Musashi
(Msi), the Drosophila neural RNA-binding protein, plays a part in eye development. Msi expression is observed in
the nuclei of all photoreceptor cells (R1-R8). Although a msi
loss-of-function mutation results in only weak abnormalities in
photoreceptor differentiation, the msi eye phenotype is
significantly enhanced in a seven in absentia (sina) background.
sina is known to be involved in the degradation of the Tramtrack
(Ttk) protein, leading to the specification of the R7 fate. Msi also functions
to regulate Ttk expression. The sina msi mutants show significantly
high ectopic expression of Ttk69 and failure in the determination of the R1,
R6, and R7 fates. Other photoreceptor cells also fail to differentiate, with
abnormalities occurring late in the differentiation process. These results
suggest that Msi and Sina function redundantly to downregulate Ttk in
developing photoreceptor cells (Hirota, 1999).
To examine the pattern of Msi expression in eye development, a
monoclonal antibody was generated against the Msi protein. The N-terminal
region of Msi (210 amino acids) was used as the antigen for immunization.
The antibody 3A5 recognizes three protein species with relative molecular
masses of 60-70 kDa in immunoblots of protein extracts from wild-type eye
discs. These sizes are compatible with the molecular weight predicted from
the cDNA sequence (63 kDa, 606 amino acids). Msi may receive three
different types of modification, resulting in the three bands observed. In the
immunoblot analysis, the intensities of these bands were reduced in
msi1/+ extracts and undetectable in msi1/msi1extracts.
Immunohistochemistry with this antibody does not detect any signals in
msi1/msi1 discs. Taken together, these results suggest that
the antibody 3A5 recognizes the Msi protein specifically. To determine the
types of cells expressing Msi in developing eye discs, double staining with
antibodies against Msi and a neuronal marker, Elav (Robinow and White,
1991), was performed. In eye imaginal discs from third-instar larvae, a low
level of Msi expression is observed in cells forming a stripe immediately
anterior to the morphogenetic furrow (MF); a more intense signal is also observed in the
developing ommatidia posterior to the MF. Msi expression is restricted to
nuclei in cells posterior to the MF, although it appears not to be restricted to
nuclei anterior to it. Compared with Elav staining, some cells anterior and
just posterior to the MF are immunopositive for Msi, but negative for Elav.
To compare Msi and Elav expression in detail, discs were stained with both
Msi and Elav antibodies using HRP labeled secondary antibodies. The
ommatidial clusters located 4 to 6 rows posterior to the MF contain three
Elav-positive neurons: R8, R2, and R5. These three cells are also positive for
Msi, and Msi expression is also detectable in the presumptive R3 and R4 cells
prior to their Elav expression. At 7 to 10 rows posterior to the MF, the
ommatidial clusters contain five Elav-positive neurons: R8, R2, R5, R3, and
R4. In addition to these five cells, three Msi-positive cells are located basally
adjacent to R2, R8, and R5; these locations correspond to the presumptive R1, R6, and
R7 cells. This result suggests that expression of Msi begins earlier than that
of Elav during the neuronal differentiation of photoreceptor cells. Staining of
pupal retinas 40 h after puparium formation (40 h APF) with the anti-Msi
antibody shows that all photoreceptor cells continue to express Msi protein
during pupal eye development. Msi is also expressed in the photoreceptor
cells of adult flies (Hirota, 1999).
To investigate the functions of Msi during eye development, the eye
phenotype of the msi1/msi1mutants was examined.
msi1/msi1eyes contain abnormal ommatidia, with deformed
rhabdomeres and/or irregular orientation, at a low frequency (3.05%).
