teashirt
Transcripts are initially detected at the beginning of the 14th nuclear division in a central ring of cells spanning 45-60% egg length (where 0% is the posterior pole). Three segments-worth of cells are present with three bands resolving in the abdominal region. By the extended germband stage [Images], cells specific to parasegments 3-13 express tsh, forming a striped pattern. At the posterior tip of older embryos, a small group of cells exhibit tsh expression, corresponding to the future anal opening. Expression of tsh can be detected in internal tissues, the trunk region of the CNS, part of the visceral mesoderm and the somatic musculature (Fasano, 1991 and Mathies, 1994).
The teashirt gene has an homeotic function which, in combination
with HOM-C genes, determines thoracic and abdominal (trunk) identities. Analysis of
Tsh protein distribution during embryogenesis using a specific polyclonal antibody
shows that it is nuclear. The protein is present with regional modulation in several
tissues within the trunk, suggesting additional tsh functions to those already studied. During gastrulation, Tsh protein is first detected at a low level in a domain located in the central part of the embryo. A transient pattern of five stripes with variable width and spacing is observed. During early germ band elongation (stage 7), the striped pattern disappears and the whole trunk region of the elongateing germ band becomes stained, although more protein is detected in the thoracic than in the abdominal segments. After germ band shortening is completed, one row of cells is more intensively stained in the anterior part of every thoracic and abdominal segment. At this stage, a longitudinal line of labelled cells can be detected at the junction between the amnioserosa and the segmented ectoderm of the trunk. During late stages 14 and 15, these labelled cells provide a good marker to follow dorsal closure and heart morphogenesis. At these stages and later, labelling is also detected in a small group of cells corresponding to the anal tuft, and in three contiguous rows of cells in the eighth abdominal segment (Alexandre, 1996).
The Drosophila visceral mesoderm (VM) is a favorite system for studying the regulation of target genes by Hox proteins. The VM is formed by cells from only the anterior subdivision of each mesodermal parasegment (PS). The VM itself acquires modular anterior-posterior subdivisions similar to those found in the ectoderm. Mesodemal cells located just under the engrailed-expressing cells in the posterior ectodermal compartment have been called the mesodermal "P domain." The dorsal-most cells of the mesodermal P domain in each PS express the homeobox gene bagpipe (bap); they detach from the mesodermal fold and move inward toward the center of the embryo. These bap-expressing cells form the VM progenitor groups. The VM subdivisions, and the metameric expression of Connectin, form in response to ectodermal production of secreted signals encoded by the segment polarity genes hedgehog and wingless and are independent of Hox gene activity. A cascade of induction from ectoderm to mesoderm to endoderm thus subdivides the gut tissues along the A-P axis. Induction of VM subdivisions may converge with Hox-mediated information to refine spatial patterning in the VM. Con patches align with ectodermal engrailed stripes, so the VM subdivisions correspond to PS 2-12 boundaries in the VM. The PS boundaries demarcated by Connectin in the VM can be used to map expression domains of Hox genes and their targets with high resolution. The resultant map suggests a model for the origins of VM-specific Hox expression in which Hox domains clonally
inherited from blastoderm ancestors are modified by diffusible signals acting on VM-specific
enhancers (Bilder, 1998).
Since Con expression marks the imprint of ectodermal PS boundaries on the VM, Con patches can be used to precisely map the domains of Hox gene transcription in relation to Con patches. teashirt is expressed in two domains. The anterior midgut domain extends from visceral mesoderm segment (VS) 4 to mid-VS 6, where it shares a posterior boundary with Antennapedia; the central midgut domain extends several cells to either side of the VS 8 boundary. dpp is also expressed in two domains: at the gastric caeca, it is found in the A domain of VS2 and the P domain of VS 3, while in the central midgut it extends from the A domain of VS 6 to terminate just anterior to the VS 8 boundary. wg is expressed just anterior to the VS 8 boundary, with some cells after stage 12 lying in VS 8. pnt is expressed throughout VS 8, although expression is not seen until early stage 13. At stage 13, the two domains of odd paired (opa) expression extend from the P domain of VS 4 to the VS 6 boundary and from VS 9 through VS 11 (Bilder, 1998).
In the ventral cord, the protein is abundantly expressed, with higher levels in the three thoracic segments. In the gut of stage 13 embryos, the visceral mesoderm surrounding the midgut in posterior parasegments 4, 5 and 8 is strongly stained, as well as that for parasegment 6 (with weaker intensity). At stage 16, the protein is present in the first and second midgut constrictions and endodermal labelling is clearly visible. The Tsh protein appears to be differentially located in the primordia of leg imaginal discs, where it seems to be present in the proximal, but not in the distal part of the disc primordium (Alexandre, 1996).
Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, attempts were made to identify genes that underlie the specification of all appendage primordia. Evidence is presented that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation (Weihe, 2004).
Evidence suggests that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal
discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna, and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do
so, when examined in detail (Weihe, 2004).
Dll expression is required for the formation of all leg and antenna
elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc.
Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh. Thus an
essential mediator of the effects of Wg and Dpp was missing. The current results present evidence that El and Noc serve this function, since their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so it is concluded that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc (Weihe, 2004).
The situation differs slightly in the wing. Repression of Tsh is the
earliest marker for specification of the distal wing region,
preceding the onset of Hth repression or of Nub induction. Loss of Tsh
and Hth are required to allow Nub expression. Ectopic expression
of Hth and Tsh and loss of Nub is observed in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll).
The vestigial gene is also important for wing development and has
been proposed to be a wing specifying gene. However, Vestigial is expressed all along the DV boundary of the wing, both in
the wing primordium and in the body wall. This led to the suggestion that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages. For this reason analysis of the relationship between El, Noc and Vestigial was not performed in this study (Weihe, 2004).
Is the repression of trunk genes needed to specify appendage, as opposed to body wall, in wing and leg discs? In the wing disc the answer appears to be yes; repression of 'trunk genes' like hth is necessary to make the
remaining part of the disc competent to form the appendage. However, in the leg the situation is more complex. Coexpression of Dll and Hth does not disrupt proximal-distal axis formation, but leads to homeotic transformation of leg tissue into antennal tissue. Hth is not repressed and limited to proximal areas in the
antenna. However, loss of el and noc activities in the leg
disc leads to loss of distal leg tissue without any evident transformation
into antennal tissue. Thus, El and Noc may regulate the expression of other 'trunk genes', whose restricted expression is required to make the remaining
leg and antenna disc competent to form the appendage (Weihe, 2004).
