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First published online 13 December 2006
doi: 10.1242/dev.02735
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1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot
76100, Israel.
2 Institut für Neurobiologie, Universität Münster, Badestrasse 9,
D-48149 Münster, Germany.
* Author for correspondence (e-mail: lgvolk{at}weizmann.ac.il)
Accepted 7 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Splicing, Tendon cells, stripe, how, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Somatic muscles
are specified in the mesoderm, while tendon precursor cells are defined as a
group of ectodermal cells expressing the Early growth response (EGR)-like
transcription factor Stripe (Frommer et
al., 1996; Hatini and DiNardo,
2001; Piepenburg et al.,
2000). Once each of these cell types has been specified, it
undergoes tissue-specific differentiation, which is tightly coordinated and
occurs following the encounter between the two cell types. Specifically, the
newly formed myotube migrates toward the tendon precursor cell in response to
guidance cues provided by the tendon cell
(Schnorrer and Dickson, 2004).
Once the approaching myotube reaches its destination, it provides a
differentiation signal (the EGFR ligand, Vein), which triggers the maturation
of the tendon cell (Yarnitzky et al.,
1997). This maturation is essential for both the formation of
adherens junctions between tendon and muscle cells to tightly hold the two
cell types together and for developing tendon resistance and elasticity to
accommodate muscle contractions during larval movements
(Subramanian et al.,
2003).
stripe, the earliest tissue-specific gene detected in the tendon
precursor cells is required for the appropriate differentiation of tendon
cells, and in its absence, tendon cells do not form. Moreover, in
stripe homozygous mutant embryos, muscle migration and adhesion is
abrogated, resulting in the disruption of the entire somatic muscle pattern
and embryonic lethality (Frommer et al.,
1996). Reciprocally, ectopic Stripe expression in the ectoderm
drives the expression of an array of tendon-specific genes outside their
normal domain of expression (Becker et al.,
1997; Vorbruggen and Jackle,
1997). Thus, stripe is a key factor in tendon cell
specification and differentiation.
At early embryonic stage 11, stripe transcription is regulated by
signaling pathways involved in ectodermal segment polarity, such as the Hh, Wg
and EGFR signaling pathways (Hatini and
DiNardo, 2001; Piepenburg et
al., 2000). However, Stripe protein expression is detected only at
later stages (11-12), and is significantly elevated at stages 14-16
(Becker et al., 1997;
Frommer et al., 1996). At these
stages, high protein levels of Stripe are maintained only in musclebound
tendon cells. In addition, the stripe gene has been shown to produce
two spliced variants, stripeA and stripeB
(Frommer et al., 1996). StripeB
coding sequence is included within StripeA, and both splice variants share a
similar 3' UTR. However, StripeA contains a unique N-terminal domain as
well as unique 5' UTR. In situ hybridization showed that
stripeB is detected already at stage 11, while stripeA
appears at later developmental stages in a subset of tendon precursor cells.
An enhancer sequence located upstream to the stripeA 5' UTR was
shown to drive expression of a reporter gene at a pattern similar to StripeB
starting at embryonic stage 10 (Piepenburg
et al., 2000). This suggests that the stripe locus is
transcriptionally active already at embryonic stage 10, and
post-transcriptional mechanisms may control its mRNA accumulation and couple
them to the state of differentiation of the tendon cell. Distinct promoters
for each of the stripe variants have not been identified yet but
could also affect their expression profile.
A post-transcriptional mechanism controlling stripe mRNA levels is
based on the activity of the RNA-binding protein Held out wing (How)
(Nabel-Rosen et al., 1999).
How is a member of the Star (Signal transduction and RNA control) family,
which includes the Caenorhabditis elegans protein Gld-1 and the
mammalian protein Quaking (Vernet and
Artzt, 1997). These proteins are essential for the control of
transition between differentiation states, including the transition from
mitosis to meiosis and sex determination mediated by Gld-1 in C.
elegans (Crittenden et al.,
2002; Crittenden et al.,
2003; Hansen et al.,
2004), and the switch to maturation of Schwann cells in the
peripheral nervous system and oligodendrocytes in the central nervous system
mediated by Quaking in mammalian species
(Ebersole et al., 1996;
Hardy, 1998;
Larocque et al., 2005). In
Drosophila, How regulates mesoderm invagination, muscle-dependent
tendon cell differentiation and heart rate
(Baehrecke, 1997;
Lo and Frasch, 1997;
Nabel-Rosen et al., 1999;
Zaffran et al., 1997).
