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Agrin isoforms with distinct amino termini: differential expression, localization, and function.

Burgess RW, Skarnes WC, Sanes JR - J. Cell Biol. (2000)

Bottom Line: The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons.Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated.Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, Missouri 63110, USA.

ABSTRACT
The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

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Expression of LN- and SN-agrin. The β-geo insertion allowed LN-agrin expression to be analyzed by lacZ staining in heterozygotes (A, B, D, E, and G–L), whereas SN-agrin RNA was detected by in situ hybridization (C, F, M, and N). (A and B) In the brains of E14 mice, LN-agrin is expressed in cerebral blood vessels and in the ventricular (vz) and intermediate zone (iz) of the cortex, but not in the cortical plate (cp; B). (C) SN-agrin is expressed in a pattern that is complementary to the LN pattern, being most abundant in the postmitotic cells of the cortical plate. (D) In E18 embryos, LN-agrin continues to have very restricted expression in the hippocampus, being present only near the ventricles and in blood vessels. (E) In the spinal cord, LN-agrin is strongly expressed by motoneurons and sensory neurons of the DRG (arrows). (F) SN-agrin is widely distributed in many neuronal cells types in both the DRG and the spinal cord, although motoneurons are not more intensely positive than other cell types (white arrows). (H) LN-agrin is also expressed by cranial nerve motor nuclei in the hindbrain (E18 horizontal section; IV, forth ventricle; arrowheads, oculomotor nuclei). (I) Motoneuron expression of LN-agrin persists into adulthood. (G and J–N) Nonneuronal cells that abut basal laminae express LN-agrin but not SN-agrin. Epidermal cells in E18 skin (G), kidney glomeruli and tubules (J), and pulmonary epithelium (K) are all positive for LN-agrin. No lacZ activity was present in littermate controls in any tissue (E18 kidney shown, L). Consistent with northern blotting, SN-agrin is not expressed at levels detectable by in situ hybridization in nonneuronal tissues such as kidney (M) or lung (N).
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Figure 6: Expression of LN- and SN-agrin. The β-geo insertion allowed LN-agrin expression to be analyzed by lacZ staining in heterozygotes (A, B, D, E, and G–L), whereas SN-agrin RNA was detected by in situ hybridization (C, F, M, and N). (A and B) In the brains of E14 mice, LN-agrin is expressed in cerebral blood vessels and in the ventricular (vz) and intermediate zone (iz) of the cortex, but not in the cortical plate (cp; B). (C) SN-agrin is expressed in a pattern that is complementary to the LN pattern, being most abundant in the postmitotic cells of the cortical plate. (D) In E18 embryos, LN-agrin continues to have very restricted expression in the hippocampus, being present only near the ventricles and in blood vessels. (E) In the spinal cord, LN-agrin is strongly expressed by motoneurons and sensory neurons of the DRG (arrows). (F) SN-agrin is widely distributed in many neuronal cells types in both the DRG and the spinal cord, although motoneurons are not more intensely positive than other cell types (white arrows). (H) LN-agrin is also expressed by cranial nerve motor nuclei in the hindbrain (E18 horizontal section; IV, forth ventricle; arrowheads, oculomotor nuclei). (I) Motoneuron expression of LN-agrin persists into adulthood. (G and J–N) Nonneuronal cells that abut basal laminae express LN-agrin but not SN-agrin. Epidermal cells in E18 skin (G), kidney glomeruli and tubules (J), and pulmonary epithelium (K) are all positive for LN-agrin. No lacZ activity was present in littermate controls in any tissue (E18 kidney shown, L). Consistent with northern blotting, SN-agrin is not expressed at levels detectable by in situ hybridization in nonneuronal tissues such as kidney (M) or lung (N).