The regional requirements for El and Noc highlight another interesting
difference between leg and wing disc development. el noc double
mutant cells are excluded from contributing to the tarsal region of the leg but not from contributing to the femur and tibia. Lineage
tracing has shown a considerable net flux of cells from the proximal
(Tsh-expressing domain) into femur and tibia. While there is no boundary of lineage restriction separating these domains,
cells must be able to change from expressing the proximal marker Hth to
expressing the distal marker Dll in order to move from one territory to the other. The wing in contrast does not appear to normally exhibit this large net flux of cells from proximal to distal and the el noc double mutant cells are excluded from contributing to
the entire wing region. Clonal analysis has suggested that el noc
double mutant cells attempt to sort out toward proximal territory, or if that fails, they can be lost from the disc, apparently by sorting out perpendicular to the epithelium. These observations suggest that El and Noc activity may contribute to the production of proximal-distal differences in cell affinities and thereby may help to maintain segregation of these cell populations during development (Weihe, 2004).
Patterning of the developing limbs by the secreted signaling
proteins Wingless, Hedgehog and Dpp takes place while the
imaginal discs are growing rapidly. Cells born in regions of
high ligand concentration may be displaced through
growth to regions of lower ligand concentration. A novel lineage-tagging method was used to address the
reversibility of cell fate specification by morphogen
gradients. Responses to Hedgehog and Dpp in
the wing disc are readily reversible. In the leg,
cells readily adopt more distal fates, but do not normally
shift from distal to proximal fate. However, they can do so
if given a growth advantage. These results indicate that cell
fate specification by morphogen gradients remains largely
reversible so long as the imaginal discs are growing. In other systems,
where growth and patterning are uncoupled, nonreversible
specification events or ‘ratchet’ effects may be of functional
significance (Weigmann, 1999).
In the developing leg disc, some responses to
Wg and Dpp are readily reversible, while others are not.
Lineage tracing of cells born in the TshGAL4 domain (proximal) suggests
that cells readily lose Tsh (and Hth) expression and instead
express Dll and Dac (distal markers). In young discs, a small
proportion of cells expressing low levels of both Dll and Tsh
are found at the edge between these domains. It is possible that these
are cells in transition between the domains. These results
suggest that cells born in the presumptive body wall readily
contribute to formation of more distal leg regions. Under
normal circumstances cells born in the Dll-expressing distal
domain of the leg do not contribute to the body wall. However,
they are not prohibited from doing so when
given a strong growth advantage.
The progeny of Dll-expressing cells in second instar are
mostly fated to give rise to the tarsus and do not contribute to
femur. In contrast, femur, tibia and tarsal
segments (the distal segments) derive from cells that have expressed Dll in early
third instar. The difference between these stages suggests that
new cells must be induced to turn on Dll in order to provide
the population of cells that contribute to the femur (the most proximal of the distal segments). These cells
must derive from the Tsh domain in second instar and acquire
Dll and Dac expression. At later stages,
Dll is expressed at very low levels in the femur, where it may
be repressed by Dac. Downregulation of Dll expression in the
femur is unlikely to be a direct response to a lowering of Wg
and Dpp signaling, because clones of cells unable to respond
to these signals do not show abnormal Dll or Dac expression. This contrasts with the situation in
the wing where removal of Dpp signaling leads to loss of Spalt
expression. The low
level of Dll expression in the femur is in part due to Dac
activity, since dac mutant clones show elevated levels of Dll. The transient induction of Dll in the
precursors of the femur is consistent with genetic analyses
showing that formation of all leg segments except coxa
depends on Dll activity in early development, whereas the low
level of Dll expressed later is apparently not required for
normal femur development (Weigmann, 1999).
When the distal part of a leg imaginal disc, or of an
amphibian or a cockroach leg is removed, distal structures will
regenerate from the cut edge. If the distal part of the leg disc
is cultured in isolation, distal structures will regenerate from the cut
edge, leading to a duplication of the fragment. The fact that
distal structures regenerate but proximal structures do not has
been termed distal transformation. These classical
experiments have shown that cells have a general tendency to
distalize, whereas their capability to proximalize is restricted.
The current experiments show that distal transformation happens
during normal development. Some proximal cells switch their
pattern of gene expression as the disc grows and they acquire distal
fate. Distal cells do not normally switch to proximal fate, but
can do so if forced during early development. The classical regeneration
studies suggest that the ability to shift from distal to proximal
fate may be lost as development proceeds (Weigmann, 1999).
The teashirt (tsh) gene has dorso-ventral (DV) asymmetric functions in Drosophila eye development: promoting eye development in dorsal and suppressing eye development in ventral regions by Wingless mediated Homothorax (HTH) induction. A search was carried out for DV spatial cues required by tsh for its asymmetric functions. The dorsal Iroquois-Complex (Iro-C) genes and Delta (Dl) are required and sufficient for the tsh dorsal functions. The ventral Serrate (Ser), but not fringe (fng) or Lobe (L), is required and sufficient for the tsh ventral function. It is proposed that DV asymmetric function of tsh represents a novel tier of DV pattern regulation, which takes place after the spatial expression patterns of early DV patterning genes are established in the eye (Singh, 2004).
The three genes of the Iro-C (ara, caup and mirr) are expressed in the dorsal domain of the eye disc and are functionally redundant. Misexpression of ara (driven by bi-GAL4 and abbreviated as bi>ara) results in eye suppression on both dorsal and ventral margins. However, coexpression of tsh and ara in bi>tsh+ara results in overall enlargement of the eye. Clonal induction of ara (abbreviated as Act>ara) and coexpression of tsh and ara (Act>tsh+ara) gives the same results. Thus, ara provides the dorsal cue for tsh to induce eye enlargement on both margins (Singh, 2004).
The requirement for ara was further confirmed by misexpressing tsh (bi>tsh) in Df(3L) iroDFM3/+ background. This deficiency uncovers ara, caup and the promoter region of mirr. In this background, bi>tsh suppresses eye development in both ventral and dorsal. Thus, when the Iro-C dosage is reduced, the dorsal function of tsh can be reversed to its ventral function. These results suggest that the dorsal function of tsh is dependent on the Iro-C genes (Singh, 2004).
Dl is expressed preferentially in the dorsal eye. Misexpression of Dl anterior to morphogenetic furrow in the hairy domain (hairy>Dl) accelerates photoreceptor differentiation but does not result in eye enlargement. bi>Dl (bifid-Gal4, UAS Delta) does not affect eye size. However, coexpression of tsh with Dl (bi>tsh+Dl) results in eye enlargements on both dorsal and ventral margins. Act>tsh+Dl clones in both dorsal- and ventral-eye also causes enlargements. These results suggest that Dl can provide the dorsal cue for tsh function (Singh, 2004).
