BACKGROUND
a fundamental aspect of nervous system development and function is the expansion of basic cellular polarity into a highly diverse array of neural phenotypes specialized for directional information processing.
morphological polarization of glia and neurons alike has long been used as a basic principle for classification of these cells, which was fundamental to the initial understanding of their functions and interactions  <cit> .
in neurons, the formation and maintenance of dendrites and axons clearly is one of the most palpable aspects of developmental polarization.
function-driven plasticity, and post-traumatic reaction of neuronal processes, document that polarization is dynamic and needs to be regulated beyond the stage of development proper; in addition, these phenomena point to the potential practical clinical significance of understanding basic mechanisms of neural polarization from molecular principles.
the cytoskeleton is center stage to the mechanistic realization of cellular polarity .
in neurons, the differential subcellular distribution of distinct subsets of these proteins has been related to neurite identity, cytoskeletal organization, and function .
mtss <dig>  is a recently identified actin-binding protein.
it has been implicated in the regulation of actin filament assembly  <cit> , in mediating interaction of the cytoskeleton with phosphatidylinositol  <dig> -bisphosphate-rich membranes, and membrane bending  <cit> .
mtss <dig> is expressed in several embryonic tissues including the developing central nervous system.
in the adult central nervous system, it is prominently expressed by cerebellar purkinje cells  <cit> .
it has also been observed to be down-regulated, or missing, in several metastatic cancer cell lines  <cit> .
in the epidermis, mtss <dig> has been identified as a sonic hedgehog -responsive gene that modulates gli-regulated transcription  <cit> .
as multiple members of the shh-gli pathway are strongly expressed in the cerebellar anlage during the key phase of neural migration and morphogenesis in this structure , and indeed are necessary for the orderly development of the cerebellum, we sought to elucidate the expression of mtss <dig> in the developing and adult cerebellum and relate it to that of members of the shh-signaling pathway on the one hand, and defined steps of cerebellar morphogenesis on the other.
our results document a transient developmental expression of mtss <dig> in granule cells which closely parallels their migration and neuritogenesis.
neuronal maturation is accompanied by a switch in splice variant expression of mtss <dig>  bioinformatic analysis of the resulting protein isoforms reveals that they may differentially interact with several proteins previously identified as critical for cerebellar development and function and suggests that continued expression of mtss <dig> in adult purkinje cells relates to their functional plasticity.
RESULTS
expression of mtss <dig> in the developing cerebellum
starting from the observation of mattila et al  <cit> , who documented expression of mtss <dig> in adult purkinje cells, we scrutinized mtss <dig> expression in the developing cerebellum, from the day of birth into adulthood.
in newborn  animals, labeled cells were arranged in a broad band which outlined the incipient cerebellar folia.
both the deep cerebellar mass and the external granule cell layer were labeled only weakly, if at all .
at postnatal day  <dig> , when individual cerebellar cortical layers could be better told apart, a clear label was detected over the purkinje cell layer .
in addition, we observed a somewhat fainter but unambiguous signal in the inner part of the outer granule cell layer and in the nascent internal granule cell layer .
this became clearly visible from p <dig> onward, when individual layers of the cerebellar cortex became more prominent and could be easily delineated .
at p <dig>  the internal granule cell layer was still positive for mtss <dig> .
in contrast, at p <dig> , and in adult specimens , the mtss <dig> signal was restricted to purkinje cells, and indeed to their perikarya.
even prolonged development of the color reaction did not result in any appreciable signal localized over purkinje cell dendrites .
we did not observe any indication of a differential expression, along the anterior-posterior axis, in vermal sections of all ages analyzed, neither in purkinje cells, nor in granule cells .
moreover, analysis of coronal sections of p <dig>  p <dig> and p <dig> animals also showed homogeneous expression along the medio-lateral axis, again in purkinje cells and in granule cells.
i.e., there was no indication that molecularly defined sagittal compartments  might differ with respect to mtss <dig> expression.
control sections hybridized with sense probe did not show any signal .
all of the above results were obtained with a probe derived from the 5'-part of mtss <dig> .
in addition, we hybridized cerebella derived from p <dig> and adult animals with a second in-situ probe derived from the last 3' utr .
except for slight differences in signal strength which probably relate to probe length, we observed identical results to the ones documented for the 5'-mtss <dig> probe .
differential splicing of mtss <dig> during cerebellar development
previous studies have revealed the existence of several variants of mtss <dig>  both in normal murine tissue  <cit>  and in tumor cell lines  <cit> ; cf also  <cit> .
we used pcr to further elaborate the molecular structure of the mtss <dig> isoform expressed in the cerebellum and purified cell populations thereof.
using primers located in exons  <dig> and  <dig> , we observed a single pcr product of the size predicted for this segment encompassing all known exons, in cerebella of p <dig> and adult mice, and also in rna prepared from immature and maturing granule cells sorted based on their level of math1-egfp expression .
specifically, we did not detect a band which might be indicative of an alternative usage of exon  <dig> .
we verified expression of exon  <dig> using primers located in exon  <dig>  and exon  <dig> under stringent annealing conditions.
