held out wings
The early expression of How protein is apparently under the control of twist. A direct role for twist in the mesodermal expression of how is further suggested by the existence of four putative Twist binding-sites in a 80 base pair intronic fragment. Also consistent with this hypothesis is the fact that How is expressed in the precursor cells of adult muscles that re-express twist. Early expression is not modified in either snail or tinman mutants. The expression is not affected in a mef2 mutant in which late differentiation stages of muscular cells is altered (Zaffran, 1997).
In order to test if how is transcriptionally regulated by ecdysone (see Ecdysone receptor), third larval instar tissues were isolated before the increase in ecdysone titer. They were cultured for 8 hours, and a physiologically high titer of ecdysone was added to cultures for varying periods of time. RNA was extracted from the tissues, electorphoresed, transferred to a membrane, and hybridized with a radiolabelled how probe. how is induced by ecdysone in cultured organs (Baehrecke, 1997).
Terminal differentiation of single cells selected from a group of equivalent precursors may be random, or may be regulated by external signals. In the Drosophila embryo, maturation of a single tendon cell from a field of competent precursors is triggered by muscle-dependent signaling. The transcription factor Stripe induces both the precursor cell phenotype, as well as the terminal differentiation of muscle-bound tendons. The mechanism by which Stripe activates these distinct differentiation programs remained unclear. This study demonstrates that each differentiation state is associated with a distinct Stripe isoform and that the Stripe isoforms direct different transcriptional outputs. Importantly, the transition to the mature differentiation state is triggered post-transcriptionally by enhanced production of the stripeA splice variant, which is typical of the tendon mature state. This elevation is mediated by the RNA-binding protein How(S), with levels sensitive to muscle-dependent signals. In how mutant embryos the expression of StripeA is significantly reduced, while overexpression of How(S) enhances StripeA protein as well as mRNA levels in embryos. Analysis of the expression of a stripeA minigene in S-2 cells suggests that this elevation may be due to enhanced splicing of stripeA. Consistently, stripeA mRNA is specifically reduced in embryos mutant for the splicing factor Crooked neck (Crn), which physically interacts with How(S). Thus, a mechanism is generated by which tendon cell terminal differentiation is maintained and reinforced by the approaching muscle (Volohonsky, 2007).
This study demonstrates the involvement of post-transcriptional control in
a cell-differentiation process that must be coupled to muscle-tendon
interaction. Terminal differentiation of tendons involves a major
reorganization of the microtubule and actin networks.
Such processes are presumably not compatible with embryonic morphogenetic
movements such as germ band retraction. Thus, it is essential to spatially and
temporally restrict differentiation to single muscle-bound tendon cells.
Indeed, the results show that premature overexpression of StripeA in the
entire ectoderm leads to severe defects in germ band retraction (Volohonsky, 2007).
Stripe mediates both the determination of precursor
cells as well as their maturation and ability to undergo specific temporal and
spatial regulation. The current findings suggest both negative and positive feedback
loops, based on post-transcriptional regulation of stripe splice
variants that on one hand maintain non-bound tendon cells at the precursor
state, and on the other hand enable irreversible differentiation of
muscle-bound tendons (Volohonsky, 2007).
Whereas some tissue differentiation processes (e.g. tracheal development)
initiate upon the expression of a key transcription factor, which
autoregulates its own expression, thus leading to a unidirectional
differentiation route, other cells (e.g. cells in the proneural region) go
through an intermediate stage of a field of competent precursors, in which
only additional local interactions lead to irreversible differentiation.
Maturation of tendon cells follows the latter path, although the selection
mechanism is based on regulation at the post-transcription level (Volohonsky, 2007).
The following model is used to explain the transition between the two
phases of tendon cell development: the initial expression of stripeB
is induced by segment polarity-dependent signals. StripeB defines a set of
tendon precursor cells. StripeB then reinforces its own expression and in
addition induces How(L) expression, which in turn suppresses stripeB
mRNA levels, thus keeping StripeB levels constant throughout embryonic
development. This is supported by experiments that show that StripeB
overexpression leads to elevation in How(L) and in StripeB itself.
Following myotube extension and adhesion to a tendon precursor cell, How(S)
levels are elevated in the muscle-adherent tendon cells, presumably due to
EGFR activation. How(S) associates with the splicing factor Crn and the
complex shuttles into the nucleus, where it binds to stripeA intronic
sequences and elevates its mRNA levels, by enhancing its splicing and
maintaining the stability of the spliced mRNA. The resulting muscle-bound
tendon cell expresses high StripeA levels, which further drive the expression
of genes required for terminal tendon differentiation (e.g. shot,
how), as inferred from StripeA overexpression experiments. This
regulatory mechanism couples muscle binding and tendon cell maturation, while
preventing differentiation of additional, non-bound, precursors (Volohonsky, 2007).
