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Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1) in normal and transformed cerebellar cells.

Glassmann A, Molly S, Surchev L, Nazwar TA, Holst M, Hartmann W, Baader SL, Oberdick J, Pietsch T, Schilling K - BMC Dev. Biol. (2007)

Bottom Line: In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells.Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum.These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Anatomisches Institut, Anatomie & Zellbiologie, University of Bonn, Bonn, Germany. alexander.glassmann@uni-bonn.de

ABSTRACT

Background: Mtss1 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 Mtss1 in the developing murine cerebellum and human medulloblastoma specimens.

Results: During development, Mtss1 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, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 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 Mtss1, and one also harbors a potential PKA and PKC phosphorylation site.

Conclusion: Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

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A: Schematic view of the organization of the murine Mtss1 gene based on Ensemble entry ENSMUSG00000022353. The 5' and 3' UTRs (dark boxes) are not drawn to scale, nor are the intronic regions. Comparison of the murine gene with that of the rat (ENSRNOG00000009001; transcript ENSRNOT00000023505) further suggests that the region labeled here as exon 15 may contain an additional, 108 bp long intron (I, marked in light gray) which would result in the division of exon 15 into two exons of 354 and 239 bps, respectively. Also, the length of exon 14 may either encompass 163 (ENSMUST00000080371) or 208 bp (ENSMUST00000036782). The 45 bps in question are located C-terminal and alternatively form part of the intron separating exons 14 and 15. Regions covered by the in situ hybridization probes used are labeled by horizontal lines A and B. Arrows mark positions of primers used (black arrows, forward primers; open arrows, reverse primers). B: Schematic view of the derived protein. The N-terminal IMD domain and the C-terminal WH2 domain are shown as gray boxes. The localization of the putative nuclear import (I) and export (E) motives are indicated as black and white boxes, respectively. C, D: Expression of Mtss1 splice variants in the early postnatal and adult murine cerebellum. Use of primers located in exons 7 and 13 (primers 2, 5; panel C) reveals the existence of 4 splice variants (the band representing exon combination 11/12/12a/13 reproduces only very weakly here) in the developing and adult cerebellum, as does the use of primers located in exons 11 and 13 (primers 4, 5; panel D). Note that the relative intensity in particular of the band representing splice variants comprising exons 11/12/13 and 11/12a/13 varies during development. The band labeled gapdh is a loading control. Numbers indicate days postnatal; Ad, adult. The arrow indicates a spurious amplificate from an unrelated sequence: This was verified by sequencing, as were the products labeled as bands 11/12/12a/13, 11/12/13, 11/12a/13 and 11/13.
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Figure 2: A: Schematic view of the organization of the murine Mtss1 gene based on Ensemble entry ENSMUSG00000022353. The 5' and 3' UTRs (dark boxes) are not drawn to scale, nor are the intronic regions. Comparison of the murine gene with that of the rat (ENSRNOG00000009001; transcript ENSRNOT00000023505) further suggests that the region labeled here as exon 15 may contain an additional, 108 bp long intron (I, marked in light gray) which would result in the division of exon 15 into two exons of 354 and 239 bps, respectively. Also, the length of exon 14 may either encompass 163 (ENSMUST00000080371) or 208 bp (ENSMUST00000036782). The 45 bps in question are located C-terminal and alternatively form part of the intron separating exons 14 and 15. Regions covered by the in situ hybridization probes used are labeled by horizontal lines A and B. Arrows mark positions of primers used (black arrows, forward primers; open arrows, reverse primers). B: Schematic view of the derived protein. The N-terminal IMD domain and the C-terminal WH2 domain are shown as gray boxes. The localization of the putative nuclear import (I) and export (E) motives are indicated as black and white boxes, respectively. C, D: Expression of Mtss1 splice variants in the early postnatal and adult murine cerebellum. Use of primers located in exons 7 and 13 (primers 2, 5; panel C) reveals the existence of 4 splice variants (the band representing exon combination 11/12/12a/13 reproduces only very weakly here) in the developing and adult cerebellum, as does the use of primers located in exons 11 and 13 (primers 4, 5; panel D). Note that the relative intensity in particular of the band representing splice variants comprising exons 11/12/13 and 11/12a/13 varies during development. The band labeled gapdh is a loading control. Numbers indicate days postnatal; Ad, adult. The arrow indicates a spurious amplificate from an unrelated sequence: This was verified by sequencing, as were the products labeled as bands 11/12/12a/13, 11/12/13, 11/12a/13 and 11/13.