Staining developing msi1/msi1-eye discs with antibodies
against several neuronal markers reveals that the number of photoreceptor
cells is not affected, suggesting that msi is involved in late processes
of photoreceptor cells differentiation, including the formation of
rhabdomeres. However, the penetrance of the
msi1/msi1phenotype is so low that it was difficult to
investigate the Msi function. Also examined were the genetic interactions of msi with
ttk, a possible target gene of Msi, and sina, a factor involved
in the degradation of Ttk. Strong genetic interactions occur
between msi and sina mutations in eye development. In
wild-type flies, the compound eye has a regular array of ommatidia, each of
which contains eight photoreceptor cells (R1-R8). At the R7 level, the
rhabdomeres of seven photoreceptor cells (R1-R7) are arranged in a
characteristic asymmetrical trapezoid. Since Sina is essential for the
differentiation of R7, R7 is missing in 90% of the ommatidia of
sina2/sina3 mutants (in which little if any functional gene products
are produced) and the external morphology of the eyes shows a slight
roughness. Notably, the eye phenotype of the double homozygous mutants
of msi and sina (sina2msi1/sina3msi1) show
synergistic, but not additive, enhancement. The external morphology of the
double homozygous mutants shows strikingly disturbed ommatidial arrays.
Most of the rhabdomeres are severely deformed and no ommatidia contain
more than five rhabdomeres, suggesting that msi and sina
have important and distinct roles in the differentiation of photoreceptor
cells. To confirm the function of Msi, rescue experiments of sina msi
double mutants by Msi were performed. A genomic region containing the
wild-type msi gene (referred to as P[msi1+]) was introduced
into the sina msi mutant flies by P element-mediated germline
transformation. The P[msi1+] fragment significantly rescues the
defects in the sina msi eyes. The external morphology of the eye and
the formation of rhabdomeres are nearly normal. This result indicates that
the sina msi eye phenotype is partially due to the loss of msi
function. Since Msi contains two RNA recognition motifs (RRMs), RRM-A and
RRM-B, the RNA-binding activity of Msi is likely to be essential for its
rescuing activity. To test this possibility, a transgenic rescue experiment was
performed in which mutant Msi proteins whose RNA-binding activities were
designed to have been abolished were expressed. The RRM domains contain a
consensus sequence that is composed of two highly conserved short
segments, referred to as RNP1 (ribonucleoprotein octamer consensus) and
RNP2. Since the aromatic side chains of RNP1 are known to be crucial for
RNA binding, mutations that change phenylalanine to alanine in three places
in the RNP1 were induced into both of the two RRMs of Msi (referred to as
P[msiA*B*]). The P[msiA*B*] fragment does not rescue the
sina msi eye phenotype. Additionally, P[msi1+] fully rescues
the weak defects in the msi1eyes described above, while
P[msiA*B*] had no effect. Taken together, it is concluded that the
RNA-binding ability of Msi is involved in normal eye development (Hirota,
1999).
To examine how the sina and msi mutations causes the
severe eye defects described above, the neuronal differentiation of this
double mutant was examined by staining with anti-Elav antibody. All the
ommatidia posterior to the eighth row from the MF contain eight
photoreceptor cells in wild-type eye discs. In the same region, 90% of the
ommatidia of sina2/sina3 lack R7. In the same region, all the
ommatidia of sina2msi1/sina3msi1contain only five
cells, consistent with the appearance of the phenotype in adult eyes. In the
sina2msi1/sina3msi1eye discs, a nearly normal
pattern of Elav staining was observed in developing ommatidia up to the
five-cell precluster stage, which is composed of R2, R3, R4, R5, and R8.
Subsequently, however, R1, R6, and R7 are never added to the ommatidia.
These results suggest that Msi and Sina have redundant functions in the cell
fate determination of R1, R6, and R7. Furthermore, in the posterior region of
the eye discs, the spatial arrangement of the five cells in each ommatidium
changes and overlaps abnormally. In the most posterior region of the eye
discs, many ommatidia with reduced numbers of Elav-positive cells are
observed. These results indicate that R2, R3, R4, R5, and R8, which had once
expressed Elav, gradually have their spatial arrangement in the ommatidia
disrupted, and that some of the photoreceptor cells fail to maintain Elav
expression, resulting in a strikingly deformed eye (Hirota, 1999).