RNA-binding proteins can function as adaptor units promoting the assembly
of large protein complexes that control the various aspects of RNA metabolism.
How, together with Quaking and GLD-1, belongs to the Star family of
RNA-binding proteins, the members of which often regulate more than one facet
of RNA metabolism. For example, GLD-1 has been suggested to regulate mRNA
stability as well as translation of some of its targets. Similarly
Quaking controls mRNA stability as well as RNA splicing, and
possibly also mRNA nuclear export and localization. It
appears that How proteins also exhibit a wide range of activities on RNA
metabolism. While the effect of How(L) and How(S) on stripe mRNA
stability has been demonstrated previously,
this study suggests that How(S) has an additional activity in regulating the
splicing of stripeA. Consistent with this study, How has been
identified in a dsRNA-based screen for alternative splicing regulators, as a
protein required for specific splicing of exons within two out of five tested
genes, paralytic (exons A/I), and Dscam (exon 4), in S-2 cells. Previous studies suggested that the ability of How proteins to stabilize stripe mRNA is mediated by the 3' UTR of stripe. However, the splicing of stripeA appears to be regulated by its specific intronic sequences (Volohonsky, 2007).
By contrast to How(L), which is localized specifically in the nucleus,
How(S) is distributed both in the nucleus and the cytoplasm. However, when
How(S) is retained in the nucleus by the addition of an NLS sequence, it loses
its effect on the mRNA levels of its target. What could be the molecular
explanation for the involvement of How(S) in splicing? It is suggested that How(S)
binds to a cytoplasmic splicing factor and recruits it to the nucleus, where
it is targeted to bind stripeA-specific intronic sequences. This may
enhance the splicing of stripeA-specific exons. A candidate splicing
factor is Crn. Crn is a general, well-conserved splicing factor that is
expressed by a wide range of cell types and is distributed both in the nucleus
and the cytoplasm. In a parallel study, it was demonstrated that crn
and how mutants exhibit closely related phenotypes, affecting glial
cell maturation. Importantly, both Crn and How(S) proteins [but
not How(S)-NLS] coprecipitate from S-2 cell extracts, indicating that both
proteins are associated in a common protein complex in the cytoplasm
(Edenfeld, 2006). In addition, when Crn is myristoylated and transfected into S-2 cells together with How(S), How(S) is relocated to the membrane
(Edenfeld, 2006). Furthermore, in crn mutants StripeA, but not StripeB, levels are
reduced, and this is reflected in the reduction of Shot levels (Volohonsky, 2007).
These results support a model in which How(S) interacts with Crn in the
cell cytoplasm, shuttles into the nucleus and facilitates stripeA
splicing, and possibly mRNA stability, leading to StripeA protein elevation. A
similar mechanism may operate in the Quaking-dependent facilitation of
myelin-associated glycoprotein splicing (Volohonsky, 2007).
In summary, a molecular mechanism has been described that is based on
post-transcriptional control, by which cell differentiation is induced and
maintained by local interactions with neighboring cells (Volohonsky, 2007).
Regulation of RNA metabolism plays a major role in controlling gene
expression during developmental processes. The Drosophila RNA-binding
protein Held out wing (HOW), regulates an array of developmental processes in
embryonic and adult growth. The primary sequence and
secondary structural requirements for the HOW response element (HRE) has been characterized; this site (ACUAA) is necessary and sufficient for HOW binding. Based on this
analysis, the Drosophila TGFß homolog,
dpp, was identified as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW(L) to the dpp
3' untranslated region (UTR) leads to a reduction of
GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE
site-dependent manner. Moreover, co-expression of HOW(L) in the wing imaginal
disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP
levels in a dpp-3'UTR-dependent manner. Conversely, a reduction
of the endogenous levels of HOW by targeted expression of HOW-specific
double-stranded RNA led to a corresponding elevation in dpp mRNA
level in the wing imaginal disc. Thus, by characterizing the RNA sequences
that bind HOW, a novel aspect has been demonstrated of regulation, at the mRNA level,
of Drosophila DPP (Israeli, 2007).