Mentions: Starting from the observation of Mattila et al [12], who documented expression of Mtss1 in adult Purkinje cells, we scrutinized Mtss1 expression in the developing cerebellum, from the day of birth into adulthood. In newborn (p0) 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 (Fig 1A, B). At postnatal day 3 (p3), when individual cerebellar cortical layers could be better told apart, a clear label was detected over the Purkinje cell layer (which, as typical for this age, was still multilayered). 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 (Fig 1C and insert in Fig 1C). This became clearly visible from p5 onward, when individual layers of the cerebellar cortex became more prominent and could be easily delineated (Fig 1D–F). At p15, the internal granule cell layer was still positive for Mtss1 (Fig 1G, H). In contrast, at p21 (Fig 1I), and in adult specimens (Fig 1J, K), the Mtss1 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 (Fig 1K and data not shown). 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 (Fig 1G, J and data not shown). Moreover, analysis of coronal sections of p3, p8 and p9 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 (e.g., [24,25]) might differ with respect to Mtss1 expression. Control sections hybridized with sense probe did not show any signal (not shown). All of the above results were obtained with a probe derived from the 5'-part of Mtss1 (probe A in Fig 2; extending from exon 1 to 9). In addition, we hybridized cerebella derived from p8 and adult animals with a second in-situ probe derived from the last 3' UTR (probe B; for primers, see additional file 1). Except for slight differences in signal strength which probably relate to probe length, we observed identical results to the ones documented for the 5'-Mtss1 probe (data not shown).


Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1) in normal and transformed cerebellar cells.

Glassmann A, Molly S, Surchev L, Nazwar TA, Holst M, Hartmann W, Baader SL, Oberdick J, Pietsch T, Schilling K - BMC Dev. Biol. (2007)

