Musashi homologs in invertebrates

A Caenorhabditis elegans Musashi homolog, MSI-1 has been identified, whose RNA-recognition motifs show extensive similarity to those of Drosophila and vertebrate Musashi proteins. A msi-1 mutant was isolated and males with this mutation have a mating defect. C. elegans male mating behavior includes a distinct series of steps: response to contact, backing, turning, vulva location, spicule insertion, and sperm transfer. msi-1 is required for the turning and vulva location steps. Like other Musashi family members, MSI-1 is expressed specifically in neural cells, including male-specific neurons required for turning and vulva location. However, msi-1 is not expressed in proliferating neural progenitors in C. elegans, unlike the Musashi family genes in other systems. These results suggest that msi-1 is expressed specifically in postmitotic neurons in C. elegans. msi-1 is required for full development of male mating behavior, possibly through regulation of msi-1 expressing neurons (Yoda, 2000).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Although neurons are dispersed throughout the epidermis in the adult, it has not been demonstrated that neurogenesis in the embryo is uniform. To determine the site of neurogenesis, the domains of expression were localized for three orthologs of pan-neural genes of chordates and Drosophila -- nrp/ musashi, sox1/2/3/ soxneuro, and hu/elav. The first two are markers of proliferating neuron precursors, whereas the third is a marker of differentiating neurons. All are expressed in the neural plate of various chordates, but not in the epidermis. nrp/musashi and sox1/2/3 /soxneuro are expressed in the entire ectoderm of the early S. kowalevskii embryo (except for the ciliated band, which all probes except emx fail to stain). In later stages, the expression remains strong in the prosome and declines in the metasome, correlating with Bullock's observation of decreasing neuron density posteriorly. In sections, weak expression of nrp/musashi can be detected in the posterior endoderm, possibly correlated with a sparse endodermal nerve net. Hu/elav exhibits similar diffuse staining throughout the ectoderm in early stages. Additionally, Hu/elav staining remains strong along the posterior dorsal midline at later stages, in a punctate pattern perhaps reflecting a concentration of early-differentiating nerves at this site. In sagittal sections of embryos, hu/elav expression appears localized toward the basal side of the ectoderm (basiepithelial); it is absent from the mesoderm. Thus, S. kowalevskii shows pervasive neurogenesis with no large, contiguous nonneurogenic subregion, as occurs in chordates (Lowe, 2003).

The enhancer trap technique, established in Drosophila, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. A Minos insertion in the Ci-Musashi gene is responsible for the altered GFP expression. Ci-Musashi shows a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis (Awazu, 2004).

Characterization of the RNA-binding protein Musashi1 in zebrafish

Musashi (Msi) is an evolutionarily conserved gene family of RNA-binding proteins (RBPs) that is preferentially expressed in the nervous system. The first member of the Msi family was identified in Drosophila. Drosophila Msi plays an important role in regulating asymmetric cell division of the sensory organ precursor cells. The mammalian orthologs, including human and mouse Musashi1 (Msi1), are neural RBPs that are strongly expressed in fetal and adult neural stem/progenitor cells (NS/PCs). Mammalian Msi1 contributes to self renewal of NS/PCs through translational regulation of several target mRNAs. The zebrafish Msi ortholog zMsi1 was identified and characterized in this study. The normal spatial and temporal expression profiles for both protein and mRNA were determined. A series of splice variants were detected. Overall, zMsi1 was strongly expressed in neural tissue in early stages of development and exhibited similarity to mammalian Msi1 expression patterns. To reveal the in vivo function of zMsi1, morpholinos against Msi1 were introduced into one-cell stage zebrafish embryos. Knock down of zmsi1 frequently resulted in aberrant formation of the Central Nervous System (CNS). These results suggest that Msi1 plays roles in CNS development in vertebrates (Shibata, 2012).