It has been shown that HOW binds directly to the 3'UTR of
stripe. To characterize the HOW-binding sites further,
the stripe 3'UTR 1.2 kb sequence was truncated into smaller fragments, which
were individually transcribed in vitro and labeled with biotin. These
fragments were tested for HOW binding by adding in vitro-translated HOW tagged
with hemagglutinin (HA) to the biotin-labeled RNA followed by precipitation of
the RNA complexes using avidin-conjugated magnetic beads. The presence of HOW
on the beads was then tested by western blot analysis using anti-HA
antibodies. As a control for non-specific binding, a mutant HOW
variant (HOWm) was used, that carries a mis-sense mutation in the KH domain
exchanging arginine at position 185 to cysteine, mimicking the severe
loss-of-function howe44 allele. HOWm does not exhibit RNA-binding activity.
This analysis allowed selection of two HOW-binding fragments
(a and b) in which the sequence ACUAA, which
was similar, but not identical, to the GLD-1 hexanucleotide-binding site in
tra-2, was identified. In fragment a, there are three repeats of this
sequence, and fragment b contains one such sequence (Israeli, 2007).
It is concluded that the sequence ACUAA represents the primary HRE.
Importantly, one of the HRE sequences (at position 766) is conserved in the
3'UTR of stripe in Drosophila pseudoobscura. Moreover,
three repeats of the pentamer AAUAA (which also binds HOW, but
to a lesser extent) were identied that are conserved
between the two Drosophila species. Thus the
HOW-binding site NA(C>A)UAA closely resembles that of STAR proteins from
other species, although it is not identical. The binding
of HOW was studied in the context of the entire stripe 3'UTR, and it was
demonstrated that deletion of these four sites indeed abrogates the
responsiveness of the stripe 3'UTR to HOW (Israeli, 2007).
Because a pentanucleotide sequence would be relatively abundant within the
3'UTRs of many RNAs, it was suspected that additional restrictions might
exist in addition to the primary sequence ACUAA. Analysis of the distinct
HOW-binding sites in the stripe 3'UTR using the Mfold program showed that
high-affinity binding for HOW occurs when the binding site (ACUAA) is included
within a single-stranded loop. However, secondary-structure predictions of
large RNA fragments (larger than 30-40 nucleotides) using the Mfold program
resulted in numerous alternatives. To test whether a loop secondary structure
is essential for the binding of HOW, HRE-containing loops of
distinct sizes were constructed, fused to the 3' end of the stripe 3'UTR
fragment (1-225), which does not bind HOW. It was found that
single-stranded loops that are larger than 12 nucleotides and contain a single
HRE site exhibit significant binding, whereas loops smaller than 12
nucleotides did not exhibit specific binding to HOW. Presumably, these
loops are too small to allow this binding (Israeli, 2007).
Structural studies helped identify a novel HOW target, namely
dpp mRNA, in the wing imaginal disc. Normally, the
repressor isoform of HOW, HOW(L), reduces dpp mRNA levels in the wing
imaginal disc and in the pupal wing, leading to reduced DPP protein levels
during the establishment of the anteroposterior axis, and later during wing
vein formation. Presumably, in the absence of HOW(L), higher DPP levels at the
source would alter the overall shape of the DPP gradient, thus expanding the
Spalt expression domain. The phenotype of ectopic veins obtained by continuous
expression of HOW(L) dsRNA in the pupal wings supports an additional role for
HOW(L) in repressing dpp mRNA at later stages of wing development (Israeli, 2007).
The sensitivity of the embryo to DPP levels has been demonstrated by the
DPP haplo-insufficient phenotype. This sensitivity is also exhibited in the
wing imaginal disc by the observation that endogenous dpp can be
replaced by UAS-GFP-dpp driven by dpp-gal4 only at
low temperatures (16°C or 19°C), at which the Gal4 protein is significantly less
active. Because the responsiveness of the cells to DPP levels is highly
sensitive, it is necessary to tightly regulate the levels of DPP protein; for
example, by constitutive reduction of its mRNA levels in DPP-secreting cells
by the HOW(L) protein (Israeli, 2007).
Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated. This study shows that the repressor isoform of the Drosophila RNA binding protein Held Out Wing [HOW(L)] is required to inhibit mesodermal cell division during gastrulation. Embryos mutant for how exhibit an excess of cell divisions, leading to delayed mesoderm invagination. The levels of the mitotic activator string/cdc25 mRNA in these embryos were significantly elevated. Protein-RNA precipitation experiments show that HOW(L) binds string/cdc25 mRNA. Overexpression of HOW(L) in Schneider cells reduces specifically the steady-state mRNA levels of a gfp reporter fused to string/cdc25 untranslated region (3'UTR). These results suggest that in wild-type embryos, string/cdc25 mRNA levels are downregulated by the repressor isoform HOW(L), which binds directly to string/cdc25 mRNA and regulates its degradation. Thus, this study proposes a novel posttranscriptional mechanism controlling cell-cycle progression in the Drosophila embryo (Nabel-Rosen, 2005; full text of article).