The how gene is spliced into two isoforms: How(L) and How(S)
(Lo and Frasch, 1997) share
the same RNA-binding domain but differ at their C-terminal region. The How(L)
isoform is maternally contributed and is expressed during earlier stages of
embryonic development. The How(S) isoform is detected at high levels in tendon
cells following muscle binding
(Nabel-Rosen et al., 1999) and
in cardiac tissue. While both How(L) and How(S) bind the same target mRNA at
the 3' UTR, their activity is in opposing directions; How(L) binding to
mRNA leads to mRNA degradation, while How(S) binding to the same target leads
to its stabilization (Nabel-Rosen et al.,
2002).
In homozygous how mutant embryos, Stripe protein (analyzed by an
antibody that recognizes both Stripe proteins) is detected in a higher number
of tendon cells at late embryonic stages, suggesting that the selection of a
single tendon precursor to undergo terminal differentiation does not occur
(Nabel-Rosen et al., 1999). We
have previously shown that How proteins bind to the 3' UTR of
stripe, which is shared by both stripe variants, and thus
affect the stability of stripe mRNA
(Nabel-Rosen et al., 2002). We
have also shown that overexpression of StripeB in the ectoderm leads to
elevation of How(L) (Nabel-Rosen et al.,
1999), and that StripeB enhances the ß-gal expression of
stripe enhancer trap where the P-element is located close to
the stripeB unique 5' UTR
(Becker et al., 1997).
Here we address the specific function of each Stripe protein isoform in tendon cell differentiation, and the contribution of How proteins to the post-transcriptional regulation of each Stripe isoform. We show that the expression of the two Stripe isoforms is tightly regulated and is linked to maturation of tendon cells. StripeB is expressed continuously from the precursor stage, while StripeA protein is detected only in the muscle-bound mature state. Consistently, each Stripe protein exhibits distinct biological activities and transcription output. Importantly, we demonstrate that the elevation of StripeA depends on the activity of How(S); embryos homozygous for how show specific reduction of the stripeA mRNA and protein levels, a phenotype that is shared with embryos lacking the splicing factor Crooked neck (Crn). Consistent with a role for How(S) in stripeA splicing, we demonstrate that How(S) promotes the splicing of stripeA-specific exons in S-2 cells. In tendon cells, How(S) is elevated only upon muscle binding, and its expression is further reinforced by StripeA activity, thus driving a single muscle-bound tendon to express high StripeA levels, leading to its irreversible maturation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
using RNA probes complementary to the unique 3' UTRs of each
how splice variant.
RT-PCR analysis of total RNA extracts
Total RNA was extracted from transfected cells 16 hours after transfection
or from embryos at stage 15-16. About 8x106 cells or 100
embryos were used for each extraction. RNA was extracted with the Nucleospin
RNA II kit (Machery Nagel). For RT-PCR, cDNA was synthesized using
Transcriptor First Strand cDNA Synthesis Kit (Roche) with 2 ugr template RNA.
RT-PCR was performed using the LightCycler FastStart DNA Master SYBR Green I
(Roche) kit according to the manufacturer's instructions. As a control,
primers specific for the ubiquitous transcription factor eIF4A or Tubulin,
were used, with a similar amount of total RNA. The following primers were used
in the RT-PCR reaction: for stripeA levels SrA504F-TGGACTACAGATGAAAATCC and
SrA726R-GCGGTTAGTTTGATTGATTC. For StripeB, SrB328F-CGCAGACCGACTACTAGGA and
SrB566R-CTTGAACAGACAGGTGTCG. For the in vivo splicing assay,
SrAint509F-CCTAGCCAGACCAGCTTTC and SrAint745R-GGTAAACGATCACTTTTTGGT were used
to detect the spliced form. To detect unspliced mRNA, a different reverse
primer, SrAint785R-GTAATAAATTCGGCCCCGC was used. All mRNA levels were
normalized to the level of a housekeeping gene in the same sample.