this resulted in  <dig> discrete bands .
the same pattern of bands was seen when primers located in exons  <dig> and  <dig> were used ; these were shifted towards lower molecular weights by what would be expected based on the sequence separating exons  <dig> and  <dig>  and encompassing all  intervening exons.
the bands obtained after amplification between exons  <dig> and  <dig> were verified by sequencing and found to comprise  <dig>   <dig>   <dig> and  <dig> nt, respectively.
thus, they correspond to splice variants comprising the exon combinations 11/ <dig>  11/12a/ <dig>  11/12/ <dig> and 11/12/12a/ <dig>  respectively.
intriguingly, the relative intensity and pattern of these bands varied for rnas prepared from different developmental stages .
a developmental pattern identical to the one shown in fig 2d was consistently obtained in rna prepared from three sets of mice in three independent experiments.
in particular, we consistently observed that band corresponding to the exon combination 11/12/ <dig> was most prominent at p <dig> and p <dig>  analysis of peripheral tissues further revealed that the splice variant 11/12a/ <dig> is found exclusively in the cns .
we further observed, in the developing p <dig> and the adult cerebellum, the expression of both the short  and the long  versions of exon  <dig> described in the ensembl databank .
in addition, we also observed very weak bands at approximately  <dig> and  <dig> nt , which probably correspond to a splice variant in which exon  <dig> is subdivided in a n- and c-terminal part separated by 108nt  <cit> .
clearly, this variant is expressed at best at very low levels in the murine cerebellum.
none of the splice variants downstream of exon  <dig> were observed to be specific to the cerebellum; nor did we obtain any indication that they might be developmentally regulated.
therefore, we did not follow up on them presently.
association of differential splicing of exons  <dig> and 12a with cellular differentiation
the developmental shift of relative intensities of the bands representing splice variants of the region between exons  <dig> and  <dig>  in conjunction with the temporal differences of mtss <dig> expression in purkinje and granule cells, prompted us to ask whether these splice variants might be expressed in a cell type-specific pattern.
to address this issue, we isolated cerebellar granule cells based on their expression of an egfp-tagged, math1-derived transgene  <cit>  by facs and assessed their expression of mtss1-splice variants by rt-pcr .
we chose this approach rather than attempting to derive splice variant-specific probes for in situ hybridization, because the sensitivity and specificity of such probes would be critically limited by the relatively small sizes of the exons concerned , and the problems inherent in quantitative cross specimen comparison of hybridized sections.
in the cerebellar anlage, math <dig> is specifically expressed in immature, proliferating granule cells, and it is rapidly down-regulated once these cells stop to proliferate and migrate to their adult position  <cit> .
we capitalized on the fact that this developmental regulation is replicated by a math1-egfp transgene, the expression of which quantitatively correlates with granule cell maturation  <cit> , to isolate mrna from two subsets of granule cells, i.e.
immature, strongly math1-egfp positive, and maturing granule cells, which express only low levels of egfp .
we assessed the efficacy of this approach by evaluating levels of cognate math <dig> mrna in the cell populations thus obtained .
we further verified that the granule cell populations obtained were not contaminated by purkinje cells by screening for the presence of the purkinje cell specific marker, l7/pcp <dig>  <cit> , which could not be detected in any of the granule cell fractions analyzed .
both immature  and maturing  granule cells expressed the same complement of splice variants seen in mrna prepared from whole cerebellum.
however, the relative intensities of individual bands varied in a systematic and consistent way .
whereas in the adult cerebellum, where mtss <dig> is expressed exclusively in purkinje cells  <cit> ; and see fig 1j, k), the band representing exon combination 11/12a/ <dig> is the most prominent one , the major band seen in immature granule cells is the one indicative of exon combination 11/12/ <dig> .
in contrast, in mrna prepared from maturing granule cells, like in that from adult cerebellum, the band indicative of the exon combination 11/12a/ <dig>  is most prominent.
such a developmentally regulated switch could also be observed in p <dig> cells induced to differentiate into neural cells .
cerebellar granule cells, or their precursors, have been identified as a cellular origin of medulloblastomas.
based on their morphology, pattern of gene expression and biology, two major subsets of medulloblastomas have been defined, referred to as classical and desmoplastic types, respectively  <cit> .
analysis of mrnas obtained from  <dig> classic and  <dig> desmoplastic tumor specimens  <cit>  revealed that all of them expressed mtss <dig>  and specifically the same basic splice variants within the region of exons 11– <dig> as described above .
among individual tumor samples, the relative intensities of these bands varied, and again as described for the developing cerebellum, the most conspicuous differences were observed for the bands representing exon combinations 11/12/ <dig> and 11/12a/ <dig>  in two cell lines derived from human medulloblastomas , exon combinations 11/ <dig> and 11/12/ <dig> were prominent, whereas the combinations 11/12/12a/ <dig> and 11/12a/ <dig> could not be observed .
as expected from the murine data, the most prominent band in mrna obtained from normal, immature human cerebellar tissue was the one indicative of the exon combination 11/12a/ <dig>  the band representing the exon combination 11/12/12a/ <dig> of mtss <dig> appeared very weak in all human specimens analyzed.