String/Cdc25 is a limiting factor that controls cell-cycle progression in early embryonic stages after cellularization. Both string/cdc25 mRNA and String/Cdc25 protein are extremely unstable (T1/2 < 15 min). The instability of the mRNA and protein allows for a sensitive response of String/Cdc25 levels to transcriptional regulation by various transcription factors operating in pattern formation in the embryo. It has been reported that, in addition to the time of initiation of string/cdc25 transcription, accumulation of string/cdc25 mRNA is slower in mitotic domain 10 (MD10) than in MD2. This is consistent with lower mRNA levels detected in MD10 in relation to MD2 in wild-type embryos. This study provides a molecular basis for string/cdc25 mRNA instability. In situ hybridization with string antisense probe as well as RT-PCR experiments demonstrated that, in how mutant embryos, string/cdc25 is upregulated. Moreover, protein-RNA binding experiments showed a direct binding between HOW and string RNA, and in Schneider cells a gfp-string3'UTR reporter mRNA is specifically degraded in the presence of HOW(L). Collectively, these experiments are consistent with HOW(L) being the major factor responsible for string instability in the early embryo (Nabel-Rosen, 2005).
A consensus RNA binding site, (U>A/C/G)ACUAA, has been recently described for the binding of the STAR protein Gld-1. The same sequence has also been characterized in as being a consensus RNA binding site for HOW. Importantly, this sequence, GACUAA, is present in the string 3'UTR. These results are consistent with the idea that HOW binds the relaxed consensus sequence described for Gld-1, which is also present in string 3′UTR. Interestingly, this sequence appears also in the C. elegans cdc25/string homolog, suggesting that Gld-1 (similarly to HOW in Drosophila) may control cdc25/string in C. elegans (Nabel-Rosen, 2005).
The arrest of cell-cycle progression in the invaginating mesoderm must be transient because immediately after the invagination process the cells undergo a round of cell division. Thus, String/Cdc25 protein levels must be downregulated to a narrow time window to enable mesoderm invagination. This time frame may be achieved in the following manner: Twist, a regulator of mesoderm fate, activates the transcription of string/cdc25 and HOW. Maternal HOW, as well as zygotic HOW shown previously to be downstream of Twist, compromises string/cdc25 mRNA levels, and Tribbles (which requires Twist and Snail to perform its activity) reduces String/Cdc25 protein levels at this stage. Thus, in parallel to string/cdc25 transcriptional activation, Twist provides a double safe mechanism that silences string at the mRNA via HOW activity and String protein via Tribbles activity in MD10. The activity of both HOW and Tribbles should enable the eventual accumulation of String/Cdc25 protein at the end of the invagination process to allow cell-cycle progression at this stage. Therefore, both HOW's and Tribbles's inhibitory effect may not be highly efficient. This, together with the constitutive transcriptional activation of string by Twist, may lead to the eventual accumulation of string mRNA and protein levels, overcoming the negative control imposed by HOW and Tribbles. Alternatively, String accumulation may be caused by a more direct inhibition of both HOW and Tribbles activities, possibly by signaling pathways that operate in the mesoderm after its invagination (Nabel-Rosen, 2005).
Maternal HOW appears to reduce string mRNA levels in the lateral ectoderm in stage-5 and -6 embryos in addition to MD10. It is therefore possible that the extra cell divisions detected in these regions may have an additional indirect effect on mesoderm invagination (Nabel-Rosen, 2005).
It is instructive to ask whether HOW regulates additional processes during mesoderm development. Although zygotic how mutants do not exhibit mesoderm defects until late developmental stages, the how germline clone embryos do show significant mesoderm aberrations. The entire somatic muscle pattern of these embryos is severely disrupted, presumably owing to accumulation of defects. At this stage, it is impossible to distinguish between primary and secondary effects induced by the complete lack of HOW. The muscle defects detected in how germline clone embryos suggest that HOW has a broader function in the mesoderm and that it may regulate the levels of an array of essential genes necessary for appropriate mesoderm development. The identification of such genes should elucidate the full regulatory range of HOW activity (Nabel-Rosen, 2005).
Finally, regulation at the level of mRNA metabolism by STAR family proteins has been shown to occur in several developmental systems, for example, C. elegans gld-1 and mammalian quaking. These proteins exhibit a wide range of activities, affecting RNA splicing, mRNA nuclear export, mRNA stability, and possibly others. The advantage of such regulation is the ability to respond rapidly to external signals by controlling the mRNA levels of an array of target genes. The synchronization between muscle-cell differentiation and cell-cycle progression may be based on the activities of both HOW and Tribbles, but the molecular link between both processes has yet to be elucidated (Nabel-Rosen, 2005).