Dl function was blocked by a dominant-negative form of DL, DLDN. bi>DlDN causes reduction of eye on both dorsal and ventral margins whereas coexpression of tsh and DlDN (bi>tsh+DlDN) further enhances this phenotype. A dorsal Act>tsh+DlDN clone suppresses eye development. Act>tsh+DlDN clones also non-autonomously suppress eye development, a phenotype also seen in Act>DlDN clones. These phenotypes suggest that Dl is also required for the dorsal function of tsh in eye. In the absence of Dl, tsh exerts its ventral function in dorsal eye (Singh, 2004).
Ser is preferentially enriched in the ventral eye until late second instar of larval development. Misexpression of Ser (bi>Ser) does not suppress eye development whereas coexpression of tsh+Ser(bi>tsh+Ser) suppresses eye development on both dorsal and ventral margins. Despite the suppression of photoreceptor differentiation, bi>tsh+Ser eye disc shows overall enlargement. The adult eyes were also enlarged and folded despite the suppression of photoreceptors differentiation on the dorsal and ventral margins. These results suggest that Ser can provide the ventral cue for the eye suppression function of tsh but does not affect its early function in promoting growth (Singh, 2004).
The dominant-negative form of Ser, SerDN was used to block Ser function. In bi>SerDN, the eyes are suppressed on both dorsal and ventral margins. This phenotype is partially blocked in bi>tsh+SerDN eye. Similar results were observed in Act>tsh+SerDN clones. Thus, tsh requires Ser for its ventral function (Singh, 2004).
These results show that tsh requires several early DV eye patterning genes for its dorsal and ventral specific functions in the eye. The requirement for these DV patterning genes is specific, because not all the DV patterning genes have similar effects. Eye suppression by tsh is prevented in the dorsal eye region. This function requires the normal dosage of both Iro-C and Dl genes, because the reduction of either Iro-C or Dl allows tsh to suppress eye development even in the dorsal eye. However, when ectopically expressed in the ventral eye, either Iro-C genes or Dl can block the ventral function of tsh, suggesting that the two genes may play similar roles (Singh, 2004).
The genes involved in early DV eye patterning can be categorized in two classes: (1) genes that are preferentially expressed in dorsal (e.g., Iro-C, Dl) or ventral (e.g. Ser, fng) and (2) genes that are uniformly expressed but function only in one domain (e.g., L). It is proposed that tsh comprises a new class of genes, which is expressed symmetrically but perform asymmetric functions in dorsal and ventral eye (Singh, 2004).
Although tsh is expressed ubiquitously in the early eye disc, the DV asymmetric functions of tsh can be uncovered only after the expression of early DV patterning genes is established. These results suggest that early expression of tsh may be responsible for its growth function only, whereas for the DV asymmetric functions the expression of early DV patterning genes is a prerequisite. Therefore, TSH function in eye represents a new tier of DV pattern regulation, which functions in interpreting the DV spatial cues in eyes. It would thus be interesting to identify other members of this class. Interestingly, two orthologs of tsh have been identified in mouse but their function in eye is not yet known. Since there is evolutionary conservation in patterns of gene expression and functions, it would be interesting to look for the role of tsh during eye development in higher organisms (Singh, 2004).
In insects, selector genes are thought to modify the development of a
default, or 'ground state', appendage into a tagma-specific appendage such as
a mouthpart, antenna or leg. In the best described example, Drosophila
melanogaster, the primary determination of leg identity is thought to
result from regulatory interactions between the Hox genes and the
antennal-specifying gene homothorax. Based on RNA-interference, a
functional analysis of the Teashirt family selector gene tiptop (see Drosophila
tiptop) and the Hox gene
Antennapedia in Oncopeltus fasciatus embryogenesis is
presented. It is shown that, in O. fasciatus, tiptop is required for
the segmentation of distal leg segments and is required to specify the
identity of the leg. The distal portions of legs with reduced tiptop
develop like antennae. Thus, tiptop can act as a regulatory switch
that chooses between antennal and leg identity. By contrast,
Antennapedia does not act as a switch between leg and antennal
identity. This observation suggests a significant difference in the mechanism
of leg specification between O. fasciatus and D.
melanogaster. These observations also suggest a significant plasticity in
the mechanism of leg specification during insect evolution that is greater
than would have been expected based on strictly morphological or molecular
comparisons. Finally, it is proposed that a tiptop-like activity is a
likely component of an ancestral leg specification mechanism. Incorporating a
tiptop-like activity into a model of the leg-specification mechanism
explains several mutant phenotypes, previously described in D.
melanogaster, and suggests a mechanism for the evolution of legs from a
ground state (Herke, 2005).
To investigate the variation and evolution of mechanisms of insect
appendage formation, the role of selector genes in the formation
of embryonic appendages of the milkweed bug O. fasciatus was examined. There are several advantages
in using this species to study leg development and evolution. (1) As a
hemimetabolous insect, O. fasciatus first instars have fully formed
legs (i.e. all segments are present). Thus, the entire process of leg
formation is readily apparent in the embryo and is not stretched out over the
process of imaginal disc formation and metamorphosis as it is in
holometabolous insects such as D. melanogaster. (2) O.
fasciatus is positioned more basally on the insect phylogeny than any
other insect for which the RNA-I technique has been successful in assaying
gene function. (3) Because O. fasciatus is more distantly related
to D. melanogaster than the more common genetically tractable model
insects (e.g., the holometabolous insects Tribolium castaneum and
Bombyx mori), there is greater potential for uncovering regulatory
variation and perhaps gaining greater insight into the evolution of the leg
specification mechanism. (4) Because O. fasciatus is generally
less derived and has an ancestral leg composition, it may also have conserved
the ancestral mechanisms of leg development allowing the direction of
evolutionary change to be inferred (Herke, 2005).
From O. fasciatus embryos, partial cDNAs were cloned that represent
a homolog of the D. melanogaster gene tiptop. tiptop is a
member of the tsh-family that is typified by zinc-finger motifs and is
presumed to be a transcription factor. Also, while present in the D.
melanogaster genome, this gene was identified solely on the basis of
molecular data. No mutations have been reported in tiptop, and its
developmental function has not been previously reported for any arthropod (Herke, 2005).
tsh-family genes have been cloned by different methods from at least
five insect species and, with the exception of D. melanogaster tsh,
all appear to have greater similarity to tiptop. Through extensive
PCR on genomic DNA and cDNA, both tiptop and
tsh were recovered from D. melanogaster, but only a
single gene was recovered from O. fasciatus or other insects. Also, a gene tree
constructed using PAUP* for the
tsh-family genes shows that the two D. melanogaster genes
cluster together. The gene tree and the absence of a tsh gene in the
other insects surveyed suggest that the two tsh-family genes in D.
melanogaster result from a recent duplication of an ancestral gene.