Mentions: In the agrinLN allele, genomic regulatory elements that normally direct expression of LN-agrin would be expected to direct expression of a LN–β-geo fusion protein, which is detectable with the histochemical stain for lacZ. We therefore used lacZ histochemistry to assess the cellular distribution of LN-agrin in the central nervous system of phenotypically normal agrinLN/+ heterozygotes at E14 and E18. Weak signals were present in numerous areas, including cerebellum, cortex, and hippocampus, but four sites of expression were especially prominent at both ages. First, small blood vessels were intensely stained throughout the nervous system (Fig. 6A and Fig. B). Second, neural progenitors were stained in the ventricular zones of the cerebral cortex (Fig. 6 B), hippocampus (Fig. 6 D), and spinal cord (Fig. 6 E). Third, sensory neurons were lacZ positive in dorsal root ganglia (Fig. 6 E) and in the trigeminal ganglion (not shown). Fourth, motoneurons were intensely stained, indicating that these cells express high levels of LN-agrin. Selective staining of motoneurons was apparent both in the spinal cord (Fig. 6 E) and in the hindbrain (Fig. 6 H). Staining was intense at both E14 and E18, and expression persisted into adulthood (Fig. 6 I). Selective expression of LN-agrin by motoneurons is noteworthy in view of functional studies reported below.


Agrin isoforms with distinct amino termini: differential expression, localization, and function.

Burgess RW, Skarnes WC, Sanes JR - J. Cell Biol. (2000)

Expression of LN- and SN-agrin. The β-geo insertion allowed LN-agrin expression to be analyzed by lacZ staining in heterozygotes (A, B, D, E, and G–L), whereas SN-agrin RNA was detected by in situ hybridization (C, F, M, and N). (A and B) In the brains of E14 mice, LN-agrin is expressed in cerebral blood vessels and in the ventricular (vz) and intermediate zone (iz) of the cortex, but not in the cortical plate (cp; B). (C) SN-agrin is expressed in a pattern that is complementary to the LN pattern, being most abundant in the postmitotic cells of the cortical plate. (D) In E18 embryos, LN-agrin continues to have very restricted expression in the hippocampus, being present only near the ventricles and in blood vessels. (E) In the spinal cord, LN-agrin is strongly expressed by motoneurons and sensory neurons of the DRG (arrows). (F) SN-agrin is widely distributed in many neuronal cells types in both the DRG and the spinal cord, although motoneurons are not more intensely positive than other cell types (white arrows). (H) LN-agrin is also expressed by cranial nerve motor nuclei in the hindbrain (E18 horizontal section; IV, forth ventricle; arrowheads, oculomotor nuclei). (I) Motoneuron expression of LN-agrin persists into adulthood. (G and J–N) Nonneuronal cells that abut basal laminae express LN-agrin but not SN-agrin. Epidermal cells in E18 skin (G), kidney glomeruli and tubules (J), and pulmonary epithelium (K) are all positive for LN-agrin. No lacZ activity was present in littermate controls in any tissue (E18 kidney shown, L). Consistent with northern blotting, SN-agrin is not expressed at levels detectable by in situ hybridization in nonneuronal tissues such as kidney (M) or lung (N).
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Related In: Results  -  Collection