The even spreading of mesoderm cells in the Drosophila embryo is
essential for its proper patterning by ectodermally derived signals. In
how germline clone embryos, defects in mesoderm spreading lead to a
partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein
that is thought to regulate diverse mRNA targets. To identify direct HOW
targets, a series of selection methods were implemented on mRNAs whose levels
were elevated in how germline clone embryos during the stage of
mesoderm spreading. Four mRNAs were found to be specifically elevated in the
mesoderm of how germline clone embryos and to exhibit specific
binding to HOW via their 3' UTRs. Importantly, overexpression of three
of these genes phenocopied the mesoderm-spreading phenotype of how
germline clone embryos. Further analysis showed that overexpressing one of
these genes, miple, a Drosophila midkine and pleiotrophin
heparin-binding growth factor, in the mesoderm leads to abnormal scattered MAPK
activation, a phenotype that might explain the abnormal mesoderm spreading. In
addition, the number of EVE-positive cells, which are responsive to receptor
tyrosine kinase (RTK) signaling, was increased following Miple overexpression
in the mesoderm and appeared to be dependent on Heartless function. In
summary, these analysis suggests that HOW downregulates the levels of a number
of mRNA species in the mesoderm in order to enable proper mesoderm spreading
during early embryogenesis (Toledano-Katchalski, 2007).
Previous analysis of how germline clones suggested that HOW is
essential for correct mesoderm spreading over the ectoderm
(Nabel-Rosen, 2005), a
process that is required for the spatial patterning of the mesoderm layer by
ectoderm-derived signals. This phenotype could not result from the earlier
effect of extra cell division during gastrulation detected in the how
germline clone embryos, because mesoderm invagination and gastrulation were
eventually completed in these embryos. Furthermore, mesoderm spreading was
unaffected in tribbles (trbl) mutant embryos, which exhibit
a similar defect of extra cell divisions during gastrulation that leads to
delayed and unsynchronized mesoderm invagination (Toledano-Katchalski, 2007).
Regulation of mesoderm-specific mRNA levels by HOW might contribute to the
spatial and temporal control of gene expression during mesoderm spreading. The
genome analysis was designed to identify mRNAs whose
levels might be directly controlled by the repressor isoform HOW(L) in the
mesoderm. Such targets should be normally repressed to enable even spreading
of the mesoderm. Three out of the four HOW targets identified in this screen,
namely falten, CG31638 and LAP (CG2520) are contributed
maternally, and
therefore HOW(L)-dependent repression in the mesoderm might be essential for
reducing their levels in this tissue to enable proper mesoderm spreading. This
scenario is supported by the defective mesoderm spreading induced by
overexpression of falten and CG31638. Miple does not appear
to be maternally contributed according to expression data and in situ analysis. It is not
clear, however, which transcription factor is responsible for miple
induction. Because miple mRNA was detected in mesoderm derivatives at
stage 11, it might be induced by mesoderm-specific transcription factors such
as Twist, MEF2 and/or Tinman, which are expressed in the mesoderm during
spreading. In that case, to abrogate the effects of Miple, it would be
necessary to block miple expression during mesoderm spreading. These
data suggest that this is the role of HOW(L), because in its absence,
miple mRNA is significantly elevated in the mesoderm. Thus, HOW(L) in
the mesoderm of gastrulating embryos is necessary to reduce maternal mRNA
expression and, in addition, to reduce the levels of gene products whose
expression is not compatible with early mesoderm development, but might be
required shortly after the process of mesoderm spreading has been completed.
Thus, HOW(L) is essential to enable temporal morphogenetic processes in the
mesoderm during its spreading over the ectoderm (Toledano-Katchalski, 2007).
Miple was further analyzed because its vertebrate homologs, midkine and
pleiotropin, are involved in cell migration and are associated with receptor
tyrosine kinase (RTK) signaling (Stoica, 2002). Therefore, its downregulation by HOW(L) might
contribute to the restricted dorsal activation of the HTL-dependent signaling
during mesoderm spreading. Moreover, the putative heparin-binding motif of
Miple could affect the affinity of the HTL ligands to the HTL receptor,
thereby modulating the strength of HTL-dependent signaling (Toledano-Katchalski, 2007).