Vertebrate Musashi homologs and neural development

Mouse-Musashi-1 RNA-recognition motif is 60% identical to Xenopus NRP-1 and 95% identical to Drosophila MSI. In the C-terminal half of m-Msi-1, which does not contain an RNA-recognition motif, m-Msi-1 shows a 30% homology to Drosophila MSI. m-Msi-1 is preferentially expressed in neural tissues, especially mitotically active neural precursor cells within the CNS. The m-Msi-1 expressing cells overlap the major population of nestin intermediate filament positive cells. Lineage analysis using single neuroepithelial cell culture systems reveal that m-Msi-1 is highly enriched in CNS cells, precursors of neuronal and glial cells. However, m-Msi-1 expression is rapidly down-regulated during neural differentiation. Expression of m-Msi-1 protein shows a pattern complementary to that of another mammalian RNA-binding protein, Hu (a mammalian homolog of the Drosophila neuron-specific RNA binding protein ELAV). Hu is exclusively expressed in postmitotic neurons in the CNS. In vitro studies indicate that these two proteins have distinct RNA target species. Therefore, it is likely that m-Msi-1 and Hu have distinct roles in neurogenesis that are relevant to those of Drosophila MSI and ELAV, respectively (Sakakibara, 1996).

There is an increasing interest in the role of RNA-binding proteins during neural development. Mouse-Musashi-1 (m-Msi-1) is a mouse neural RNA-binding protein with sequence similarity to Drosophila musashi, an essential gene for neural development. m-Msi-1 is highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic CNS development. The present study characterizes m-Msi-1-expressing cells in the postnatal and adult CNS. Postnatally, m-Msi-1 is expressed in proliferative neuronal precursors in the external granule cell layer of the cerebellum and in the anterior corner of the subventricular zone of the lateral ventricles. In gliogenesis, the persistent expression of m-Msi-1 is observed in cells of the astrocyte lineage ranging from proliferative glial precursors in the subventricular zone (SVZ) to differentiated astrocytes in the parenchyma. m-Msi-1 is still expressed in proliferating cells in the adult SVZ, which may contain neural precursor or stem cells. Another neural RNA-binding protein Hu (the mammalian homolog of a Drosophila neuronal RNA-binding protein Elav) is present in postmitotic neurons throughout the development of the CNS; its pattern of expression was compared with that of m-Msi-1. These observations imply that these two RNA-binding proteins may be involved in the development of neurons and glia by regulating gene expression at the post-transcriptional level (Sakakibara, 1997).

Musashi1 (Msi1) is a mammalian neural RNA-binding protein highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic and postnatal CNS development. Musashi2 (Msi2), a novel mammalian RNA-binding protein that exhibits high sequence similarity to Msi1 has been identified. The Msi2 transcript appears to be distributed ubiquitously in a wide variety of tissues, consistent with the mRNA distribution of its Xenopus homolog, xrp1. However, the present study reveals cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. Interestingly, Msi2 is expressed prominently in precursor cells in the ventricular zone and subventricular zone with the same pattern as Msi1 throughout CNS development. In the postnatal and adult CNS, this concurrent expression of Msi2 and Msi1 is seen in cells of the astrocyte lineage, including ependymal cells, a possible source for postnatal CNS stem cells. During neurogenesis, the expression of both Msi2 and Msi1 is lost in most postmitotic neurons, whereas Msi2 expression persists in a subset of neuronal lineage cells, such as parvalbumin-containing GABA neurons in the neocortex and neurons in several nuclei of the basal ganglia. Msi2 may have a unique role that is required for the generation and/or maintenance of specific neuronal lineages. Furthermore, in vitro studies show that Msi2 and Msi1 have similar RNA-binding specificity. These two RNA-binding proteins may exert common functions in neural precursor cells by regulating gene expression at the post-transcriptional level (Sakakibara, 2001).

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U(n)AGU (n = 1 to 3) sequences which are essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammalian numb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3'-untranslated region (UTR) and binds endogenous m-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays have demonstrated that Msi1 decreases the reporter activity without altering the reporter mRNA level. Thus, these results suggest that Msi1 regulates the expression of its target gene at the translational level. Furthermore, Notch signaling activity is increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene (Imai, 2001).