This ancestral gene has been called tiptop because of its greater similarity to
that gene and not to imply a closer evolutionary relationship of the ancestral
gene to either the D. melanogaster tsh or tiptop (Herke, 2005).
tsh has a variety of developmental functions in the cuticle of the
Drosophila larva and adult. Specifically, tsh is thought to
specify trunk identity in the larva through interactions with Hox genes, is
required for the formation of proximal regions of appendages in adults, and plays
a role in restricting the development of the adult eye. There is
little similarity between any of these activities of the Drosophila
tsh gene and O. fasciatus tiptop. Thus, these activities appear
to have been acquired by the tsh-family relatively recently.
Significant to the discussion here is that the function tsh has in
the formation of the proximal region of the leg in D. melanogaster
cannot be provided by tsh in O. fasciatus because the gene
is not present. These roles may be provided by other proximally expressed
genes such as hth and exd (Herke, 2005).
A model of the leg specification mechanisms in D. melanogaster and
the proposed differences from O. fasciatus is presented. In contrast to D.
melanogaster, where loss of Hox (Antp, Scr, Ubx) function
produces dramatic transformations of leg to antenna, no
transformation toward antennae of Antp-phenocopy legs is detected that could be
interpreted as an expansion of hth activity. Thus, although it is
possible that some residual Antp function remains in these animals, it is
suggested that it is tiptop and not Antp that represses the
activity of hth (or other antennal specifier) in the O.
fasciatus leg. A role for Antp in the segmentation of the distal
region is absent in D. melanogaster while its role in medial
segmentation is conserved between the two insects. Also, given that neither
tiptop nor the Hox genes act as specifiers of proximal identity (leg
vs. antenna) in the leg specification mechanism of milkweed bugs, additional
undetermined genes are implicated. This further distances the mechanism of
appendage specification in milkweed bugs from the relatively simple two-gene
(Hox, hth) system evident in D. melanogaster (Herke, 2005).
A tiptop-like activity is also evident in D.
melanogaster. This is illustrated most convincingly by the persistent
pretarsi formed on legs that are otherwise transformed to antennae in the
absence of Antp activity. Also, the leg-like appendage (composed primarily of tarsi
and pretarsi) produced by hth Antp null clones in D.
melanogaster is what might be predicted if an independent
tiptop-like activity for distal segmentation and specification
remained active in these appendages. Genetic analysis of Drosophila
tiptop has not revealed a role in distal specification or segmentation of
the adult leg (Laurent Fasano, personal communication to Herke, 2005). However, due to the
technical difficulties of determining the role embryonic gene activities have
in adult structures in D. melanogaster, it has not been possible to
rule out that embryonic activities of either tiptop or tsh
affect the adult leg. Thus, it remains a possibility that a
tiptop-like activity could be provided by tiptop or
tsh, as well as by other genes in D. melanogaster (Herke, 2005).
Interestingly, the defects induced by reduced Antp activity in
O. fasciatus are in striking contrast to the transformations of
mouthparts to antennae seen when Scr and Dfd activity are
reduced. These latter transformations have been used as evidence for a universal
mechanism of Hox specification of insect appendages.
However, in O. fasciatus, Scr and Dfd apparently repress the
activity of antennal specification in gnathal appendages while Antp does
not repress this activity in thoracic appendages. Thus, in O.
fasciatus, two mechanisms (one Hox-dependent and one Hox-independent) exist for
specifying the identity of appendages. Additional factors (including
tiptop) must mediate the differences in the active mechanisms in
these tagma (Herke, 2005).
It is possible to describe the genetic changes required for the O.
fasciatus mechanism of leg development to evolve into that of D.
melanogaster. (1) Antp acquired the ability to repress the
antennal specifier (hth) in the distal leg and lost its role in
distal segmentation. These changes might have been relatively simple. A
mechanism for Hox genes (e.g. Scr and Dfd) to repress the
antennal specifier already existed and the segmentation functions of
Antp might be partially provided by tiptop. (2) The
change in Antp function relaxed the constraints on tiptop,
thereby allowing its function (including hth repression) to diverge.
(3) Duplication and further divergence of the ancestral tiptop
gene produced the tsh and tiptop genes of D.
melanogaster (Herke, 2005).
Revertants of embryonic lethal mutants show normal head and tail regions but disrupted trunk regions. Monoclonal antibody to teashirt ventral neuronal clusters in the trunk are disrupted and axons are misrouted. Dorsal parts of the embryos, with the exception of the prothorax, are morphologically normal (Fasano, 1991).
teashirt was initially identified as a gene required for the specification of the trunk segments in Drosophila
embryogenesis. Targeted
expression of teashirt in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a
phenotype similar to that produced from targeted expression of eyeless, dachshund, and eyes absent. The expression of so and dac are induced in the antennal disc by the ectopic expression of tsh, suggesting that tsh may act
upstream of these genes in eye development.
Furthermore, teashirt and eyeless induce the expression of one another, suggesting that teashirt is part of the
gene network that functions to specify eye identity (Pan, 1998).
However, these results do not prove that tsh does
play a role in specifying the eye identity during normal development. To address this issue, an examination was carried out to see if tsh is expressed at the right time and the right place to have a role in specifying the eye
identity. Indeed, TSH mRNA is expressed in the eye disc, with the strongest expression anterior to the
morphogenetic furrow. This pattern of expression is similar to that of ey, a gene that is
known to play an essential role in specifying eye identity. An examination was carried out to see if loss-of-function
mutations of tsh affect eye development. Several weak loss-of-function tsh alleles were examined and no eye defects were found. X-ray-induced mitotic recombination was used to generate mutant clones of a null tsh allele.
tsh mutant clones were recovered at a frequency similar to the wild-type control, and sections through
the mutant clones revealed a normal ommatidial organization. These data suggest that tsh may
play a redundant role during normal eye development, and the requirement for tsh may be masked by
other factor(s) that play a role similar to tsh (Pan, 1998).