mtss <dig> exon 12a is conserved in mammals
a blast search of the ncbi nr database using the murine mtss <dig> exon 12a as input identified highly conserved, full-length matching sequences in the mtss <dig> genes of several mammals , but not in non-mammalian genomes.
indeed, the sequence and the exon/intron structure of mtss <dig> in xenopus tropicalis and danio rerio  substantially divergence from that of mammal and chick, over the region comprising exons  <dig> and 12a in these species, such that an alignment of these regions seem not sensibly possible.
this contrasts with the highly conserved structure over the imd, and also over more c-terminal parts of mtss <dig> across species.
analysis of mtss <dig> in silico
to get a first clue as to the potential functional consequences of differential splicing of mtss <dig>  we compared the various splice variants of mtss <dig> observed presently using a bioinformatic approach.
neither of the two known functional domains of mtss <dig>  i.e.
the n-terminal, actin bundling imd domain  <cit> , nor the actin monomer-binding wh <dig> domain located close to its c-terminus  <cit>  would be  affected in the splice variants reported here.
in addition to these well characterized functional domains, mtss <dig> contains a serine-rich region, spanning amino acids  242– <dig> in the exon 11/12/ <dig> splice variant, i.e.
ending in the n-terminal part of exon  <dig> .
as exon 12a encodes a rather serine-rich sequence, this serine-rich region is somewhat extended in the exon 11/12a/ <dig> splice variant, where it spans aa 242– <dig> .
the translation of the short sequence in exon  <dig> that may variably be interpreted as an exonic or intronic sequence would result in an extension of the proline rich domain in the c-terminal part of mtss <dig>
analysis of the mtss <dig> sequence for protein-protein interaction motifs using the algorithms implemented in ispot, scansite, and minimotif .
intriguingly, two out of a total of four motifs in mtss <dig> predicted to bind to the non-receptor tyrosine kinases fyn and src are encoded by exon  <dig>  one of these motifs  is predicted to bind to the sh <dig> domains, the other  to sh <dig> domains of fyn and src, and both received reasonably high prediction scores for these interactions .
there is only one additional sh3-binding motif outside of exon  <dig> in mtss <dig> predicted to bind fyn and src , and one sh2-binding motif predicted to bind fyn .
exon 12a is predicted to form one  class iv ww binding motif, and one  fha-domain binding motif .
the functionality of these motifs, which need to be phosphorylated for binding, remains unresolved as we have no information about their phosphorylation.
we complemented this prediction of potential protein binding motifs in mtss <dig> by an extensive search of the literature and the bgem  <cit>  and allan brain atlas  <cit>  databases for information about the cerebellar expression, potential function, and pathophysiological changes of the highest scoring potential mtss1-interaction partners thus identified.
the results are summarized in supplemental tables  <dig> and  <dig> .
lastly, comparative analysis of the mtss <dig> sequence also revealed the existence of two potential nuclear export signals .
in addition, two bona fide, bipartite nuclear localization signals  could be recognized in mtss <dig> .
both of these nuclear localization signals are located in the imd domain .
intriguingly, both nls comprise amino acid residues previously found critical for actin filament binding by mtss <dig> , some of which are also conserved in the imd of irsp <dig>   <cit> .
in this context, it seems worth mentioning that s <dig>  s <dig>  as well as t <dig> are predicted as potential phosphorylation sites for pkc, pka, and calmodulin dependent protein kinase  <dig> .
both predicted basic nuclear localization signals, and the presumed nuclear export signal in the imd, are highly conserved in mtts <dig> from various species, and also in a set of proteins from species which express proteins that share a considerable degree of homology with mtss <dig> in imd domain, but not beyond .
similarly, the nuclear export signal localized to exon  <dig> of the murine sequence is also highly conserved in mtss <dig> across species .
no comparable sequences are found in the imd-domain containing proteins irsp <dig> and baiap2l <dig> .
discussion
expression and splicing of mtss <dig> during neuronal differentiation
the protracted time course of cerebellar histogenesis and the spatial segregation of progressively differentiated neurons within the cerebellar cortex allowed us to relate the developmental expression of mtss <dig>  in cerebellar granule cells, to the postmitotic migration and neuritogenesis of these cells  <cit> .
once they settle in the internal granule cell layer and get synaptically integrated, they cease to express mtss <dig>  progressive granule cell differentiation is further accompanied by a switch in mtss <dig> splicing, such that usage of exon  <dig> is down-regulated, whereas expression of exon 12a increases strongy.
an identical switch can be triggered in p <dig> cells upon their induction to differentiate into neurons.
together with the observation that exon 12a is not expressed in peripheral tissues, this indicates that the switch from exon  <dig> to 12a usage occurs specifically in neurons and is linked to their terminal differentiation.
as a cautionary note, we would like to stress that differences in the intensities of the pcr bands representing splice variants comprising exons  <dig> and 12a respectively cannot be directly equated with absolute mrna levels, let alone levels of the corresponding protein isoforms.