In vitro binding assay
The protein-RNA binding assay was performed as described previously
(Nabel-Rosen et al., 1999). An
intron sequence unique to SrA, with and without the How response element, was
used as a template, mixed with invitro-translated How(S) or
How(S)e44 HA-tagged proteins (TNT Coupled Reticulocyte Lysate
System, Promega). Binding was performed by adding 
1 µg biotin-labeled
RNA to 5 µl translated How proteins. The magnetic beads were then isolated,
washed, boiled in sample buffer and analyzed by western analysis with anti-HA
antibodies (monoclonal, 1:2000 dilution, Covance). As a nonspecific RNA
control, we used RNA transcribed from GFP cDNA and stripe intronic
sequence without the putative How-binding site.
Fly strains
yw (wild type), howstru, howe44
(Baehrecke, 1997),
mysXG43, UAS-How(S)
(Nabel-Rosen et al., 1999),
UAS-Hid, ptc-gal4, en-gal4 and 69B-gal4, mefgal4.
UAS-StripeA, and UAS-StripeB (created in our lab).
UAS-Crn (Klaembt Lab, Muenster, Germany).
Constructs
All HA-tagged proteins were created by PCR with a reverse primer containing
the HA sequence
(5'-TCATGCGTAATCTGGAACATCGTATGGGTAN18-3') and 18 bp
overlapping the 3' end of the specific gene. Howe44 mutant
constructs were created by PCR. The nuclear localization signal (NLS) of SV40
large T was added to HA-tagged How constructs by using a 3' primer that
contains the sequence encoding the NLS
(5'-TCAATCTACCTTTCTCTTCTTTTTTGGATCTGCGTAATCTGGAACATC-3') or the
mutated NLS
(5'-TCAATCTACTGCTCTTGCTGCTGCTGGATCTGCGTAATCTGGAACATC-3').
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) raised against the coding region of StripeB, recognizes both
Stripe isoforms. We compared the pattern of expression of each of the Stripe
isoform in embryos before, and following, muscle binding
(Fig. 1). At stage 14, in
regions where muscle binding to tendon cells did not take place, StripeB was
detected in all the tendon precursor cells, while StripeA levels were
extremely low (Fig. 1A-D). At
stage 15, StripeA expression gradually increased and was observed in
muscle-bound tendon cells (Fig.
1E-H). At stage 17, following the establishment of the entire
somatic muscle patterning, StripeA was significantly elevated in all the
muscle-bound tendon cells. StripeB was still detected in the neighboring
tendon precursor cells (Fig.
1I-L). This result supports a model in which StripeA is
significantly elevated only in muscle-bound tendon cells.
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We thus conclude that the two Stripe isoforms exhibit differential expression patterns; StripeB is expressed following the initial specification of the tendon precursors at stage 12 of embryonic development, whereas StripeA expression is uniquely expressed only following muscle binding to the tendon cells at later embryonic stages.
Ectopic expression of Stripe isoforms leads to distinct effects on somatic muscle patterning
To assess the biological activity of the distinct Stripe proteins, we
tested the effect of each Stripe isoform on the overall pattern of the somatic
musculature, following overexpression in the entire ectoderm. Previous studies
showed that overexpression of StripeB leads to a subtle effect on muscle
migration toward their attachment sites
(Becker et al., 1997;
Vorbruggen and Jackle, 1997).
We produced transgenic flies expressing UAS-stripeA, and repeated
this experiment using the pan-ectodermal 69B-gal4 driver, in
combination with either UAS-stripeA or UAS-stripeB,
comparing their effect on muscle patterning. Staining of embryos
overexpressing each of the Stripe isoforms with anti-Stripe antibody
(recognizing both Stripe isoforms) revealed that the levels of the ectopic
Stripe directed by both UAS constructs were comparable
(Fig. 3). Overexpression of
StripeA led to a severe disruption of the somatic muscle pattern
(Fig. 3C-F). Moreover, 52%
(n=89) of the embryos exhibited an additional phenotype in which germ
band retraction was arrested. We also noted that in these embryos,
overexpression of StripeA altered the shape of the cells in the ectoderm, and
they looked narrower. Notably, embryos that did undergo germ band retraction
also showed severe muscle pattern defects, suggesting that this phenotype did
not stem from germ band retraction defects per se. By contrast, overexpression
of StripeB did not affect germ band retraction and cell shape, and the effect
on muscle path-finding was less severe
(Fig. 3G,H). Similarly, a more
severe effect of StripeA overexpression relative to StripeB, was also detected
using the en-gal4 driver (not shown).