Localized transcription factors specify the identity of developmental domains. The function of the Teashirt zinc finger protein, which is expressed in
the proximal domain of the Drosophila leg, has been analyzed. At stage 10 of embryogenesis, Distal-less is detected in the putative distal part of the primordia of the leg in each of the thoracic hemisegments of the embryo. Tsh is coexpressed with Dll at this stage. By stage 15 the cells of the presumptive leg imaginal discs have invaginated inside the embryo and Tsh is not detected in the most distal part of the leg primordium, where Dll is expressed alone. However, Tsh is still coexpressed with Dll in a ring of cells at the periphery of the Dll domain. At the beginning of the third instar, Dll occupies a distinct distal ring of cells in the disc; Tsh is expressed in a proximal ring. These territories are separated by 2 or 3 cells in ventral and lateral regions and up to 10 cells in dorsal parts. The Dachshund transcription factor is expressed in the intermediate ring of cells overlapping the Dll expression domain by, at the most, 1 or 2 cells. By mid-third instar Dll is expressed in a new 4-cell-wide proximal ring that is destined to make the proximal femur and possibly the distal edge of the trochanter. Tsh overlaps with this new Dll domain at the proximal edge, which persists until the late third instar stage. By ectopic expression of a teashirt transgene it has been shown that Teashirt contributes to the differences in cell-cell adhesion
between proximal and distal leg cells. Whereas clones of cells expressing the teashirt transgene survive in the endogenous Teashirt domain, most cells expressing
Teashirt in an ectopic distal position are lost from the epithelium. In clones that were recovered in the distal domain, different effects were seen, dependent on
position with respect to the dorsal-ventral axis. In the ventral region, where Wingless is signaling, surviving clones express Teashirt and cause abnormalities in the
adult leg. In contrast, lateral and dorsal clones generally do not accumulate Teashirt and have no effect on patterning. One exception to the differential dorsal-ventral
effects occurs at the boundary between Teashirt-expressing and -nonexpressing cells. Both ectopic and hypomorphic loss of teashirt affects patterning at all positions
at the boundary, suggesting that Teashirt plays a crucial role in boundary formation. The results are discussed with respect to the roles of transcriptional and
posttranscriptional mechanisms in proximal-distal axis patterning of the Drosophila legs. It is suggested that because Arm binds to Tsh, this binding stablizes Tsh in cells within the Wg signaling domain (Erkner, 1999).
The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the
axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and
proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. Evidence is presented that Teashirt is required for the formation of proximal leg segments, but Tsh has no role in boundary formation (Wu, 2000).
The leg disc consists of a single epithelial sheet in which
the presumptive distal segments are specified in the center
and the presumptive proximal segments are specified in the
periphery. Cross-sections show that proximal segments,
which express Hth and Tsh, fold back over the distal
segments, which express Dll and Dac. Hth
and Tsh expression is limited to the proximal region of
the disc through repression by the combined activities of
Wg and Dpp. Although the Hth and Tsh expression
domains overlap through much of the proximal region,
Hth expression extends more distally than Tsh. This is visible as a band of Hth expression that does not overlap Tsh in
a basal optical section. This band coincides with the outer ring of Dll
expression. The Tsh domain overlaps the proximal edge of the Dll ring
by one or two cells. Tsh expressing cells are
also found beneath the disc epithelium. Their location suggests that these may be adepithelial cells. Hth functions as a repressor to modulate Tsh expression.
More distally located hth mutant
clones lose Tsh expression. Loss of Tsh expression in hth
correlates with ectopic expression of Dachshund. hth mutant clones cause ectopic expression of Dac close to the endogenous Dac domain,
but do not do so in more proximal regions. The differential
effect on Dac expression of hth clones located at different
positions along the PD axis has been attributed to a role of
Hth as a repressor of Wg and Dpp signaling. Thus the paradoxical loss of
Tsh in more distal hth clones can be explained as an
indirect effect of Hth on Dac expression. Dac can repress
both Tsh and Hth when overexpressed. Thus the
different distal limits of the Hth and Tsh expression domains
presumably reflect a difference in their sensitivity to repression by Dac.
The observation that Tsh levels increase in proximal
hth clones suggests that Hth serves as a repressor of
Tsh. Thus Hth modulates Tsh expression levels in the proximal
leg in two ways. Hth may act directly to reduce Tsh expression levels in the proximal leg, and indirectly via repression
of Dac to define the distal limit of Tsh expression (Wu, 2000).
To assess the role of Tsh in development of the proximal leg the phenotypes of adult viable mutant alleles of tsh were examined. tshGAL4
is a weak allele caused by insertion of the GAL4 enhancer-trap P-element. The trochanter is strongly reduced in legs of flies homozygous for tshGAL4. The coxa is reduced and lacks most of the bristles and sense organs found in wild-type. The femur is short, but contains the normal complement of proximal sense organs (including the sc11 group of campaniform sensillae that is normally located at the joint between
femur and trochanter. Although the trochanter is reduced, joints can still be
seen between coxa, trochanter and femur segments. One or two sensilla trichodea are
generally found at the articulation between the reduced
trochanter and coxa segments (there are normally two groups of 5-7
sensilla trichodea at this position in wild-type). The tibia
and tarsal segments appear to be normal in tsh mutants.
In a stronger mutant combination, tshGAL4/tshHD1 the trochanter is no longer detectable as a discrete segment
and the coxa appears to articulate directly with the femur. Both coxa and femur are reduced in size. No sense organs can be recognized on the coxa and trochanter rudiment, but the sc11 group of campaniform sensillae
was reliably found on the proximal femur where it articulates with the coxa. The shortening of the femur in the strong tsh mutant combination suggests
that defects in the trochanter and coxa may have non-autonomous effects on femur development. This may reflect the finding that many of the cells that contribute to femur development originate in the Tsh-expression domain at earlier stages of development and are displaced distally as the disc grows (Wu, 2000).
Reducing Tsh activity produces a phenotype quite
distinct from that of removing Hth activity. Tsh is required
for the development of trochanter and coxa but does not
appear to have a role in segment boundary formation.
Hth and its partner Extradenticle are required to prevent
fusion of coxa and trochanter with the femur. To better understand the basis for the defects
in tsh mutant legs, Hth, Dll and Dac expression
were studied in tshGAL4/tshHD1 and tshGAL4/tshGAL4
mutant discs. In wild-type discs Hth and Dac expression overlap in the proximal ring of Dll expression. Hth function in this ring is required for the affinity boundary between proximal and distal regions of the leg. This basic relationship
holds in the tshGAL4/tshHD1leg disc. Hth, Dll and Dac expression overlap, and the affinity boundary between
proximal and distal leg segments appears to be intact. The principal difference in these discs is expansion
of the Dll domain into the proximal, Hth-expressing region.
The spatial relationship between Hth and Dac is normal. Ectopic Dll expression is
not sufficient to repress Hth but does appear to reduce the size of the coxa
and trochanter and to cause problems that result in loss of
pattern elements from the remaining portions of these
segments. Even slight reductions in Tsh activity causes
loss of sensory bristles from the coxa. In contrast, small
clones of hth mutant cells are capable of differentiating
bristles (Wu, 2000).