rather, comparative assesment of band intensities as used here may be conceptually and methodologically likened to aproaches using exogenous competitors to quantify mrnas .
differences in pcr efficacy, and in vivo, potential differences in translational regulation related to the differentially spliced exons need to be considered.
these issues will require the development of reliable tools to distinguish, at the protein level, mtss <dig> isoforms arising consequent to differential splicing.
still, our data document that exon 12a is expressed, at detectable levels, only in nervous tissue, and that its expression increases with neuronal differentiation; in contrast in cerebellar granule cells, as in the forebrain , expression of mtss <dig> comprising exon  <dig> ceases concomitant with neuronal differentiation.
we directly relate relative band-intensities of defined splice variants to each other, an approach that is equivalent to
the continued, life-long expression of mtss <dig> in purkinje neurons, which contrasts to its differentiation-associated down-regulation in cerebellar granule cells, and indeed throughout the cns , suggests a role, in purkinje cells, beyond neural migration and neuritogenesis.
it is tempting to relate continued mtss <dig> expression in purkinje cells to their functional plasticity, realized at the level of their dendritic spines, which remain mobile throughout life  <cit> .
this mobility is known to be actin-dependent  <cit> ; see also  <cit> .
possible consequences of the differential usage of exons  <dig> and 12a
the precisely timed and tissue-specific change in the usage of exons 12a and <dig> during neuronal differentiation begs the question as to its potential significance.
the part of mtss <dig> encoded by the region encompassing these exons is evolutionary quite variable, in contrast to the rather strong conservation of the imd domain, and also the c-terminal part of mtss <dig>  indeed, from an evolutionary perspective, exon 12a appears to be rather novel, as no homologous sequences are found in the mtss <dig> genes of xenopus and zebrafish.
of the several motifs predicted to mediate protein-protein interactions encoded by exons  <dig> and 12a, those predicted to mediate interaction with fyn and src, which are only encoded by exon  <dig>  but not by exon 12a, appear particularly intriguing.
indeed, the combined ablation of fyn and src in the cns has been reported to cause a reeler-like phenotype  <cit> , i.e.
a migratory defect that also affects cerebellar histogenesis.
thus, mtss <dig> might constitute part of the link that relates activity/subcellular localization of these kinases to morphogenesis.
the developmental switch in the use of exons  <dig> and 12a may be expected to substantially affect interaction of mtss <dig> with src and fyn, or at least lead to the generation of two isoforms with differential.
finally, we note that the exchange of exon  <dig> for 12a may also affect interaction with gli, as exon  <dig>  codes part of a broad region  to which the interaction site for gli <dig> has been localized  <cit> .
a structural rationale to coordinate actin bundling and nuclear translocation
mtss <dig> has been observed to localize both to the cytoplasm and nuclei in fibroblasts  <cit> , and indeed the latter is a key prerequisite for its interaction with gli proteins in transcriptional regulation as observed by callahan et al  <cit> .
the identification, here, of two evolutionary highly conserved nuclear localization motifs and an equally conserved nuclear export motif suggests a structural rationale for a twin role of mtss <dig> as a cytoskeleton associated protein and a transcriptional modulator/regulator.
as detailed above, the actin-binding "basic patch"  <cit>  of the mtss1-imd domain is integrated in a bona fide bipartite nuclear localization signal, which suggests dual use of this element.
several potential phosphorylation sites intercalated within this nuclear localization motif further suggests that use of this element might be dynamically regulated.
finally, phosphorylation of t <dig>  which is part of a predicted pkcα/β/γ phosphorylation site , would transform the actin basic patch/nuclear localization motif in a perfect fha domain-binding motif .
this domain is found in many transport-associated and nuclear proteins .
that this view is conceptually on target is corroborated by the recent observation that deletion of a larger part of the imd domain , which included the actin binding basic patch/nuclear localization motif/fha-binding motif identified here, indeed affects mtss <dig> subcellular distribution  <cit> .
thus, if we allow for a somewhat speculative perspective for the moment, we might envision the basic patch/nuclear localization motif in the imd domain of mtss <dig> could be critical for a dynamic, activity-dependent relay, triggered by  input at the spine, and formed by the spine actin cytoskeleton, kinesins, the nuclear import machinery, and lastly nuclear partners of mtss <dig>  this perspective is also based on the observations that rac, a known binding partner of mtss <dig>  <cit>   is critical for purkinje cell spine morphogenesis  <cit> ; so is irsp <dig>  <cit> , which also functions as a downstream signal tranducer of insulin-like growth factor  <dig>  an exemplary regulator of purkinje cell dendritic plasticity  <cit> .
the imd domain shard between irsp <dig> and mtss <dig> begs the question whether they might interact, or compete for binding partners.
finally, our in silico approach suggests crk and esp <dig> as potential interacting proteins for mtss <dig>  both of which have been linked to cerebellar development and function  <cit>  .
