Increasing numbers of sna-related genes have been identified in diverse species. These proteins have been assigned to the Sna family
based mostly on the similarity of the sequences in the zinc-finger domains. The expression patterns and some functional
studies of the vertebrate proteins suggest a role in regulating cell movement. However, gene
knock-out experiments have demonstrated that mutating a mouse Slug homolog does not lead to a detectable cell movement
defect. Such a result suggests a possible redundant function provided by other genes, similar
to this report. If the vertebrate homologs do have a function in controlling cell movement, it would be reminiscent of
the control of cell movement during gastrulation by Drosophila Sna. However, the expression of vertebrate Sna proteins in
developing CNS has not been demonstrated. A careful examination of the expression and function in the
CNS is needed to reveal the importance of Sna expression. The analysis of the functions of Sna, Esg and Wor in Drosophila CNS
development will certainly provide a foundation for similar analysis in other species (Ashraf, 1999 and references therein).
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, inscP49 and inscP72 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).
Suppressor mutations provide potentially powerful tools for examining mechanisms underlying neurological disorders and identifying novel targets for pharmacological intervention. Mutations are described that suppress seizures in a Drosophila model of human epilepsy. A screen utilizing the Drosophila easily shocked (eas) 'epilepsy' mutant identified dominant suppressors of seizure sensitivity. Among several mutations identified, neuronal escargot (esg) reduced eas seizures almost 90%. The esg gene encodes a member of the snail family of transcription factors. Whereas esg is normally expressed in a limited number of neurons during a defined period of nervous system development, the suppressor mutation caused normal esg to be expressed in all neurons and throughout development. This greatly ameliorates both the electrophysiological and the behavioral epilepsy phenotypes of eas. Neuronal esg appears to act as a general seizure suppressor in the Drosophila epilepsy model, since esg reduces the susceptibility of several seizure-prone mutants. esg must be ectopically expressed during nervous system development to reduce seizure susceptibility in adults. Furthermore, induction of esg in a small subset of neurons (interneurons) will reduce seizure susceptibility. A combination of microarray and computational analyses revealed 100 genes that represent possible targets of neuronal esg. It is anticipated that some of these genes may ultimately serve as targets for novel antiepileptic drugs (Hekmat-Scafe, 2005).
esg is defined as a seizure-suppressor gene on the basis of gain-of-function mutations that (1) revert the bang-sensitive behavioral phenotype associated with eas, sda, and bss/+ flies and (2) cause an increase in the seizure threshold of eas mutants. This conclusion is bolstered by the identification of five different esgEP alleles (all with independently derived P-element insertions) and one UAS-esg construct (located in a distinct cytological location) that all act as sda suppressors. On the basis of the lack of allele specificity of the esg-sda interaction, mutations of esg appear to be general seizure suppressors. This is expected since neither the esgEP alleles nor UAS-GAL4 would be expected to produce structurally altered gene products; suppression is presumably due to the ectopic expression of a structurally normal protein. It is expected that the five esgEP mutations identified in this study, as well as the UAS-esg insertion, show similar ectopic expression patterns under elav-GAL4 control and thereby produce seizure suppression in a similar gain-of-function manner (Hekmat-Scafe, 2005).
Neuronal induction of esgEP appears to reduce the fly's overall seizure susceptibility. This assertion is supported by the observation that wild-type, non-BS flies carrying elav-GAL4-activated esgEP display an increased seizure threshold. A general reduction in seizure sensitivity would also explain why neuronal esgEP suppresses a variety of BS mutations. Two of the BS mutations examined (eas and sda) encode very different products: eas is an ethanolamine kinase involved in synthesis of the phosphatidyl ethanolamine in neuronal membranes, and sda encodes an aminopeptidase. The third BS mutation is likely to encode yet another very different product. These three BS mutations may well reduce the fly's seizure threshold by different mechanisms. The elav-GAL4 activation of esgEP suppresses sda best of all; suppression of eas is intermediate and suppression of bss is the weakest. This is consistent with previous observations on general seizure suppressors that bss is the strongest of the three mutations (in terms of both its reduction of seizure threshold and the facility with which it can be suppressed by secondary mutations that reduce nervous system excitability) and sda is the weakest, with eas being intermediate (Hekmat-Scafe, 2005).
The gain-of-function esgEP mutations that act to suppress seizures cause no other obvious phenotypes whether in a wild-type or an eas background. Thus, esg mutant flies show no obvious nervous system excitability defects: they are not temperature-sensitive paralytics (hypoexcitability) and they do not shake their legs under ether anesthesia (hyperexcitability). Other behaviors also appear to be normal: flies groom, court, mate, jump, and fly. Flies that are eas+; esgEP2009; elav-GAL4 have a seizure threshold that is near the wild-type range (Hekmat-Scafe, 2005).