Taken together, these observations suggest that Tsh and
Hth have distinct functions in the proximal leg. Hth limits
the proximal extent of Dac expression, and is required for
the affinity boundary between trochanter and femur. Tsh
limits the proximal extent of Dll expression and is required
for proper growth and differentiation of proximal segments, but does not appear to have a role in PD boundary
formation (Wu, 2000).
Split ends (Spen) is a protein that acts in parallel with Hox proteins to regulate different segmental morphologies. spen plays two important segment identity roles. One is to promote sclerite development in the head region, in
parallel with Hox genes; the other is to cooperate with
Antennapedia and teashirt to suppress head-like sclerite
development in the thorax. Without spen and teashirt functions, Antennapedia loses its ability to specify thoracic identity in the epidermis. Spen is the only known homeotic protein with RNP binding
motifs: this indicates that splicing, transport, or other
RNA regulatory steps are involved in the diversification of
segmental morphology. Other studies have identified spen
as a gene that acts downstream of Raf to suppress Raf
signaling in a manner similar to the ETS transcription
factor Aop/Yan. This raises the intriguing possibility that the Spen RNP protein might integrate signals from both the Raf and Hox pathways (Wiellette, 1999).
A Drosophila gene involved in distinguishing head
from body is teashirt (tsh). Tsh protein is expressed only in the
labial segment and trunk region of embryos, where it is
required to repress head identity and to promote thoracic and
abdominal segment identities. tsh transcription levels in the thorax are maintained by Antp, but a variety of genetic
interaction tests have shown that Antp and Tsh have
independent functions in repressing head development. Is spen integrated into the Antp;tsh pathways by regulation of the tsh or Antp expression patterns? Experiments show that (1) expression pattern of Tsh protein is unchanged in spen mutant embryos; (2)
protein expression patterns of Scr or Antp in spen-;tsh- double
mutant embryos are unchanged from the pattern seen in tsh
mutants alone, and (3) the spen transcript
pattern is normal in tsh mutants. Therefore,
Spen suppression of head-like sclerites is not exerted by a
regulatory effect on the Tsh protein expression pattern, nor by
Tsh effects on the spen transcript pattern, nor through
combinatorial effects of spen and tsh on Scr or Antp protein abundance (Wiellette, 1999).
The phenotype of tsh - , spen- mutant embryos suggests that
the two genes act to promote thoracic development. In tsh
mutant embryos, the T1 denticle belt is absent and although
the remaining denticle belts appear to have the appropriate
segmental identities, the denticles themselves are disorganized
and smaller than in wild type. In contrast, tsh-;spen- double mutants
completely lack denticle belts in the thorax. This may
be due to the the death of cells in the denticle field in the thorax
of the double mutants, or to the inability of Antp protein, still
expressed in the remaining cells, to promote the development
of thorax-specific structures (Wiellette, 1999).
As to whether tsh and spen collaborate in repressing head-like
sclerites, it was found that the tsh-, spen- double mutants still
have bits of sclerite in the 'thorax' of the double mutants, so
this phenotype is not enhanced. However, the
effects of Tsh overexpression on the ectopic head-like sclerites
was also examined in spen mutants. In wild-type embryos, overexpression of Tsh protein throughout the embryo results in transformation of
head regions toward thoracic identity,
as well as poorly differentiated denticle belts, especially in the
thorax. In the thorax of spen mutant embryos that also
overexpress Tsh, the ectopic ventral head-like sclerites are strongly suppressed. Taken together, these results
suggest that spen, tsh and Antp function in a combinatorial
manner to repress the development of head-like sclerites and
promote the development of thoracic identity (Wiellette, 1999).
During animal development, the HOM-C/HOX proteins direct axial patterning by regulating region-specific expression of downstream target genes. Though much is known about these pathways, significant questions remain regarding the mechanisms of specific target gene recognition and regulation, and the role of co-factors. From studies of the gnathal and trunk-specification proteins Disconnected (Disco) and Teashirt (Tsh), respectively, evidence is presented for a network of zinc-finger transcription factors that regionalize the Drosophila embryo. Not only do these proteins establish specific regions within the embryo, but their distribution also establishes where specific HOM-C proteins can function. In this manner, these factors function in parallel to the HOM-C proteins during axial specification. In tsh mutants, disco is expressed in the trunk segments, probably explaining the partial trunk to head transformation reported in these mutants, but more importantly demonstrating interactions between members of this regionalization network. It is concluded that a combination of regionalizing factors, in concert with the HOM-C proteins, promotes the specification of individual segment identity (Robertson, 2004).
disco was initially identified in a screen for mutations affecting neural development. It was not until the discovery of disco-related (disco-r) that a patterning role was uncovered (Mahaffey, 2001). The phenotype of terminal embryos lacking disco and disco-r is similar to those lacking the gnathal HOM-C genes Dfd and Scr; that is, structures from the gnathal segments (mandibular, maxillary and labial) are missing. This phenotype is due to reduced expression of Dfd and Scr target genes. Since HOM-C
protein distribution is normal in disco, disco-r null
embryos, and vice versa, these factors appear to act in parallel pathways (Robertson, 2004).
These studies have been extended and it is shown that: (1) Dfd can only direct maxillary developmental when Disco and/or Disco-R are present; (2) Tsh represses disco (and disco-r), helping to distinguish
between trunk and gnathal segment types, and thereby establishing domains for appropriate HOM-C protein function, and (3) when ectopically expressed in the trunk, Disco represses trunk development and may transform these segments towards a gnathal segment type (Robertson, 2004).
Though HOM-C genes have a clear role in establishing segment
identities, ectopic expression often has only a limited effect. The data
indicate that, for Dfd, this restriction arises because of the limited
distribution of Disco in the trunk segments. There are two important
conclusions from these observations: (1) the spatial distribution of Disco establishes where cells can respond to Dfd, and this is probably true for Scr as well. Cells expressing disco develop a maxillary identity when provided with Dfd, even though this may not have been their original HOM-C-specified fate. This highlights (2) -- the combination of Disco and Dfd overrides normal trunk patterning, without altering expression of tsh and trunk HOM-C genes. As with the maxillary segment, identity is lost in the mandibular and labial segments when embryos lack disco and disco-r. This indicates that Disco and Disco-R may have similar roles in all gnathal segments. That co-expression of Disco and Scr in the trunk activates the Scr gnathal target gene pb strengthens this conclusion. Therefore, it is proposed that Disco defines the gnathal region, and establishes where the gnathal HOM-C proteins Dfd and Scr can function (Robertson, 2004).