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Stripe proteins differ in their transcriptional output
To address whether Stripe proteins activate/repress a differential set of
downstream genes, we further examined the induction of a set of
tendon-specific genes following overexpression of each Stripe isoform. To this
end, the expression of short stop, slit and how was
analyzed. We used ptc-gal4, 69B-gal4 or en-gal4 to drive
either UAS-stripeB or UAS-stripeA in the ectoderm of embryos
at different stages.
A differential effect of each of the Stripe isoforms is detected on the
expression levels of Short stop (Shot), a cytoskeletal protein required for
late stages of tendon cell maturation
(Strumpf and Volk, 1998;
Subramanian et al., 2003).
Using the ptc-gal4 driver, we detected a significant elevation of
Shot protein as induced by StripeA, and no elevation by StripeB
(Fig. 4). Notably, although the
embryos were at the same developmental stage (stage 14-15), the overall shape
of the ectodermal cells in embryos overexpressing StripeA was more elongated
relative to control embryos or embryos overexpressing StripeB (see the
outlines of the cells, as marked by staining with the membrane-associated
marker Shot, in high magnification in Fig.
4D',E',F'). Using the en-gal4 driver we
consistently detected high induction of Shot by StripeA, and a very low Shot
induction by StripeB (not shown). The levels of the ectopic Stripe in the
embryos overexpressing either StripeA or StripeB were comparable (as shown in
Fig. 3), supporting a
significant positive effect of stripeA, and almost no effect of StripeB, on
Shot levels.
Slit, a secreted protein essential for guiding neuronal and muscle
migration toward target tissues is expressed by midline glial cells as well as
by tendon cells (Kramer et al.,
2001). Importantly, Slit activity in the segmental border tendon
cells takes place before muscle attachment to the tendon cell, and thus may be
induced by the early StripeB activity. To address the effect of Stripe
proteins on Slit expression, we tested Slit expression following ectopic
expression of Stripe proteins driven by 69B-gal4. In a wild-type
embryo, Slit protein expression is somewhat diffuse around the
Stripe-expressing cells. Following ectopic expression of either Stripe B or
StripeA, the expression of Slit was strongly induced (verified by microarray
analysis, not shown) and appeared in a spike-like structure in the entire
ectoderm (Fig. 5). As StripeB
is expressed earlier in tendon precursors, it appears that the relatively
early expression of Slit surrounding the tendon cells is probably due to
StripeB activity, while StripeA may be essential for the maintenance of Slit
in the muscle-tendon junction site.
Next, we tested the effect of Stripe proteins on the levels of the RNA-binding protein, How (Fig. 5). We performed in situ hybridization with probes specific to each of the How variants in embryos overexpressing Stripe using the en-gal4 driver. The results show that whereas both Stripe isoforms induced elevation of How(L), only embryos overexpressing StripeA showed elevation in How(S) mRNA (Fig. 5H,I,K,L), indicating that StripeA, but not StripeB, elevates How(S) levels.
Tissue-specific transcription factors often exhibit autoregulatory
activities that ensure their constitutive expression following an initial
external input. StripeB was shown previously to autoregulate its own
expression as inferred from its ability to induce ß-gal expression
through the stripe enhancer trap
(Becker et al., 1997), thus
creating a positive transcriptional autoregulatory control of its expression.
To test whether StripeB controls StripeA, or vice versa, we performed an
RT-PCR analysis on embryos overexpressing Stripe proteins under the
ptc-gal4 driver. To monitor endogenous stripe levels, we
used primers specific for their unique 5' UTR, which are not included
within the pUAST stripe cDNA constructs, used to produce the
transgenic flies. While this analysis demonstrated that StripeB positively
regulates its own expression, and elevated endogenous stripeB levels
to about fourfold (confirming previous data), it induced a 50% reduction of
stripeA levels (Fig.
5M). Conversely, overexpression of stripeA induced no
significant changes in stripeA levels, or in stripeB
levels.