CONCLUSIONS
the present findings document the developmentally regulated splicing and expression of mtss <dig> in cerebellar granule and purkinje neurons.
they suggest that these isoforms of mtss <dig>  which differ in  domain predicted to mediate interaction with a set of proteins known to affect the physiology of purkinje and granule cells, may be related to specific functional characteristics and properties of these cells, in particular developmental migration and adult synaptic plasticity.
lastly, bioinformatic analysis of mtss <dig> suggests that the basic patch of the imd domain, which mediates actin binding, overlaps with a nuclear localization signal and suggests a key structural motif that may be regulated in an activity-dependent manner to define subcellular distribution, and hence function, of mtss <dig>  our findings define the cerebellum, and purkinje cells in particular, as a paradigm to further test the function of mtss <dig>  to unravel how its functional properties are structurally encoded, and how they may relate to tumor cell biology.

 a fundamental aspect of nervous system development and function is the expansion of basic cellular polarity into a highly diverse array of neural phenotypes specialized for directional information processing. morphological polarization of glia and neurons alike has long been used as a basic principle for classification of these cells, which was fundamental to the initial understanding of their functions and interactions. in neurons, the formation and maintenance of dendrites and axons clearly is one of the most palpable aspects of developmental polarization. function-driven plasticity, and post-traumatic reaction of neuronal processes, document that polarization is dynamic and needs to be regulated beyond the stage of development proper; in addition, these phenomena point to the potential practical clinical significance of understanding basic mechanisms of neural polarization from molecular principles. the cytoskeleton is center stage to the mechanistic realization of cellular polarity. in neurons, the differential subcellular distribution of distinct subsets of these proteins has been related to neurite identity, cytoskeletal organization, and function. mtss is a recently identified actin-binding protein. it has been implicated in the regulation of actin filament assembly, in mediating interaction of the cytoskeleton with phosphatidylinositol -bisphosphate-rich membranes, and membrane bending. mtss is expressed in several embryonic tissues including the developing central nervous system. in the adult central nervous system, it is prominently expressed by cerebellar purkinje cells. it has also been observed to be down-regulated, or missing, in several metastatic cancer cell lines. in the epidermis, mtss has been identified as a sonic hedgehog -responsive gene that modulates gli-regulated transcription. as multiple members of the shh-gli pathway are strongly expressed in the cerebellar anlage during the key phase of neural migration and morphogenesis in this structure, and indeed are necessary for the orderly development of the cerebellum, we sought to elucidate the expression of mtss in the developing and adult cerebellum and relate it to that of members of the shh-signaling pathway on the one hand, and defined steps of cerebellar morphogenesis on the other. our results document a transient developmental expression of mtss in granule cells which closely parallels their migration and neuritogenesis. neuronal maturation is accompanied by a switch in splice variant expression of mtss bioinformatic analysis of the resulting protein isoforms reveals that they may differentially interact with several proteins previously identified as critical for cerebellar development and function and suggests that continued expression of mtss in adult purkinje cells relates to their functional plasticity.  expression of mtss in the developing cerebellum starting from the observation of mattila et al, who documented expression of mtss in adult purkinje cells, we scrutinized mtss expression in the developing cerebellum, from the day of birth into adulthood. in newborn animals, labeled cells were arranged in a broad band which outlined the incipient cerebellar folia. both the deep cerebellar mass and the external granule cell layer were labeled only weakly, if at all. at postnatal day, when individual cerebellar cortical layers could be better told apart, a clear label was detected over the purkinje cell layer. in addition, we observed a somewhat fainter but unambiguous signal in the inner part of the outer granule cell layer and in the nascent internal granule cell layer. this became clearly visible from p onward, when individual layers of the cerebellar cortex became more prominent and could be easily delineated. at p the internal granule cell layer was still positive for mtss. in contrast, at p, and in adult specimens, the mtss signal was restricted to purkinje cells, and indeed to their perikarya. even prolonged development of the color reaction did not result in any appreciable signal localized over purkinje cell dendrites. we did not observe any indication of a differential expression, along the anterior-posterior axis, in vermal sections of all ages analyzed, neither in purkinje cells, nor in granule cells. moreover, analysis of coronal sections of p p and p animals also showed homogeneous expression along the medio-lateral axis, again in purkinje cells and in granule cells. i.e., there was no indication that molecularly defined sagittal compartments might differ with respect to mtss expression. control sections hybridized with sense probe did not show any signal. all of the above results were obtained with a probe derived from the 5'-part of mtss. in addition, we hybridized cerebella derived from p and adult animals with a second in-situ probe derived from the last 3' utr. except for slight differences in signal strength which probably relate to probe length, we observed identical results to the ones documented for the 5'-mtss probe. differential splicing of mtss during cerebellar development previous studies have revealed the existence of several variants of mtss both in normal murine tissue and in tumor cell lines; cf also. we used pcr to further elaborate the molecular structure of the mtss isoform expressed in the cerebellum and purified cell populations thereof. using primers located in exons and, we observed a single pcr product of the size predicted for this segment encompassing all known exons, in cerebella of p and adult mice, and also in rna prepared