To confirm the requirement of Sina and Msi for the cell fate
determination of R1 and R6, the sina msi eye discs were stained with
antibody against Bar, an R1/R6-specific marker. Bar is expressed in R1 and
R6 in wild-type eye discs. In 40% of the ommatidia of sina2/sina3 eye
discs, the number of Bar-positive cells is reduced to one or zero,
suggesting that Sina has some roles in the cell fate determination of R1 and
R6, where Sina is known to be expressed. Consistent with anti-Elav staining,
Bar-positive cells completely disappear in the
sina2msi1/sina3msi1eye discs. In combination with
sina1, a hypomorphic allele, instead of sina2, one or two
Bar-positive cells per ommatidium are detected in 50% of the
sina2msi1/sina3msi1ommatidia, confirming that Msi
and Sina have redundant functions required for the cell fate determination
of R1 and R6 (Hirota, 1999).
Sina has been suggested to be involved in the degradation of Ttk, a
general inhibitor of neuronal differentiation. Since sina msi double
mutants show defects in neuronal differentiation, the possibility that the
expression pattern of Ttk69 is different in these animals was tested by
examining the Ttk expression pattern in eye discs stained with anti-Ttk69
antibody. In wild-type eye discs, Ttk69 is detected in four cone cells per
ommatidium. The expression pattern of Ttk69 in the
msi1/msi1eye discs is indistinguishable from that of
wild-type. In sina2/sina3 eye discs, five cells were labeled in 5% of
the ommatidia, suggesting that degradation of Ttk69 is reduced. Notably,
50% of the ommatidia in the sina2msi1/sina3msi1eye
discs contain additional Ttk69-expressing cells, indicating that the average
number of cells per ommatidium that express Ttk69 ectopically is larger in
sina2msi1/sina3msi1 than in sina2/sina3 eye discs.
Since both photoreceptor and cone cells are deformed at later stages of
development the cell types ectopically expressing Ttk69 could not be
identified. This result suggests that the Ttk69 expression is negatively
regulated by both Msi and Sina. Consistent with this idea, the morphology of
sina msi double mutants is significantly recovered in a heterozygous
background for ttkosn (sina2msi1ttk osn/sina3msi1), a
mutation that disrupts expression of both the Ttk69 and Ttk88 proteins.
The external morphology of the eye and the formation of the rhabdomeres
are nearly normal. Thirty percent of the
sina2msi1ttkosn/sina3msi1ommatidia show the normal
number and arrangement of photoreceptor cells and are indistinguishable
from wild-type ommatidia. These results suggest that the severe eye
phenotype of the sina2msi1/sina3msi1 double mutant may result
from an elevation in Ttk expression levels (Hirota, 1999).
These results demonstrate that Msi and Sina redundantly function as
factors required for the downregulation of Ttk69; however, the mechanism
remains unknown. A recent study, exploring the target RNA for Msi,
indicates that TTK69 mRNA contains multiple sites that could
potentially be recognized by Msi. Therefore, it is likely that Msi binds to the
TTK69 mRNA to inhibit its translation or reduce its stability. In
contrast, Sina has been shown to function with Phyl to target Ttk protein for
degradation. Thus, Msi and Sina are likely to function to down-regulate Ttk
at different levels in independent manners. If one functions via the other, the
phenotype of the sina msi double null mutants would be identical to
the phenotype of single null mutants for the gene that functions downstream
of the other. Instead, the sina msi mutants show a synergistically
enhanced eye phenotype that is much more severe than that of the single
mutants. Furthermore, the finding that a half-reduction in the gene dosage
of ttk suppresses the sina msi double null mutants indicates
that ttk is downstream of msi and sina. Taken
together, the expression of Ttk is likely to be regulated posttranscriptionally
by factors including Msi, and posttranslationally by Sina, Phyl, and Ebi (a WD
repeats protein). The negative regulation of Ttk by Msi and Sina is required
for both the early processes of R1, R6, and R7 differentiation and the late
processes of the differentiation of other photoreceptor cells in eye
development. Further studies will extend knowledge of how the
posttranscriptional regulation of gene expression by Msi controls ommatidial
development (Hirota, 1999).
A conserved three-nucleotide core motif defines Musashi RNA-binding specificity
Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are over-expressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. This study analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. It was found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila Msi is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins (Zearfoss, 2014).