These results are consistent with the notion that the Stripe proteins may share a set of genes that are commonly activated, e.g. slit and thrombospondin (A. Subramanian and T.V., unpublished), as well as an additional set of genes that differ in their sensitivity to each of the Stripe isoforms. Moreover, it appears that distinct mechanisms control the expression of each of the Stripe proteins. While StripeB is autoregulatory, StripeA does not exhibit such activity.
Taken together, these experiments demonstrate that in addition to commonly induced genes, StripeA induces the expression of genes characteristic of the mature tendon state, such as shot and how(S). The strong muscle phenotype observed following the expression of StripeA might result from the combination of overexpressing both the early and late genes.
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).
These results suggest that in wild-type embryos How is required for elevation
of stripeA mRNA levels. Previously, we showed that when expressed ectopically in the entire ectoderm, How(L) represses stripeB levels. To further address whether How proteins exhibit differential effects on StripeA levels, we overexpressed each How isoform in the ectoderm and followed StripeA expression in the segmental border tendon cells. We found that whereas How(L) repressed StripeA levels (similar to the case for StripeB), How(S) induced elevation of StripeA levels (Fig. 6I-K). This effect was also demonstrated at the mRNA level by performing quantitative RT-PCR on mRNA extracted from embryos overexpressing How(S) with stripeA-specific primers Fig. 6M). By contrast to an expected elevation in stripeB mRNA levels following overexpression of How(S), we detected a slight decrease in the mRNA levels (Fig. 6M). This may be explained by an indirect positive effect of How(S) on a negative regulator of stripeB.
We conclude that How is required for the elevation of StripeA levels following muscle-dependent maturation of tendon cells, and that How(S) appears to be the isoform required for this elevation.
The splicing of stripeA might be mediated by How(S)
We next wished to elucidate the level of mRNA metabolism at which How(S)
controls the elevation of stripeA mRNA. Quaking, the mammalian
homolog of How, had been shown to affect myelin basic protein mRNA nuclear
export and stability (Larocque et al.,
2002; Li et al.,
2000), as well as the splicing of myelin-associated glycoprotein
in the nervous system (Wu et al.,
2002).
To address the possible involvement of How in the splicing of stripeA, a 5 kb minigene composed of a genomic fragment that includes part of the 3' end of the stripeA first exon and part of the 5' end of the second exon, together with the entire intronic sequence in between these exons, was sub-cloned into a pUAST expression vector (Fig. 7A). Importantly, the stripeA minigene did not contain the 3' UTR that is common to stripeA and stripeB and was shown previously to be responsive to How activity, thus eliminating any possible effect of How on the stability of the spliced RNA of the minigene through How binding to the 3' UTR. The efficiency of the splicing reaction of the two exons was monitored in Schneider cells (S-2 cells) transfected with this construct, using a set of primers representing the expected spliced (599F, 5020R) RNA species (see the scheme in Fig. 7A). The levels of the spliced and unspliced RNAs were measured by RT-PCR in the presence of various How isoforms that were co-transfected into S-2 cells. The results showed that How(S) induced a threefold elevation of the stripeA-specific fragment, whereas in the presence of How(L), the levels of this fragment were slightly reduced. This is consistent with the active involvement of How(S) in the splicing of stripeA-specific exons.
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Previously, we showed that a mutated form of How(L), How(L)YtoG, in which the nuclear retention of How(L) is abrogated, does not exhibit repressive activity. When tested in the splicing assay, we found that this cytoplasmic How(L)YtoG slightly elevated the levels of the spliced fragment (Fig. 7B). Western analysis showed that the levels of all the various transfected How proteins in each transfection experiment were comparable (Fig. 7C).
The stripeA intronic sequence contains a potential binding site
for How, as characterized recently in our lab (Israeli and T.V., unpublished).
We therefore tested for the possible physical association of How(S) with
stripeA intronic sequences. A fragment of 200 bp from the first
intron of stripeA, containing the putative How-binding site, was
transcribed, labeled with biotin and added to a mixture containing HA-tagged
How(S). As a negative control we used a mutated How(S)e44 form,
which imitates the ethane methyl sulfonateinduced severe
howe44 allele. The molecular defect in this allele is a
missense mutation (R to C) in the RNA-binding domain. In our protein-RNA
binding assay the mutated Howe44 protein does not bind RNA
(Nabel-Rosen et al., 1999).