from immature and maturing granule cells sorted based on their level of math1-egfp expression. specifically, we did not detect a band which might be indicative of an alternative usage of exon. we verified expression of exon using primers located in exon and exon under stringent annealing conditions. this resulted in discrete bands. the same pattern of bands was seen when primers located in exons and were used; these were shifted towards lower molecular weights by what would be expected based on the sequence separating exons and and encompassing all intervening exons. the bands obtained after amplification between exons and were verified by sequencing and found to comprise and nt, respectively. thus, they correspond to splice variants comprising the exon combinations 11/ 11/12a/ 11/12/ and 11/12/12a/ respectively. intriguingly, the relative intensity and pattern of these bands varied for rnas prepared from different developmental stages. a developmental pattern identical to the one shown in fig 2d was consistently obtained in rna prepared from three sets of mice in three independent experiments. in particular, we consistently observed that band corresponding to the exon combination 11/12/ was most prominent at p and p analysis of peripheral tissues further revealed that the splice variant 11/12a/ is found exclusively in the cns. we further observed, in the developing p and the adult cerebellum, the expression of both the short and the long versions of exon described in the ensembl databank. in addition, we also observed very weak bands at approximately and nt, which probably correspond to a splice variant in which exon is subdivided in a n- and c-terminal part separated by 108nt. clearly, this variant is expressed at best at very low levels in the murine cerebellum. none of the splice variants downstream of exon were observed to be specific to the cerebellum; nor did we obtain any indication that they might be developmentally regulated. therefore, we did not follow up on them presently. association of differential splicing of exons and 12a with cellular differentiation the developmental shift of relative intensities of the bands representing splice variants of the region between exons and in conjunction with the temporal differences of mtss expression in purkinje and granule cells, prompted us to ask whether these splice variants might be expressed in a cell type-specific pattern. to address this issue, we isolated cerebellar granule cells based on their expression of an egfp-tagged, math1-derived transgene by facs and assessed their expression of mtss1-splice variants by rt-pcr. we chose this approach rather than attempting to derive splice variant-specific probes for in situ hybridization, because the sensitivity and specificity of such probes would be critically limited by the relatively small sizes of the exons concerned, and the problems inherent in quantitative cross specimen comparison of hybridized sections. in the cerebellar anlage, math is specifically expressed in immature, proliferating granule cells, and it is rapidly down-regulated once these cells stop to proliferate and migrate to their adult position. we capitalized on the fact that this developmental regulation is replicated by a math1-egfp transgene, the expression of which quantitatively correlates with granule cell maturation, to isolate mrna from two subsets of granule cells, i.e. immature, strongly math1-egfp positive, and maturing granule cells, which express only low levels of egfp. we assessed the efficacy of this approach by evaluating levels of cognate math mrna in the cell populations thus obtained. we further verified that the granule cell populations obtained were not contaminated by purkinje cells by screening for the presence of the purkinje cell specific marker, l7/pcp, which could not be detected in any of the granule cell fractions analyzed. both immature and maturing granule cells expressed the same complement of splice variants seen in mrna prepared from whole cerebellum. however, the relative intensities of individual bands varied in a systematic and consistent way. whereas in the adult cerebellum, where mtss is expressed exclusively in purkinje cells; and see fig 1j, k), the band representing exon combination 11/12a/ is the most prominent one, the major band seen in immature granule cells is the one indicative of exon combination 11/12/. in contrast, in mrna prepared from maturing granule cells, like in that from adult cerebellum, the band indicative of the exon combination 11/12a/ is most prominent. such a developmentally regulated switch could also be observed in p cells induced to differentiate into neural cells. cerebellar granule cells, or their precursors, have been identified as a cellular origin of medulloblastomas. based on their morphology, pattern of gene expression and biology, two major subsets of medulloblastomas have been defined, referred to as classical and desmoplastic types, respectively. analysis of mrnas obtained from classic and desmoplastic tumor specimens revealed that all of them expressed mtss and specifically the same basic splice variants within the region of exons 11– as described above. among individual tumor samples, the relative intensities of these bands varied, and again as described for the developing cerebellum, the most conspicuous differences were observed for the bands representing exon combinations 11/12/ and 11/12a/ in two cell lines derived from human medulloblastomas, exon combinations 11/ and 11/12/ were prominent, whereas the combinations 11/12/12a/ and 11/12a/ could not be observed. as expected from the murine data, the most prominent band in mrna obtained from normal, immature human cerebellar tissue was the one indicative of the exon combination 11/12a/ the band representing the exon combination 11/12/12a/ of mtss appeared very weak in all human specimens analyzed. mtss exon 12a is conserved in mammals a blast search of the ncbi nr database using the murine mtss exon 12a as input identified highly conserved, full-length matching sequences in the mtss genes of several mammals, but not in non-mammalian genomes. indeed, the sequence and the exon/intron structure of mtss in xenopus tropicalis and danio rerio substantially divergence from that of mammal and chick, over the region comprising exons and 12a in these species, such that an alignment of these regions seem not sensibly possible. this contrasts with the highly conserved structure over the imd, and also over more c-terminal parts of mtss across species. analysis of mtss in silico to get a first clue as