In the experiments presented in this study, the combined features of esgEP, elav-GAL4, and GeneSwitch (a conditional, RU486-dependent GAL4-progesterone fusion protein) begin to give a picture of seizure suppression. It is suggested that esgEP produces seizure suppression via its effect on immature postmitotic larval neurons that differentiate into interneurons of the adult CNS. It is further suggested that this could be due to cytoskeletal organization or reorganization that underlies the elaboration and strengthening of synaptic interconnections (Hekmat-Scafe, 2005).
Several other possible explanations for seizure suppression are not supported by the experiments presented here. For example, seizure suppression cannot occur by esgEP ameliorating some acute property of mature neurons in adults. Thus, esgEP suppression is probably not by manipulation of neurotransmitter metabolism, ion channel maintenance, or by other steady-state mechanisms used for maintaining or sustaining nervous system function or structure. This is because the bang-sensitive phenotype of eas is not suppressed by expression of esgEP in the adult nervous system as shown by the GeneSwitch experiment. Another alternative explanation is also not supported by the experiments presented in this study: esgEP-mediated seizure suppression must be unrelated to esg's normal role in facilitating neurogenesis or its role in polyploidization. This is because elav-GAL4 does not induce esgEP expression in either embryonic or larval neuroblasts. The GeneSwitch experiment also shows that embryonic expression of esgEP most likely does not account for its seizure suppression in adults. Furthermore, reducing the dosage of esg, which is normally expressed only in neuroblasts, has no effect on bang sensitivity (Hekmat-Scafe, 2005).
The GeneSwitch experiments show that seizure suppression in adult eas flies is apparently due primarily to esgEP induction in the larval stage. In larvae, there are four main classes of neurons, all of which should have esgEP expression driven by elav-GAL4:
Seizure suppression by esgEP could be due to its effects in several of these classes; however, the class of adult-specific neurons (class 4) is an especially attractive candidate. These interneurons are involved in integrating sensory signals such as those arising from mechanical 'bang' stimulation. Interneurons in the sensory system are numerous and are probably the neurons most greatly affected by electrical stimuli delivered to the brain by HFS. The observation that induction of esgEP in larval interneurons, but not motoneurons, produces adults with reduced seizure susceptibility is also consistent with the notion that esgEP is acting primarily in the class 4 neurons. Interneurons of class 4 undergo considerable development late in third instar lavae, but can also continue development after eclosion. This development may account for the continued progression of seizure suppression through days 2-3 of the adult stage (Hekmat-Scafe, 2005).
One possibility is that larval esg induction in developing interneurons affects their synaptic connections and thereby interferes with the spread of seizures. Class 3 larval neurons undergo both new outgrowth and pruning of their dendritic and axonal processes during metamorphosis, and arrested adult-specific neurons (class 4) begin to extend processes after pupariation. Since esgEP induced in larvae may well persist in pupae, it could affect the expression of genes whose products influence synapse formation or strengthening shortly after pupariation. Pupae do not take up the RU486, which might explain why the degree of suppression by esgEP is lower when activated by elav-GeneSwitch than when activated by elav-GAL4 (Hekmat-Scafe, 2005).
An intriguing possibility is that cytoskeletal elements are playing an important role in seizure sensitivity and resistance in Drosophila. Snail family transcription factors such as esg normally promote cell-cell separation during development, at least in part by inhibiting the expression of cadherin, a homophylic cell-cell adhesion molecule linked to components of the actin network. Filamin mutants are seizure sensitive in flies and humans with periventricular heterotopia; ß-integrin has been identified as a suppressor of eas; collagen type IV is a prominent target of aminopeptidase N (sda) in metastasis, and E-cadherin is downregulated by sna and potentially esg. Indeed, cytoskeletal reorganization is probably critical in morphological changes in dendritic spines associated with synaptic plasticit and epilepsy. Cytoskeletal organization and reorganization also plays a prominent role as scaffolding for proteins subserving membrane excitability. Thus, signaling efficiency, reliability, and stability appear to be greatly influenced by the subcellular colocalization of excitability proteins in the photoreceptor and the presynaptic terminal and at the postsynaptic membrane of the neuromuscular junction. Defects in this scaffolding function via abnormal cytoskeletal elements may contribute substantially to the kinds of excitability instability thought to underlie seizure disorders (Hekmat-Scafe, 2005).