The RNA-protein complexes were precipitated on avidin beads, and the presence
of How was detected by western analysis with anti-HA antibodies. Specific
binding of How(S) but not of the mutant How(S)e44 to the
stripeA-specific intron was detected
(Fig. 7D). Thus, How(S) may
mediate its positive effect on the RNA levels of the stripeA genomic
fragment following a physical association between these two elements.
The results of these experiments support the involvement of How(S) in facilitating the splicing of stripeA, and suggest that the correct subcellular distribution of How is crucial for its positive effect on stripeA. We cannot, however, exclude a possible additional effect of How(S) on stripeA stability.
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). Moreover, our results suggest that the interaction
between How(S) and Crn occurs in the cytoplasm, consistent with our analysis
(Fig. 7) that only cytoplasmic
How(S) exhibits a positive enhancement of the spliced stripeA
minigene. Based on these observations, we went on to test the contribution of
Crn to the specific elevation of stripeA during tendon cell
maturation. To this end, we tested the relative levels of stripeA and
stripeB by quantitative RT-PCR 14-16 hours AEL on crn
homozygous mutant embryos relative to wild-type embryos at the same age. We
compared the level of each of the stripe mRNA variants to
tubulin (
Tub84B) mRNA levels in each sample. This
eliminates a non-specific reduction in the total mRNA levels caused by the
lack of Crn. We detected about 40% reduction of stripeA levels in the
crn homozygous mutant embryos. No reduction was detected in
stripeB levels (Fig.
8A). The reduction of stripeA in crn mutant
embryos is consistent with the idea that the production of stripeA
during tendon cell maturation depends on stripeA splicing. Further
support for the requirement of Crn to tendon cell maturation is a consistent
reduction in the levels of Shot in tendon cells of stage 16 crn
mutant embryos (Fig. 8D). We
showed above that StripeA is sufficient to induce Shot expression, and its
expression is characteristic of the mature state of tendon cells
(Fig. 4). Here we show that
Shot levels are also reduced in crn mutants, consistent with the
reduction of stripeA in these embryos. Staining of crn
mutant embryos with antibodies to Stripe (recognizing both isoforms) showed no
reduction in total Stripe levels (not shown), supporting a specific effect of
Crn on Shot levels. Importantly, the reduction of Shot in crn mutants
is comparable with the reduction of Shot in how mutants (see
Fig. 6F).
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These results support the idea that a Crn-How-dependent splicing event is essential for tendon cell maturation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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, our results show that premature overexpression of StripeA in the
entire ectoderm leads to severe defects in germ band retraction.
Stripe was shown previously to mediate both the determination of precursor
cells as well as their maturation and ability to undergo specific temporal and
spatial regulation (Becker et al.,
1997; Frommer et al.,
1996). Our 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.
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.
We suggest the following model 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 our experiments, which 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 (Nabel-Rosen et al.,
1999). 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.
The activity of How proteins in regulating Stripe levels
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
(Jan et al., 1999). Similarly
Quaking controls mRNA stability (Li et
al., 2000) as well as RNA splicing
(Wu et al., 1999), and
possibly also mRNA nuclear export and localization
(Larocque et al., 2002). 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
(Nabel-Rosen et al., 2002),
this study suggests that How(S) has an additional activity in regulating the
splicing of stripeA. Consistent with our 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 (Park et al., 2004). Our
previous studies suggested that the ability of How proteins to stabilize
stripe mRNA is mediated by the 3' UTR of stripe
(Nabel-Rosen et al., 2002).
However, the splicing of stripeA appears to be regulated by its
specific intronic sequences.
|
). In a parallel study, we have demonstrated that crn
and how mutants exhibit closely related phenotypes, affecting glial
cell maturation. Importantly, we show that both Crn and How(S) proteins [but
not How(S)-NLS] coprecipitated from S-2 cell extracts, indicating that both
proteins are associated in a common protein complex in the cytoplasm
(Edenfeld et al., 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 et al., 2006).
Furthermore, in crn mutants StripeA, but not StripeB, levels are
reduced, and this is reflected in the reduction of Shot levels. 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.
In summary, we describe a molecular mechanism that is based on post-transcriptional control, by which cell differentiation is induced and maintained by local interactions with neighboring cells.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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