Alone, ectopic Disco significantly alters development, indicating that
Disco has a morphogenetic ability, separate from gnathal HOM-C input. Since Disco is required for normal gnathal development, it is suspected that disco specifies a general gnathal segment type. Definitive identification is difficult because of the lack of morphological or molecular markers that denote a general gnathal segment type. Yet, there is support for the conclusion that disco expression establishes a gnathal segment type. Ectopic Disco can, to some extent, override the trunk specification system and repress trunk development (repressing denticles, oenocytes and trachea). Furthermore, ectopic Disco blocks dorsal closure, which is similar to the role of endogenous Disco in the gnathal segments (Robertson, 2004).
Perhaps the most compelling evidence that Disco specifies a gnathal segment
type comes from the observation that disco is activated in the trunk
segments when embryos lack Tsh. The identity of the trunk segments in
tsh mutant embryos is somewhat uncertain. It has been suggested that some aspects of the tsh phenotype indicate the trunk segments acquire gnathal characteristics; for
example, the ventral neural clusters appear to be transformed to a
gnathal-like identity. Mutations in the tsh gene can therefore be interpreted in two ways --
either they partially transform the trunk segments into a gnathal-like
identity, and in particular the prothoracic segment into a labial one, or they cause a non-specific change in segmental identity perhaps due to cell death. However, the loss of tsh and the trunk HOM-C genes may transform the trunk cuticle toward anterior head
cuticle. Again, the difficulty in assigning an identity is due to the lack of
a readily discernable gnathal morphological or molecular marker. Evidence is presented that disco and disco-r are reliable molecular
markers for gnathal identity, and disco mRNA is shown to be present in the ventral and lateral regions of the trunk segments in tsh mutant embryos. This expression of disco coincides, spatially, with
the region of the trunk that is transformed in tsh mutant embryos.
UAS-driven disco does mimic some aspects of tsh mutants,
denticles are reduced and the ventral chordotonal neurons do not develop, but
since Tsh is still present, the transformation caused by ectopic disco may be incomplete. Finally, Dfd cannot induce maxillary structures, even in
tsh mutants, when disco and disco-r are absent.
This reinforces the role for Disco in establishing gnathal identity, and
indicates that the ectopic Disco present in embryos lacking Tsh is functional.
Therefore, considering these arguments, it is proposed that Disco and Disco-R establish the gnathal region of the Drosophila embryo, and in this regard, they function similarly to Tsh, which specifies the trunk region (Robertson, 2004).
There are other parallels between Disco/Disco-R and Tsh. They are
regionally expressed zinc-finger transcription factors, and they are required
in parallel with the HOM-C proteins for proper segment identity. Furthermore,
the distribution of these proteins establishes domains in which specific HOM-C proteins can properly direct embryonic development. The data reveal a regulatory relationship between Tsh and disco (and disco-r), indicating they are part of an interacting network that helps regionalize the Drosophila embryo. The HOM-C proteins then establish specific segmental identities in the appropriate region. In the
trunk segments, Tsh, along with the trunk HOM-C proteins, specifies the trunk
segment characteristics, in part by repressing disco and, thereby,
preventing gnathal characteristics from arising in the trunk segments. The presented model requires that tsh expression be limited to the trunk segments,
and it is proposed this is accomplished by another C2H2 zinc-finger protein, Salm. tsh expression has been shown to expand into the
posterior gnathal and posterior abdominal segments in embryos lacking Salm.
Therefore, Salm establishes the boundary between the Tsh and Disco domains. It is
stressed that, at this time, it is not known what parts of this regulation are
direct. Interestingly, other zinc-finger transcription factors are responsible
for positioning salm expression, so that
a more extensive hierarchy of zinc-finger transcription factors leads to
regionalization, eventually establishing the domains of HOM-C protein
function. It is also noted that Tsh has other roles than just repressing
disco. Tsh actively establishes the trunk region, just as Disco does
the gnathal. It is also noteworthy that ectopic Tsh activates disco
in the labial sense organ primordia, leading to a Keilin's Organs fate, as
occurs in the thoracic segments. Therefore, for unknown reasons, Tsh changes from a repressor of disco to an activator in these cells. This observation highlights the complex interplay between factors like Tsh and Disco, and it will be interesting to determine what causes these opposing roles (Robertson, 2004).
Many other questions remain. For example, how are the expression domains
for these factors established? It is clear that Salm could form a boundary
separating gnathal from trunk, but in salm mutants, tsh is
only ectopically activated in the posterior labial segment, not in every
gnathal segment. This implies that Salm forms a boundary, not by repressing tsh throughout the head, but by, in a sense, drawing a line between
the head and trunk regions. What then prevents tsh expression from
crossing that line and extending further into the gnathal segments in
salm mutants? Is there an activator of tsh that is limiting,
another gnathal repressor, or is something else involved? Likewise, what
activates tsh and disco? It is unlikely that lack of Tsh is
the only requirement for disco expression. More likely, this relies
on the prior segmentation pathway. With regard to the HOM-C specification of segment identity, questions remain as to how the zinc-finger proteins
establish where specific HOM-C proteins can function. Are the zinc-finger
proteins co-factors or simply a parallel pathway? Furthermore, if they are
co-factors for the HOM-C proteins, how can different HOM-C proteins establish
different segment identities with the same co-factor (for example, Dfd and Scr with Disco), or how can different co-factors alter the role of a HOM-C protein (Scr with Disco or Tsh) (Robertson, 2004)?
However, while these genes appear to play similar roles in the developing eye there is evidence to suggest that they do influence both eye development and tissue proliferation to varying degrees. For example, while null Tio mutants have no visible eye phenotype, dorsal margin clones of tsh are transformed into head cuticle while ventral margin clones show overgrowth phenotypes. Additionally, in forced expression assays Tio appears to be a more proficient inducer of both ectopic eyes and cell proliferation (Datta, 2009). This study set out to understand the biochemical mechanisms that underlie functional differences between the zinc finger transcription factors Tsh and Tio, to ascertain if any of the zinc fingers mediate interactions with Hth and to determine if interactions with CtBP are required for eye specification and cell proliferation (Datta, 2011b).
To investigate the first question, focused was placed on the major structural difference that exists between these two zinc finger transcription factors: Tsh contains three such domains while Tio possesses four motifs and is thus more similar to the mouse and basal insect Tsh/Tio proteins. A series of deletion constructs was generated in which individual zinc fingers were removed from either Tsh or Tio. These constructs were then used in forced expression assays to assess the relative requirements for each DNA binding domain in promoting eye development and inducing tissue proliferation. In a companion experiment a chimeric protein was generated in which the fourth (and unique) zinc finger from Tio was fused to the full-length Tsh protein. For comparison the effects that the deletion and chimeric constructs had on ectopic eye development and tissue growth were measured against the more ancient Tribolium Tsh/Tio protein. It was found that in the case of Tsh, Zn1 and Zn2 are required for both the promotion of eye development and tissue growth. Countering the activities of these two domains is Zn3, which appears to play a role in suppressing both proliferation and differentiation. This dissection of Tio revealed a similar mechanism for regulating its activity during eye induction and retinal differentiation: Zn1, Zn2 and Z4 act to promote retinal differentiation while Zn3 appears to inhibit this process. This balancing act represents a novel mechanism for regulating the activity of a transcription factor (Datta, 2011b).