Indeed, the findings suggest that downregulation of Miple levels in the
mesoderm is essential for correct mesoderm spreading, because Miple
overexpression leads to impaired mesoderm spreading. The disordered pattern of
MAPK phosphorylation (detected by anti-dpERK antibody) observed following
Miple overexpression might be the primary cause for the mesoderm spreading
defect. In wild-type embryos, MAPK activation is detected only at the most
dorsal cells of the spreading mesodermal cells. The mechanism by which this
spatial MAPK activation is achieved is not clear. It has been suggested that
MAPK activation takes place only in cells that directly contact the ectoderm. In that
case, Miple might trigger prolonged mesoderm-ectoderm cell contacts and this
could delay mesoderm spreading. Indirect evidence, especially the observation
that overexpression of an activated form of HTL does not lead to an ectopic
dpERK signal in the entire mesoderm, led to the suggestion that a constitutive
inhibitory input of MAPK activation is present in mesoderm cells
(Wilson, 2005). This
inhibitory activity was suggested to be overcome only in cells that form close
contact with the ectoderm. It is unlikely that the role of Miple is to
counteract this inhibitory signal, because overexpression of Miple has an
effect not only on MAPK activation in early mesoderm spreading but also on the late HTL-dependent signaling in the dorsal EVE-positive cells, in which this inhibitory signal has not been implicated. Therefore the possibility is favored that Miple enhances HTL signaling, and that this enhancement is reflected by MAPK activation in both early and later stages of mesoderm development (Toledano-Katchalski, 2007).
The elevation of the dpERK signal detected following overexpression of
Miple might be mediated by HTL activation, because no other RTK has been shown
to be expressed in the mesoderm at the stage of gastrulation. Although the
increased number of EVE-expressing cells detected in the dorsal mesoderm
clusters following overexpression of Miple is eliminated in embryos lacking
active HTL receptor, the possibility cannot be excluded that the lack of
EVE-positive cells in the dorsal mesoderm might stem from the failure of the
htl mutant mesoderm cells to reach the most dorsal locations (Toledano-Katchalski, 2007).
In vertebrates, midkine and pleiotrophin have been identified by phage
display as potential high-affinity ligands for the human receptor tyrosine
kinase ALK (Stoica,
2002). Although the possible role of Miple in
ALK-dependent signaling cannot be excluded, ALK is not expressed in the early stages of mesoderm spreading, and does not overlap with the EVE-expressing clusters; thus, it is unlikely to affect the increased number of EVE-expressing cells. Receptor
tyrosine phosphatase-zeta has been implicated as a putative pleiotrophin
receptor (Milev, 1998). If a similar receptor exists in Drosophila, it might respond to Miple overexpression by altering MAPK levels (Toledano-Katchalski, 2007).
It is possible that the heparin-binding domain of Miple enhances the
activity of the HTL ligands. In vertebrates, heparin-containing proteins act
as co-ligands to FGFs by inducing their dimerization. Miple is a heparin-binding protein, because it binds
specifically to a heparin column. The contribution of heparan sulfate
proteoglycans to proper mesoderm spreading in Drosophila had been
demonstrated by the requirement of two enzymes, Sugarless and Sulfateless, for
this process. Moreover, a genetic interaction between mutations in each
of these enzymes and the two FGF receptors HTL and Breathless (BTL) was
demonstrated. Overexpression of Miple during mesoderm spreading might, on
the one hand, compete with endogenous heparan sulfate proteoglycan for Thisbe
and Pyramus binding, and thus could inhibit their ability to activate the
HTL-dependent signaling. In contrast, Miple might also activate the HTL
pathway by replacing the endogenous heparan sulfate proteoglycan that is
normally involved in activation of the FGF8-like ligands. These dual
activities might interfere with the normal dorsal-restricted MAPK activation
in the mesoderm (Toledano-Katchalski, 2007).
In wild-type embryos, miple is downregulated by HOW(L) in the
mesoderm; however, its mRNA expression is detected at later developmental
stages, including in the ventral midline and in the brain
(Englund, 2006). In
midline glial cells, a second FGF receptor, Breathless, has been implicated in
the promotion of cell migration at stages 12-13 of embryonic development. At this
stage, Miple might contribute to the spatial and temporal control of
Breathless activation. Such a scenario must be tested directly in
miple mutant embryos (Toledano-Katchalski, 2007).
Although the mesoderm spreading phenotype of how germline clone
embryos is not fully penetrant and is detected in only a few segments, the
contribution of HOW activity is crucial because of the secondary effect that
non-homogenous mesoderm spreading exhibits on the development of the heart and
dorsal somatic mesoderm. HOW(L) appears to function in the mesoderm as a
safety mechanism to prevent mis-expression of either maternally contributed
genes or genes whose early transcriptional activation in the mesoderm might
interfere with the normal development of the mesoderm. An example of similar
repressive activity of HOW(L) is its activity in the reduction of
string levels (Nabel-Rosen, 2005) in the gastrulating embryo to prevent premature cell division during mesoderm invagination (Toledano-Katchalski, 2007).