Homologs of the Musashi family of RNA-binding proteins are evolutionarily conserved across species. In mammals, two members of this family, Musashi1 (Msi1) and Musashi2 (Msi2), are strongly coexpressed in neural precursor cells, including CNS stem cells. To address the in vivo roles of msi in neural development, mice were generated with a targeted disruption of the gene encoding Msi1. Homozygous newborn mice frequently develop obstructive hydrocephalus with aberrant proliferation of ependymal cells in a restricted area surrounding the Sylvius aqueduct. These observations indicate a vital role for msi1 in the normal development of this subpopulation of ependymal cells, which has been speculated to be a source of postnatal CNS stem cells. Histological examination and an in vitro neurosphere assay showed, however, that neither the embryonic CNS development nor the self-renewal activity of CNS stem cells in embryonic forebrains appears to be affected by the disruption of msi1, but the diversity of the cell types produced by the stem cells is moderately reduced by the msi1 deficiency. Therefore, antisense ablation experiments were performed to target both msi1 and msi2 in embryonic neural precursor cells. Administration of the antisense peptide-nucleotides, which were designed to specifically down-regulate msi2 expression, to msi1- CNS stem cell cultures drastically suppresses the formation of neurospheres in a dose-dependent manner. Antisense-treated msi1- CNS stem cells show a reduced proliferative activity. These data suggest that msi1 and msi2 are cooperatively involved in the proliferation and maintenance of CNS stem cell populations (Sakakibara, 2002).

Vertebrate Musashi homologs and functions in non-neural development

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 is present in ovary; therefore, an Msi1-specific cRNA and monoclonal antibody was used to investigate Msi1 expression. Msi1 mRNA is expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrate that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein is expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 is detected in granulosa cells and their precursors. In Sertoli cells, protein is detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining is stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells (Saunders, 2002).

In the amphibian gastrointestine during metamorphosis, the primary (larval) epithelium undergoes apoptosis. By contrast, a small number of undifferentiated cells including stem cells actively proliferate and differentiate into the secondary (adult) epithelium that resembles the mammalian counterpart. In the present study, to clarify whether Musashi-1 (Msi-1), an RNA-binding protein, serves as a marker for progenitor cells of the adult epithelium, Msi-1 expression was examined in the Xenopus laevis gastrointestine by using in situ hybridization and immunohistochemistry. Similar expression profiles of Msi-1 were observed at both mRNA and protein levels. In both the small intestine and the stomach, the transient expression of Msi-1 during metamorphosis spatio-temporally correlates well with active proliferation of the progenitor cells including stem cells of the adult epithelium but expression does not with apoptosis of the larval epithelium. As the adult progenitor cells differentiate into organ-specific epithelial cells after active proliferation, Msi-1 expression is rapidly downregulated. Therefore, Msi-1 is useful to identify the adult progenitor cells that actively proliferate before final differentiation in the amphibian gastrointestine. Furthermore, culture experiments have shown that thyroid hormone (TH) organ-autonomously induces Msi-1 expression only in the adult progenitor cells of the X. laevis intestine in vitro as in vivo. However, TH can not induce Msi-1 expression in the intestinal epithelium separated from the connective tissue; under these circumstances the adult epithelium never develops. These results suggest that Msi-1 expression is upregulated by TH in the adult progenitor cells under the control of the connective tissue and plays important roles in their maintenance and/or active proliferation during amphibian gastrointestinal remodeling (Ishizuya-Oka, 2003).