to the potential functional consequences of differential splicing of mtss we compared the various splice variants of mtss observed presently using a bioinformatic approach. neither of the two known functional domains of mtss i.e. the n-terminal, actin bundling imd domain, nor the actin monomer-binding wh domain located close to its c-terminus would be affected in the splice variants reported here. in addition to these well characterized functional domains, mtss contains a serine-rich region, spanning amino acids 242– in the exon 11/12/ splice variant, i.e. ending in the n-terminal part of exon. as exon 12a encodes a rather serine-rich sequence, this serine-rich region is somewhat extended in the exon 11/12a/ splice variant, where it spans aa 242–. the translation of the short sequence in exon that may variably be interpreted as an exonic or intronic sequence would result in an extension of the proline rich domain in the c-terminal part of mtss analysis of the mtss sequence for protein-protein interaction motifs using the algorithms implemented in ispot, scansite, and minimotif. intriguingly, two out of a total of four motifs in mtss predicted to bind to the non-receptor tyrosine kinases fyn and src are encoded by exon one of these motifs is predicted to bind to the sh domains, the other to sh domains of fyn and src, and both received reasonably high prediction scores for these interactions. there is only one additional sh3-binding motif outside of exon in mtss predicted to bind fyn and src, and one sh2-binding motif predicted to bind fyn. exon 12a is predicted to form one class iv ww binding motif, and one fha-domain binding motif. the functionality of these motifs, which need to be phosphorylated for binding, remains unresolved as we have no information about their phosphorylation. we complemented this prediction of potential protein binding motifs in mtss by an extensive search of the literature and the bgem and allan brain atlas databases for information about the cerebellar expression, potential function, and pathophysiological changes of the highest scoring potential mtss1-interaction partners thus identified. the results are summarized in supplemental tables and. lastly, comparative analysis of the mtss sequence also revealed the existence of two potential nuclear export signals. in addition, two bona fide, bipartite nuclear localization signals could be recognized in mtss. both of these nuclear localization signals are located in the imd domain. intriguingly, both nls comprise amino acid residues previously found critical for actin filament binding by mtss, some of which are also conserved in the imd of irsp. in this context, it seems worth mentioning that s s as well as t are predicted as potential phosphorylation sites for pkc, pka, and calmodulin dependent protein kinase. both predicted basic nuclear localization signals, and the presumed nuclear export signal in the imd, are highly conserved in mtts from various species, and also in a set of proteins from species which express proteins that share a considerable degree of homology with mtss in imd domain, but not beyond. similarly, the nuclear export signal localized to exon of the murine sequence is also highly conserved in mtss across species. no comparable sequences are found in the imd-domain containing proteins irsp and baiap2l. discussion expression and splicing of mtss during neuronal differentiation the protracted time course of cerebellar histogenesis and the spatial segregation of progressively differentiated neurons within the cerebellar cortex allowed us to relate the developmental expression of mtss in cerebellar granule cells, to the postmitotic migration and neuritogenesis of these cells. once they settle in the internal granule cell layer and get synaptically integrated, they cease to express mtss progressive granule cell differentiation is further accompanied by a switch in mtss splicing, such that usage of exon is down-regulated, whereas expression of exon 12a increases strongy. an identical switch can be triggered in p cells upon their induction to differentiate into neurons. together with the observation that exon 12a is not expressed in peripheral tissues, this indicates that the switch from exon to 12a usage occurs specifically in neurons and is linked to their terminal differentiation. as a cautionary note, we would like to stress that differences in the intensities of the pcr bands representing splice variants comprising exons and 12a respectively cannot be directly equated with absolute mrna levels, let alone levels of the corresponding protein isoforms. rather, comparative assesment of band intensities as used here may be conceptually and methodologically likened to aproaches using exogenous competitors to quantify mrnas. differences in pcr efficacy, and in vivo, potential differences in translational regulation related to the differentially spliced exons need to be considered. these issues will require the development of reliable tools to distinguish, at the protein level, mtss isoforms arising consequent to differential splicing. still, our data document that exon 12a is expressed, at detectable levels, only in nervous tissue, and that its expression increases with neuronal differentiation; in contrast in cerebellar granule cells, as in the forebrain, expression of mtss comprising exon ceases concomitant with neuronal differentiation. we directly relate relative band-intensities of defined splice variants to each other, an approach that is equivalent to the continued, life-long expression of mtss in purkinje neurons, which contrasts to its differentiation-associated down-regulation in cerebellar granule cells, and indeed throughout the cns, suggests a role, in purkinje cells, beyond neural migration and neuritogenesis. it is tempting to relate continued mtss expression in purkinje cells to their functional plasticity, realized at the level of their dendritic spines, which remain mobile throughout life. this mobility is known to be actin-dependent; see also. possible consequences of the differential usage of exons and 12a the precisely timed and tissue-specific change in the usage of exons 12a and during neuronal differentiation begs the question as to its potential significance. the part of mtss encoded by the region encompassing these exons is evolutionary quite variable, in contrast to the rather strong conservation of the imd domain, and also the c-terminal part of mtss indeed, from an evolutionary perspective, exon 12a appears to be rather novel, as no homologous sequences are found in the mtss genes of