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Figure 6: Expression of LN- and SN-agrin. The β-geo insertion allowed LN-agrin expression to be analyzed by lacZ staining in heterozygotes (A, B, D, E, and G–L), whereas SN-agrin RNA was detected by in situ hybridization (C, F, M, and N). (A and B) In the brains of E14 mice, LN-agrin is expressed in cerebral blood vessels and in the ventricular (vz) and intermediate zone (iz) of the cortex, but not in the cortical plate (cp; B). (C) SN-agrin is expressed in a pattern that is complementary to the LN pattern, being most abundant in the postmitotic cells of the cortical plate. (D) In E18 embryos, LN-agrin continues to have very restricted expression in the hippocampus, being present only near the ventricles and in blood vessels. (E) In the spinal cord, LN-agrin is strongly expressed by motoneurons and sensory neurons of the DRG (arrows). (F) SN-agrin is widely distributed in many neuronal cells types in both the DRG and the spinal cord, although motoneurons are not more intensely positive than other cell types (white arrows). (H) LN-agrin is also expressed by cranial nerve motor nuclei in the hindbrain (E18 horizontal section; IV, forth ventricle; arrowheads, oculomotor nuclei). (I) Motoneuron expression of LN-agrin persists into adulthood. (G and J–N) Nonneuronal cells that abut basal laminae express LN-agrin but not SN-agrin. Epidermal cells in E18 skin (G), kidney glomeruli and tubules (J), and pulmonary epithelium (K) are all positive for LN-agrin. No lacZ activity was present in littermate controls in any tissue (E18 kidney shown, L). Consistent with northern blotting, SN-agrin is not expressed at levels detectable by in situ hybridization in nonneuronal tissues such as kidney (M) or lung (N).
Mentions: In the agrinLN allele, genomic regulatory elements that normally direct expression of LN-agrin would be expected to direct expression of a LN–β-geo fusion protein, which is detectable with the histochemical stain for lacZ. We therefore used lacZ histochemistry to assess the cellular distribution of LN-agrin in the central nervous system of phenotypically normal agrinLN/+ heterozygotes at E14 and E18. Weak signals were present in numerous areas, including cerebellum, cortex, and hippocampus, but four sites of expression were especially prominent at both ages. First, small blood vessels were intensely stained throughout the nervous system (Fig. 6A and Fig. B). Second, neural progenitors were stained in the ventricular zones of the cerebral cortex (Fig. 6 B), hippocampus (Fig. 6 D), and spinal cord (Fig. 6 E). Third, sensory neurons were lacZ positive in dorsal root ganglia (Fig. 6 E) and in the trigeminal ganglion (not shown). Fourth, motoneurons were intensely stained, indicating that these cells express high levels of LN-agrin. Selective staining of motoneurons was apparent both in the spinal cord (Fig. 6 E) and in the hindbrain (Fig. 6 H). Staining was intense at both E14 and E18, and expression persisted into adulthood (Fig. 6 I). Selective expression of LN-agrin by motoneurons is noteworthy in view of functional studies reported below.

Bottom Line: The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons.Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated.Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, Missouri 63110, USA.

ABSTRACT
The proteoglycan agrin is required for postsynaptic differentiation at the skeletal neuromuscular junction, but is also associated with basal laminae in numerous other tissues, and with the surfaces of some neurons. Little is known about its roles at sites other than the neuromuscular junction, or about how its expression and subcellular localization are regulated in any tissue. Here we demonstrate that the murine agrin gene generates two proteins with different NH(2) termini, and present evidence that these isoforms differ in subcellular localization, tissue distribution, and function. The two isoforms share approximately 1,900 amino acids (aa) of common sequence following unique NH(2) termini of 49 or 150 aa; we therefore call them short NH(2)-terminal (SN) and long NH(2)-terminal (LN) isoforms. In the mouse genome, LN-specific exons are upstream of an SN-specific exon, which is in turn upstream of common exons. LN-agrin is expressed in both neural and nonneural tissues. In spinal cord it is expressed in discrete subsets of cells, including motoneurons. In contrast, SN-agrin is selectively expressed in the nervous system but is widely distributed in many neuronal cell types. Both isoforms are externalized from cells but LN-agrin assembles into basal laminae whereas SN-agrin remains cell associated. Differential expression of the two isoforms appears to be transcriptionally regulated, whereas the unique SN and LN sequences direct their distinct subcellular localizations. Insertion of a "gene trap" construct into the mouse genome between the LN and SN exons abolished expression of LN-agrin with no detectable effect on expression levels of SN-agrin or on SN-agrin bioactivity in vitro. Agrin protein was absent from all basal laminae in mice lacking LN-agrin transcripts. The formation of the neuromuscular junctions was as drastically impaired in these mutants as in mice lacking all forms of agrin. Thus, basal lamina-associated LN-agrin is required for neuromuscular synaptogenesis, whereas cell-associated SN-agrin may play distinct roles in the central nervous system.

Show MeSH
Related in: MedlinePlus