In summary, this study reveals the crucial function of the STAR family
member HOW(L) in enabling proper mesoderm development via the repression of
specific mRNAs provided either maternally, or expressed prematurely in a
specific tissue. HOW(L) and its vertebrate homolog, QKI5, are expressed in
wide range of tissues during early developmental stages, and might function in
these tissues in a similar fashion to enable proper embryonic and tissue
development (Toledano-Katchalski, 2007).
In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, directs peripheral glial cell maturation. crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, some of the in vivo target genes were identified that encode proteins localized in autocellular septate junctions. Thus, glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions (Edenfeld, 2006).
Most glial cells of the Drosophila PNS are born in the CNS and migrate toward their final destination. Subsequently, the cell body follows to then initiate wrapping of the axons. crn and how are both required for the initiation of axonal wrapping. The proteins encoded by crn and how are involved in the regulation of splicing of components of the septate junctions that are required for glial cell differentiation. Crn and HOW(S) interact in the cytosol to control their nuclear import, providing a simple mechanism to couple glial and neuronal cell differentiation (Edenfeld, 2006).
crn encodes an unusual TPR-containing protein whose function is essential for embryonic development. The Crn protein is found in the cytosol and in nuclear “speckles” (Raisin-Tani, 2002). Previous genetic and biochemical evidence has already suggested that Crn and its homologs participate in the assembly and the control of the splicing machinery. A mutation of the yeast crooked neck ortholog results in the accumulation of unspliced pre-mRNAs and, furthermore, Crn-like proteins are needed for pre-mRNA splicing in vitro. Crn is found in two functional complexes with and without snRNA and via its N-terminal TRPs helps to assemble the intact spliceosome (Wang, 2003). Within the spliceosome, the Crn homolog assists in the initial spliceosome assembly and also binds the phospho-CTD of the RNA polymerase II (Gasch, 2005). In Drosophila crn mutants, changes were observed in the splicing pattern suggesting that Crn modulates splicing preferences during alternative splicing (Edenfeld, 2006).
Alternative splicing employs differential use of 5' or 3' splice sites and has evolved as an efficient way to achieve a functional diversification and regulation of gene products. The basic splicing mechanism first requires the correct choice of 5' and 3' splice junctions and subsequently the assembly of the spliceosome. While Crn can facilitate spliceosome assembly, it does not directly participate in the selection of specific splice junctions, since the Crn protein is not able to bind to RNA. However, in Drosophila, Crn does regulate alternative splicing of few specific target genes, implying the existence of interaction partners that direct the Crn protein to these target RNAs (Burnette, 1999; Park, 2004; Edenfeld, 2006 and references therein).
This study has identified the HOW(S) protein as such an interaction partner that is likely able to recruit Crn to specific splicing targets. The HOW proteins contain an hnRNP K homology (KH) motif and exhibit specific RNA-binding activities. The KH motif is found in the GSG domain (GRP33, Sam68, GLD) shared by the Signal Transduction and Activation of RNA (STAR) family of proteins. The how locus is genetically complex: it encodes two antagonizing splice variants, HOW(S) and HOW(L), the functions of which were thoroughly analyzed in tendon cells. This study shows that HOW(L) is involved in the instability of stripe mRNAs, whereas HOW(S) is involved in mRNA stability as well as in the control of stripe A splicing (Volohonsky, 2007). Since HOW(S) associates with Crn, a direct influence on splicing can be anticipated (Edenfeld, 2006).
The proposed cytoplasmic-nuclear shuttling of a Crn/HOW(S) complex furthermore allows the linking of extracellular signals to a direct control of splicing. Concerning glial cell differentiation, this suggests that wrapping of axonal fascicles is not only dependent on a transcriptional control. It is rather likely that neuronal signals help to efficiently couple glial and neuronal differentiation by directly influencing the splicing pattern. The nature of such a signal is still elusive, however. As described for the HOW-related protein Sam68, phosphorylation may be important to control the interaction of Crn and HOW in the cytoplasm and thus the transport of the complex into the nucleus. In this respect, it is interesting to note that the development of oenocytes, which is impaired in both crn and how mutants, requires EGF-receptor signaling and that neuronal EGF-receptor signaling has been shown to regulate glial expression of neuroglian (Edenfeld, 2006).