It was not possible to uncover a mechanism that would account for the higher proficiency in inducing ectopic eyes. Removal of Zn4 from Tio (Tio γZn4) or its addition to Tsh (Tsh/Tio Zn4) did not alter the specificity with which Tio or Tsh can induce ectopic eyes nor did manipulations of any other zinc finger for that matter. That topological difference may reside within the remaining non-conserved protein segments and may involve interactions with different sets of binding proteins. The data supporting the second half of this hypothesis is somewhat mixed. Previous efforts to identify binding factors using a genetic screen failed to yield paralog specific binding partners (Datta, 2009). In contrast yeast two-hybrid screen did identify a number of potential paralog specific interacting proteins. Further biochemical studies and in vivo functional assays will be required to confirm or reject this line of reasoning (Datta, 2011b).
Analysis of Tio revealed an interesting difference that may provide a mechanistic explanation for the higher proficiency of Tio in inducing tissue proliferation. While Zn1, Zn2 and Zn4 are all required to promote tissue proliferation, Zn3 appears to effect neither the location of tissue overgrowths nor the size of the induced ectopic eye. Without the use of an inhibitory zinc finger Tio has three such motifs being used to promote tissue growth while Tsh has only two such motifs being used for this same purpose. It is proposed that the use of higher numbers of zinc fingers may allow for Tio to form more stable protein-nucleic acid complexes that have longer resident times on genes that directly affect the cell cycle (Datta, 2011b).
It is also possible that each of the zinc fingers has different binding partners and/or transcriptional binding sites. It was shown that Tsh and Hth act together, along with Yki and Ey to promote anterior eye disc proliferation and suppress eye development. It was hypothesized that Hth might bind to Tsh and Tio through Zn2 to fulfill these functions. However, results from immunoprecipitation experiments suggest that the interactions between Hth and both Tsh and Tio occur through the non-zinc finger domains. Additionally, Tsh and Tio may have different targets in the dorsal and ventral regions of the antenna and this may allow for increased proliferation. And finally, each zinc finger might bind to distinct binding sites. Genomic and biochemical analyses will be required to fully investigate these possible mechanistic options (Datta, 2011b).
The tight regulation of RD genes is presumably linked to genetic and protein interactions, which is then related to gene structure. Different functional domains of Tsh and Tio are able to affect different regions of the eye antennal disc when over-expressed. While most developmental genes are highly pleiotropic, it is especially intriguing that Tsh and Tio are able to carry out all of their functions with the same C2H2 zinc finger domain. Previous studies have shown that there is differential selection acting along the lengths of the genes and that relaxed selection acting on specific motifs contributes to changes in function in highly pleiotropic gene (Datta, 2011a). It is possible that a coding sub-functionalization event has taken place, where one domain within Tsh and Tio acts as a repressor and the other acts as a promoter of eye development. A comparison with the Pax6 family can be made. Eyg acts as a repressor of eye development, while Ey promotes eye development. The duplication event giving rise to Tsh/Tio may have occurred at the same time as the Pax6/Pa65a split, allowing for the same amount of time for nucleotide substitution and sequence evolution. The only difference here is that the division of function has taken place between genes in the Pax family, while sub-functionalization may have taken place within a gene itself (Datta, 2011b).
Finally, the binding of Tsh and Tio to the transcriptional co-repressor CtBP appears crucial for both proteins, since removal of this domain interferes with the ability of Tsh and Tio to induce ectopic eyes and promote proliferation. Furthermore, the loss of CtBP expression ahead of the furrow relieves the repression of dac expression by Tsh. These results indicate that CtBP is a member of transcriptional repressive complexes that include Tsh and Tio and plays a critical role within the retinal determination network (Datta, 2011b).
Gene duplication, expansion and subsequent diversification are features of the evolutionary process. Duplicated genes can be lost, modified or altered to generate novel functions over evolutionary time scales. These features make gene duplication a powerful engine of evolutionary change. This study explores these features in the MADF-BESS family of transcriptional regulators. In Drosophila melanogaster, the family contains sixteen similar members, each containing an N-terminal, DNA binding MADF domain and a C-terminal, protein interacting, BESS domain. Phylogenetic analysis shows that members of the MADF-BESS family are expanded in the Drosophila lineage. Three members, that were named hinge1 (CG9437), hinge2 (CG8359) and hinge3 (CG13897) are required for wing development, with a critical role in the wing-hinge. hinge1 is a negative regulator of Wingless expression and interacts with core wing-hinge patterning genes such as teashirt, homothorax and jing. Double knockdowns along with heterologous rescue experiments are used to demonstrate that members of the MADF-BESS family retain function in the wing-hinge, in spite of expansion and diversification over 40 Million years. The wing-hinge connects the blade to the thorax and has critical roles in fluttering during flight. MADF-BESS family genes appear to retain redundant functions to shape and form elements of the wing-hinge in a robust and failsafe manner (Shukla, 2013).
Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Abeta42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive. A transgenic Drosophila eye model was generated where high levels of human Abeta42 was misexpressed in the differentiating photoreceptor neurons of the developing eye; this phenocopies Alzheimer's like neuropathology in the neural retina. This model was used for a gain of function screen using members of various signaling pathways involved in the development of the fly eye to identify downstream targets or modifiers of Abeta42 mediated neurodegeneration. The homeotic gene teashirt (tsh) was identified as a suppressor of the Abeta42 mediated neurodegenerative phenotype. Targeted misexpression of tsh with Abeta42 in the differentiating retina can significantly rescue neurodegeneration by blocking cell death. Tsh protein was found to be absent/downregulated in the neural retina at this stage. The structure function analysis revealed that the PLDLS domain of Tsh acts as an inhibitor of the neuroprotective function of tsh in the Drosophila eye model. Lastly, the tsh paralog, tiptop (tio) can also rescue Abeta42 mediated neurodegeneration. This study has identified tsh and tio as new genetic modifiers of Abeta42 mediated neurodegeneration. These studies demonstrate a novel neuroprotective function of tsh and its paralog tio in Abeta42 mediated neurodegeneration. The neuroprotective function of tsh is independent of its role in retinal determination (Moran, 2013).
teashirt:
Biological Overview
| Regulation
| Developmental Biology
| References
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