Targets of Activity

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).

musashi: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.