In agreement with such a model is the finding that the how mutant phenotype resembles the phenotype caused by the loss of crn. Furthermore, Crn is able to bind only cytosolic HOW(S). If HOW(L) expression is forced to the cytosol, it can also bind to Crn, confirming that the interaction of Crn and HOW occurs in the cytosol. The assembly of the Crn/HOW complex is crucial to precisely regulate the nuclear concentration of these splice factors, which in turn is relevant for alternative splicing. Within the nucleus, HOW(S) binds to a consensus sequence with a length of only five nucleotides, making the quest for specific target genes difficult. To nevertheless get insight into this important functional aspect, a collection of strains was utilized in which endogenous genes were tagged by the insertion of a GFP-exon. In this study two components of the septate junctions were identified that form important autocellular junctions needed to stabilize glial cell morphology as candidate targets for Crn. Glial septate junctions are morphologically established by the end of embryogenesis. In line with the notion that neurexinIV is a target gene of crn, no septate junctions were detected in crn mutant glial cells. Furthermore, dye-penetration experiments show that the blood-brain barrier, which crucially depends on the presence of septate junctions, is not established in crn mutants (Edenfeld, 2006).
In addition to regulating splicing, Crn and HOW proteins may also have additional functions. For example, members of the hnRNP-A/B family of RNA-binding proteins are able to regulate alternative splicing of the Drosophila P element transposase and the Ubx gene. However, the function of the hnRNP-A/B family member Hrp48 is not restricted to the control of RNA splicing since it is also involved in the control of oskar mRNA localization in the Drosophila oocyte. Such a dual specificity of the Hrp48 RNA-binding protein in regulating RNA splicing and RNA transport has also been suggested for the Crn-binding partner HOW. In addition, it was recently demonstrated that the yeast Crn homolog also affects DNA replication, and first phenotypic analyses of the Drosophila crn mutant led to the proposal that Crn might play a role in regulation of cell divisions (K. Zhang, 1991). However, no abnormal cell number was observed for the peripheral glial cells, suggesting that at least during glial development crn has no function during the cell cycle (Edenfeld, 2006).
The data lead to a model underlying glial cell differentiation that may not only be applicable for Drosophila. The vertebrate homolog of how is the quaking gene, which is also required for glial differentiation. quaking viable mutants initially develop normally but then show tremors due to severe myelination defects. The mutant phenotype is caused by a deletion in the promoter region of the quaking gene that encodes several alternatively spliced mRNAs. The quaking viable deletion abrogates the expression of QKI-6 and QKI-7 in myelinating cells of the brain. The complete loss of quaking transcripts results in early lethality. quaking and how mutants not only share a defect in axonal wrapping. Moreover, the corresponding gene products appear to have different functions in the nucleus and the cytosol. Whereas QKI-5 is strictly nuclear, QKI-6 and QKI-7 are able to shuttle between the cytosol and the nucleus as it has been observed for the HOW(S) protein. The position of putative QKI binding sites close to tissue-regulated exons was found to be conserved in mice and is similar to what was found for the putative HOW binding sites in neurexinIV. Thus, although invertebrates and vertebrates have long been thought to follow very different routes toward glial differentiation, the underlying molecular control of glial wrapping may be conserved (Edenfeld, 2006).
In spite of the strong maternal expression of the 3.6 kb transcript, no protein is detected before the onset of gastrulation, suggesting that the maternally supplied mRNA is either not translated or that the translated protein is highly unstable (Zaffran, 1997).
how mRNA is first detected in mesodermal precursors on the ventral side of the embryo at the onset of gastrulation. how transcription continues to be detected in presumptive mesoderm cells at the germ band extension stage. As germ band retraction occurs, how transcription is detected in the cells destined to form somatic and visceral muscle cells. Expression is also detected in the pharyngeal muscles at the anterior end of the embryo. how transcription is detected in cardiac precursors and muscle attachment cells of the epidermis as dorsal closure occurs. Late during embyogenesis, how transcription is restricted to the heart and muscle attachment sites of the epidermis (Baehrecke, 1997).
When the different mesodermal lineages segregate, the expression of How becomes
restricted to the myogenic lineage, including the cardioblasts and probably all the myoblasts.
Antibodies directed against the protein demonstrate that How is localized to the nucleus (Zaffran, 1997).
how transcription increases at metamorphosis. Similar to the pattern of expression during embryogenesis, how transcription is detected in muscle and muscle attachment cells during the onset of metamorphosis. how is detected in adult muscle cell precursors that are attached to the wing imaginal disc (Baehrecke, 1997).
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held out wings:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 25 February 2008
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