xenopus and zebrafish. of the several motifs predicted to mediate protein-protein interactions encoded by exons and 12a, those predicted to mediate interaction with fyn and src, which are only encoded by exon but not by exon 12a, appear particularly intriguing. indeed, the combined ablation of fyn and src in the cns has been reported to cause a reeler-like phenotype, i.e. a migratory defect that also affects cerebellar histogenesis. thus, mtss might constitute part of the link that relates activity/subcellular localization of these kinases to morphogenesis. the developmental switch in the use of exons and 12a may be expected to substantially affect interaction of mtss with src and fyn, or at least lead to the generation of two isoforms with differential. finally, we note that the exchange of exon for 12a may also affect interaction with gli, as exon codes part of a broad region to which the interaction site for gli has been localized. a structural rationale to coordinate actin bundling and nuclear translocation mtss has been observed to localize both to the cytoplasm and nuclei in fibroblasts, and indeed the latter is a key prerequisite for its interaction with gli proteins in transcriptional regulation as observed by callahan et al. the identification, here, of two evolutionary highly conserved nuclear localization motifs and an equally conserved nuclear export motif suggests a structural rationale for a twin role of mtss as a cytoskeleton associated protein and a transcriptional modulator/regulator. as detailed above, the actin-binding "basic patch" of the mtss1-imd domain is integrated in a bona fide bipartite nuclear localization signal, which suggests dual use of this element. several potential phosphorylation sites intercalated within this nuclear localization motif further suggests that use of this element might be dynamically regulated. finally, phosphorylation of t which is part of a predicted pkcα/β/γ phosphorylation site, would transform the actin basic patch/nuclear localization motif in a perfect fha domain-binding motif. this domain is found in many transport-associated and nuclear proteins. that this view is conceptually on target is corroborated by the recent observation that deletion of a larger part of the imd domain, which included the actin binding basic patch/nuclear localization motif/fha-binding motif identified here, indeed affects mtss subcellular distribution. thus, if we allow for a somewhat speculative perspective for the moment, we might envision the basic patch/nuclear localization motif in the imd domain of mtss could be critical for a dynamic, activity-dependent relay, triggered by input at the spine, and formed by the spine actin cytoskeleton, kinesins, the nuclear import machinery, and lastly nuclear partners of mtss this perspective is also based on the observations that rac, a known binding partner of mtss is critical for purkinje cell spine morphogenesis; so is irsp, which also functions as a downstream signal tranducer of insulin-like growth factor an exemplary regulator of purkinje cell dendritic plasticity. the imd domain shard between irsp and mtss begs the question whether they might interact, or compete for binding partners. finally, our in silico approach suggests crk and esp as potential interacting proteins for mtss both of which have been linked to cerebellar development and function.  the present findings document the developmentally regulated splicing and expression of mtss in cerebellar granule and purkinje neurons. they suggest that these isoforms of mtss which differ in domain predicted to mediate interaction with a set of proteins known to affect the physiology of purkinje and granule cells, may be related to specific functional characteristics and properties of these cells, in particular developmental migration and adult synaptic plasticity. lastly, bioinformatic analysis of mtss suggests that the basic patch of the imd domain, which mediates actin binding, overlaps with a nuclear localization signal and suggests a key structural motif that may be regulated in an activity-dependent manner to define subcellular distribution, and hence function, of mtss our findings define the cerebellum, and purkinje cells in particular, as a paradigm to further test the function of mtss to unravel how its functional properties are structurally encoded, and how they may relate to tumor cell biology. 
bioinformatic analysis of mtss suggests that differential exon usage may affect interaction with fyn and src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. these overlap with the actin filament binding site of mtss and one also harbors a potential pka and pkc phosphorylation site. during development, mtss is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. whereas immature granule cells express a mtss variant observed also in peripheral tissues and comprising exon this exon is replaced by a cns-specific exon, 12a, in more mature granule cells and in adult purkinje cells. given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of mtss in the developing murine cerebellum and human medulloblastoma specimens. both the pattern of expression and splicing of mtss is developmentally regulated in the murine cerebellum. neuronal differentiation is accompanied by a switch in mtss splicing.
 mtss encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/gli signaling in epidermal cells. given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of mtss in the developing murine cerebellum and human medulloblastoma specimens.  during development, mtss is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. it is also expressed by granule cell precursor-derived medulloblastomas. in the adult cns, mtss is found exclusively in cerebellar purkinje cells. neuronal differentiation is accompanied by a switch in mtss splicing. whereas immature granule cells express a mtss variant observed also in peripheral tissues and comprising exon this exon is replaced by a cns-specific exon, 12a, in more mature granule cells and in adult purkinje cells. bioinformatic analysis of mtss suggests that differential exon usage may affect interaction with fyn and src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. these overlap with the actin filament binding site of mtss and one also harbors a potential pka and pkc phosphorylation site.  both the pattern of expression and splicing of mtss is developmentally regulated in the murine cerebellum. these findings are discussed with a view on the potential role of mtss for cytoskeletal dynamics in developing and mature cerebellar neurons. 