A: Schematic view of the organization of the murine Mtss1 gene based on Ensemble entry ENSMUSG00000022353. The 5' and 3' UTRs (dark boxes) are not drawn to scale, nor are the intronic regions. Comparison of the murine gene with that of the rat (ENSRNOG00000009001; transcript ENSRNOT00000023505) further suggests that the region labeled here as exon 15 may contain an additional, 108 bp long intron (I, marked in light gray) which would result in the division of exon 15 into two exons of 354 and 239 bps, respectively. Also, the length of exon 14 may either encompass 163 (ENSMUST00000080371) or 208 bp (ENSMUST00000036782). The 45 bps in question are located C-terminal and alternatively form part of the intron separating exons 14 and 15. Regions covered by the in situ hybridization probes used are labeled by horizontal lines A and B. Arrows mark positions of primers used (black arrows, forward primers; open arrows, reverse primers). B: Schematic view of the derived protein. The N-terminal IMD domain and the C-terminal WH2 domain are shown as gray boxes. The localization of the putative nuclear import (I) and export (E) motives are indicated as black and white boxes, respectively. C, D: Expression of Mtss1 splice variants in the early postnatal and adult murine cerebellum. Use of primers located in exons 7 and 13 (primers 2, 5; panel C) reveals the existence of 4 splice variants (the band representing exon combination 11/12/12a/13 reproduces only very weakly here) in the developing and adult cerebellum, as does the use of primers located in exons 11 and 13 (primers 4, 5; panel D). Note that the relative intensity in particular of the band representing splice variants comprising exons 11/12/13 and 11/12a/13 varies during development. The band labeled gapdh is a loading control. Numbers indicate days postnatal; Ad, adult. The arrow indicates a spurious amplificate from an unrelated sequence: This was verified by sequencing, as were the products labeled as bands 11/12/12a/13, 11/12/13, 11/12a/13 and 11/13.
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Figure 2: A: Schematic view of the organization of the murine Mtss1 gene based on Ensemble entry ENSMUSG00000022353. The 5' and 3' UTRs (dark boxes) are not drawn to scale, nor are the intronic regions. Comparison of the murine gene with that of the rat (ENSRNOG00000009001; transcript ENSRNOT00000023505) further suggests that the region labeled here as exon 15 may contain an additional, 108 bp long intron (I, marked in light gray) which would result in the division of exon 15 into two exons of 354 and 239 bps, respectively. Also, the length of exon 14 may either encompass 163 (ENSMUST00000080371) or 208 bp (ENSMUST00000036782). The 45 bps in question are located C-terminal and alternatively form part of the intron separating exons 14 and 15. Regions covered by the in situ hybridization probes used are labeled by horizontal lines A and B. Arrows mark positions of primers used (black arrows, forward primers; open arrows, reverse primers). B: Schematic view of the derived protein. The N-terminal IMD domain and the C-terminal WH2 domain are shown as gray boxes. The localization of the putative nuclear import (I) and export (E) motives are indicated as black and white boxes, respectively. C, D: Expression of Mtss1 splice variants in the early postnatal and adult murine cerebellum. Use of primers located in exons 7 and 13 (primers 2, 5; panel C) reveals the existence of 4 splice variants (the band representing exon combination 11/12/12a/13 reproduces only very weakly here) in the developing and adult cerebellum, as does the use of primers located in exons 11 and 13 (primers 4, 5; panel D). Note that the relative intensity in particular of the band representing splice variants comprising exons 11/12/13 and 11/12a/13 varies during development. The band labeled gapdh is a loading control. Numbers indicate days postnatal; Ad, adult. The arrow indicates a spurious amplificate from an unrelated sequence: This was verified by sequencing, as were the products labeled as bands 11/12/12a/13, 11/12/13, 11/12a/13 and 11/13.
Mentions: Starting from the observation of Mattila et al [12], who documented expression of Mtss1 in adult Purkinje cells, we scrutinized Mtss1 expression in the developing cerebellum, from the day of birth into adulthood. In newborn (p0) 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 (Fig 1A, B). At postnatal day 3 (p3), when individual cerebellar cortical layers could be better told apart, a clear label was detected over the Purkinje cell layer (which, as typical for this age, was still multilayered). 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 (Fig 1C and insert in Fig 1C). This became clearly visible from p5 onward, when individual layers of the cerebellar cortex became more prominent and could be easily delineated (Fig 1D–F). At p15, the internal granule cell layer was still positive for Mtss1 (Fig 1G, H). In contrast, at p21 (Fig 1I), and in adult specimens (Fig 1J, K), the Mtss1 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 (Fig 1K and data not shown). 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 (Fig 1G, J and data not shown). Moreover, analysis of coronal sections of p3, p8 and p9 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 (e.g., [24,25]) might differ with respect to Mtss1 expression. Control sections hybridized with sense probe did not show any signal (not shown). All of the above results were obtained with a probe derived from the 5'-part of Mtss1 (probe A in Fig 2; extending from exon 1 to 9). In addition, we hybridized cerebella derived from p8 and adult animals with a second in-situ probe derived from the last 3' UTR (probe B; for primers, see additional file 1). Except for slight differences in signal strength which probably relate to probe length, we observed identical results to the ones documented for the 5'-Mtss1 probe (data not shown).

Bottom Line: In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells.Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum.These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Anatomisches Institut, Anatomie & Zellbiologie, University of Bonn, Bonn, Germany. alexander.glassmann@uni-bonn.de

ABSTRACT

Background: Mtss1 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 Mtss1 in the developing murine cerebellum and human medulloblastoma specimens.

Results: During development, Mtss1 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, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 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 Mtss1, and one also harbors a potential PKA and PKC phosphorylation site.

Conclusion: Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

Show MeSH
Related in: MedlinePlus