The esg gene encodes a presumptive transcriptional repressor with restricted temporal and spatial expression in the nervous system. Presumably, ectopic expression of esg in larval interneurons mediates seizure suppression in adults via the activation or repression of one or more target genes. A combination of microarray and computational analyses revealed 100 genes that represent possible targets of neuronal esg. Although none of these genes encode cytoskeletal elements, a number of them encode serine protease inhibitors (induced by neuronal esg) or serine proteases (repressed by neuronal esg) that could act on cytoskeletal elements or other proteins that influence synaptic interconnections. Further experiments will be needed to determine which of the 100 genes actually mediates seizure suppression. Such a gene could ultimately serve as target for novel antiepileptic drugs (Hekmat-Scafe, 2005).
Isolation and examination of suppressor mutations is a potentially powerful approach to seizure disorders. It allows the identification of biological processes not previously associated with seizures through genes such as esg. In addition, esg has several properties that make it attractive as a candidate for new AED development: it reduces seizure susceptibility without apparent side effects. While it appears to be effective only during a window of time during neuronal development, this window may serve as an advantage in treating cases in which epilepsy develops early. Nevertheless, esg, or a seizure-suppressor gene with similar properties identified in this or future screens, may allow the development of powerful new treatments for the devastating effects of intractable epilepsy (Hekmat-Scafe, 2005).
Stem cells are found in specialized microenvironments, or 'niches', which
regulate stem cell identity and behavior. The adult testis and ovary in
Drosophila contain germline stem cells (GSCs) with well-defined niches, and are
excellent models for studying niche development. This study investigates the
formation of the testis GSC niche, or 'hub', during the late stages of
embryogenesis. By morphological and molecular criteria, the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) were identified and followed in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad (Le Bras, 2006).
The evidence indicates that an embryonic hub, which appears to give rise to
the adult hub and create the male GSC niche, forms during the late stages of
embryogenesis. A subset of anterior SGPs initiates expression of several
molecular markers that are also expressed in the adult hub. These SGPs segregate
into a tight cluster in a distinct region of the gonad, and a subset of germ
cells organizes around these SGPs in a manner similar to the organization of
GSCs around the adult hub. Since spermatogenesis begins by early larval stages,
it is possible that
the embryonic hub already forms a functional GSC niche. The formation of the
hub, or indeed any stem cell niche, can be divided into the distinct issues of
niche cell identity, niche morphogenesis, and stem cell recruitment (Le Bras, 2006).
The data indicate that the specification of hub cell identity occurs in two stages. During the first stage, some SGPs acquire an anterior identity that is sexually dimorphic, as indicated by the male-specific expression of esg and upd. Anterior SGP identity is positively regulated by abd-A, and is repressed by Abd-B, while sexual identity is regulated by tra and dsx. During the second stage of hub cell specification, a subset of these anterior SGPs acquires hub cell identity during stage 17 of embryogenesis. Only some anterior SGPs maintain esg expression, and the control of late gene expression in the hub appears to be distinct from early expression in anterior SGPs, since some esg and upd enhancer traps only exhibit gonad expression in the hub at this later stage. Furthermore, cells that maintain esg expression
during stage 17 also express every other marker of adult hub identity tested,
including Fasciclin 3, cdi, DN-cadherin and DE-cadherin. It is concluded that
these cells are specified as hub cells at this time. The fate of the anterior
SGPs that lose esg expression and do not form part of the hub is unknown.
An intriguing possibility is that these cells could form another important
somatic cell type: the cyst progenitor cells (somatic stem cells) that associate
with the hub along with the GSCs (Le Bras, 2006).
Based on its expression pattern, the transcription factor esg would seem to be an excellent candidate for specifying hub cell identity. However, no changes were observed in the expression of other hub markers in esg null mutants; this includes expression of DE-cadherin, which is known to
be regulated by esg in other tissues. It has been reported, however,
that esg is required for hub maintenance, and that the hub is severely
defective at later stages in esg mutants that survive embryogenesis.
Thus, esg is critical for
the male GSC niche, but is either not important for the initial formation of
this structure, or acts redundantly with another factor (Le Bras, 2006).
It has been possible to follow the
morphogenesis of the hub from the time of gonad formation until the embryonic
hub is fully formed. At the time of gonad coalescence, anterior SGPs interact
with other SGPs, and with the germ cells, in a manner that is indistinguishable
from posterior SGPs. However, during stage 17, the hub cells undergo dramatic changes in
their relationship to other SGPs and germ cells. Hub cells segregate away from
other SGPs to one pole of the gonad, and coalesce tightly with one another.
In addition, hub cells do not ensheath the germ cells
at this stage. Instead, a defined
interface between hub cells and germ cells forms which is labeled by DE- and
DN-cadherin, but not Fasciclin 3. Thus, hub cells
appear to maximize their interactions with one another, and minimize their
interactions with other cells in the gonad, although they clearly still contact
a subset of germ cells (Le Bras, 2006).