Musashi homologs in tumors and pathology

Tumor cells arising from a particular tissue may exhibit the same gene expression patterns as their precursor cells. To test this proposition, the expression of a neural RNA-binding protein, Musashi1, was analyzed in primary human central nervous system (CNS) tumors. In rodents, Musashi1 is expressed predominantly in proliferating multipotent neural precursor cells, but not in newly generated postmitotic neurons. The expression of Musashi1 is downregulated with the successive progression of neurogenesis. In normal adult human tissues, low levels of Musashi1 expression were detected in brain and testis by RT-PCR analysis. In an RNA panel of 32 cancer tissues and cell lines, elevated expression of Musashi1 was seen in all five malignant gliomas studied, in contrast to the slight expression seen in other tumor cells, including those in several melanomas and a prostate cancer. Western blot analysis shows strong Musashi1 expression in malignant gliomas compared with nonneoplastic brain tissue. By immunohistochemical analysis, glioblastomas, the most malignant form of glioma, show higher Musashi1 expression than less malignant gliomas. Tumors with strong Musashi1 expression tend to have high proliferative activity. Thus, the expression of Musashi1 correlates with the grade of the malignancy and proliferative activity in gliomas. These results suggest that primary CNS tumors may share gene expression patterns with primitive, undifferentiated CNS cells and that Musashi1 may be a useful marker for the diagnosis of CNS tumors (Toda, 2001).

The pathogenetic role of the P210 BCR/ABL1 fusion gene in the chronic phase of chronic myeloid leukemia (CML) has been well established. In contrast, the genetic mechanisms underlying the disease progression into the accelerated phase (AP) and the final blast crisis (BC) remain poorly understood. Two cryptic balanced translocations, t(7;17)(p15;q23) and t(7;17)(q32-34;q23), have been identified in CML AP/BC, using multicolor fluorescence in situ hybridization. A novel gene in 17q23, Musashi-2 (MSI2), encoding a putative RNA-binding protein, is rearranged in both cases and a MSI2/HOXA9 fusion gene is formed in the case with the 7p15 breakpoint. The identified in-frame MSI2/HOXA9 fusion transcript retains both of the RNA recognition motif domains of MSI2, which is fused to the homeobox domain of HOXA9, and is likely to play an important role in the disease progression of CML (Barbouti, 2003).

The tau gene encodes a microtubule-associated protein expressed by neuronal and glial cells. Abnormal deposits of Tau protein are characteristic of several neurodegenerative disorders. Additionally, mutations affecting tau pre-mRNA alternative splicing of exon 10 are associated with frontotemporal dementia and Parkinsonism linked to chromosome 17. In rodents, this process is developmentally regulated by thyroid hormone (T3) causing the predominance of exon 10-containing transcripts. musashi-1 (msi-1) gene is induced by T3 during rat brain development and in N2a cells. T3 increases msi-1 mRNA level in an actinomycin D-sensitive, cycloheximide-resistant fashion without affecting its half-life, which suggests a transcriptional effect. Both ectopic Msi-1 expression and T3 treatment increases the proportion of exon 10-containing tau transcripts. Furthermore, antisense msi-1 expression inhibits T3 action. These results show that msi-1 mediates the posttranscriptional regulation of tau expression by T3 (Cuadrado, 2000).

Tumor cells often express phenotypic markers that are specific to the cells from which they originated. A neural RNA-binding protein, Musashil, is an evolutionarily well-conserved marker for neural stem cells/ progenitor cells. To examine the origin of gliomas, the expression was examined of the human Musashil homolog, MSI1, in human glioma tissues and in normal human adult and fetal brains. In the normal human brain MSI1 is expressed in cells located in the ventricular and subventricular zones, in GFAP-negative glial cells, and in GFAP-positive astrocytes. In glioblastomas, MSI1 is expressed in GFAP-negative tumor cells forming foci that are clearly demarcated and surrounded by GFAP-positive cells. Tumor cells arranged in pseudopalisades were also strongly immunoreactive with MSI1 antibodies. The percentage of MSI1-labeled tumor cells increases in higher-grade astrocytomas and correlates with proliferative activity. These results indicate that MSI1 is an excellent marker for neural progenitor cells including neural stem cells in normal human brains. Furthermore, the expression of MSI1 correlates well with the immature nature as well as the malignancy of tumor cells in human gliomas. Thus, it is expected that the analysis of MSI1 expression will contribute to the understanding of the cellular origin and biology of human gliomas (Kanemura, 2001).

The Musashi 1 controls the splicing of photoreceptor-specific exons in the vertebrate retina

Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. This study shows that photoreceptor cells (see photoreceptors in Drosophila), a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such "switch-like" exons. These results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments (Murphy, 2016).

musashi: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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