It is apparent that the changes in cell–cell
contact and morphology that occur during hub formation require changes in cell
adhesion. Indeed, characteristic changes have been found in expression of the
homophilic adhesion molecules Fasciclin 3, DN-cadherin and DE-cadherin occur
during hub formation; all three are significantly upregulated in the embryonic
and adult hub. Increased homophilic adhesion among hub cells could account for
their ability to maximize their contacts with one another, and sort away from
other SGPs. However, no changes were observed in embryonic hub formation
in mutants for these cell adhesion molecules.
Thus, these proteins, and possibly others, may act redundantly in
this process (Le Bras, 2006).
It is clear that a subset of germ
cells organizes specifically with the developing hub as it forms. During the
last stage of hub formation, germ cells become oriented in a rosette
distribution around the developing hub in a manner characteristic of GSCs in the
adult. These may
represent the subset of germ cells that will become GSCs. The presence of DE-
and DN-cadherin at sites of hub–germ cell contact suggests that
cadherin-mediated adhesion may be important for niche–GSC interaction in
the testis, as has been observed in the ovary. Interestingly, germ cells are not required
for hub formation. Analysis of a number of hub identity markers indicates that these
cell form normally from a subset of anterior SGPs in embryos that lack germ
cells. The hub does not appear as well compacted in
these embryos, consistent with observations of the adult hub,
indicating that hub–germ cell contact (or hub–germ cell signaling)
affects the final shape of the hub. Nevertheless, the GSC niche can form in the
absence of one of its stem cell populations (somatic stem cells may still be
present). It will be of great interest in the future to determine if the subset
of germ cells organized around the male embryonic hub are, indeed, developing
GSCs, and to study how their transition to stem cell identity might be regulated
by the niche (Le Bras, 2006).
The formation of the male GSC niche is a sex-specific
characteristic of anterior SGPs. Male-specific expression of esg and hub
formation both require the sex determination genes tra and dsx.
In some tissues, DSXM is required to
promote male development and repress female development, while the opposite is
true for DSXF. Interestingly, it was found
that embryonic hub development is entirely masculinized in dsx null
mutants; XX and XY individuals appear identical when mutant for dsx and
both resemble wild type males. Thus, no role is seen for DSXM in
promoting embryonic hub formation, while DSXF is required in females
to repress hub formation. Since esg is expressed male-specifically, it is
one candidate for being directly regulated by DSX (Le Bras, 2006).
We can compare the development of the anterior SGPs and hub with the development of another sexually dimorphic cell type, the msSGPs that join the posterior of the male gonad. First of all, these two cell types are distinct and do not depend on one another for their proper development. The hub still forms in Abd-B mutants that lack msSGPs, while msSGPs are still found in the gonad in Pc mutants, in which no anterior SGPs or hub cells form. Second, the
mechanism for how sexual dimorphism is created differs between the two cell
types. msSGPs are present only in males because they have undergone sex-specific
apoptosis in females. In contrast, no apoptosis was observed in anterior SGPs.
These cells appear to remain present in both sexes,
but only form a hub in males. Thus, although the sex determination genes
tra and dsx regulate sex-specific development of both cell types,
the cellular mechanisms employed are different. Finally, as was observed for the
hub, development of the msSGPs is completely masculinized in dsx mutant
embryos. Thus,
for both of these cell types, the male pattern of development in the embryonic
gonad is the default state in the absence of dsx function, and it is the
role of DSXF to repress male development in females. However,
DSXM may well play a role in development of one or both of these
gonad cell types at later stages, since proper testis development in males
clearly requires dsx (Le Bras, 2006).
The sex determination pathway must also ensure that GSC niches form
in females and are different from those in males. Recently, it has been shown
that germ cells populating the anterior of the gonad in female embryos are
predisposed to become GSCs in the adult ovary, while germ cells populating the
posterior rarely become GSCs. This suggests that anterior SGPs in the female embryonic gonad
may promote GSC identity, similar to what is proposed to happen in the male during
hub formation. One possibility is that anterior SGPs give rise to GSC niches in
both sexes, while genes such as tra and dsx control whether these
niches will be male or female (Le Bras, 2006).
In conclusion, the development has been followed of the embryonic hub, which may represent the nascent GSC niche for the testis. This work provides a basis for further understanding the mechanisms controlling niche formation and GSC recruitment in Drosophila, and determining if these mechanisms are conserved in other
stem cell systems, including the GSC niche of the mammalian testis (Le Bras, 2006).
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escargot:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 10 April 2008
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