- Email: [email protected]
Punc, a novel mouse gene of the immunoglobulin superfamily, is expressed predominantly in the developing nervous system
Mechanisms of Development 71 (1998) 201–204
Gene expression pattern
Punc, a novel mouse gene of the immunoglobulin superfamily, is expressed predominantly in the developing nervous system J. Michael Salbaum* The Neurosciences Institute, 10640 John J. Hopkins Drive, San Diego, CA 92121, USA Received 25 November 1997; revised version received 12 December 1997; accepted 22 December 1997
Abstract A search for genes regulated by the LlM/homeodomain transcription factor islet-1 during development identified the novel mouse gene punc (putative neuronal cell adhesion molecule). Punc is a member of the immunoglobulin superfamily, exhibits a novel configuration of four immunoglobulin domains and two fibronectin-type III repeats, and resembles proteins involved in axon guidance. Punc expression in the embryo occurs in restricted patterns in both the nervous system and limb mesoderm. A sharp decrease in punc expression after embryonic day 11.5 suggests a function for punc early in mouse embryogenesis. 1998 Elsevier Science Ireland Ltd. Keywords: Axon guidance; Cell adhesion molecule; Cell differentiation; Cell proliferation; Cell surface; Development; Differential display; Downregulation; Embryo; Gene expression; Homeodomain; Immunoglobulin superfamily; Islet-1; Limb bud; Mesoderm; Midbrain; Motorneuron; Mouse; Neural plate; Neuroepithelium; Transcription factor; Transgenic mice
1. Introduction Transcription factors play key roles in orchestrating programs of gene expression as the basis for cell differentiation during development (Tanabe and Jessell, 1996). Understanding how such programs generate cellular phenotypes therefore requires elucidating the target genes that are regulated by particular transcription factors. Focusing on motor neuron differentiation, I have set up a screen to identify target genes for the LIM/homeodomain transcription factor isl-1 (Fig. 1A) (Ericson et al., 1992), which is essential for motor neuron development (Pfaff et al., 1996). Using overexpression of isl-1 in transgenic mice, mRNA differential display, and in situ hybridization, I have identified a novel mouse gene whose expression in the embryonic spinal cord was inversely correlated with the expression of isl-1 (Fig. 1B). Sequence analysis of cDNA clones predicted a protein (Fig. 1E) with a signal peptide, four immunoglobulin-like (lg) domains, two fibronectin-type III (FnIII) repeats, a transmembrane and a cytoplasmic domain. This organization (Fig. 1C) is reminiscent of cell adhesion mole* Tel.: +1 619 626 2162; fax: +1 619 626 2199; e-mail: [email protected]
0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (98 )0 0005-7
cules such as NCAM (Cunningham et al., 1987), but constitutes a new subclass within the lg superfamily. Due to the predominantly neuronal expression, the new gene was designated punc, an acronym for putative neuronal cell adhesion molecule. Punc is most similar to the Deleted in Colorectal Cancer gene DCC (Fearon et al., 1990), with 41, 42, and 47% identity in the second, third and fourth lg domains, respectively (Fig. 1C,F). However, the FnIII repeats and the cytoplasmic regions differed significantly. This suggested that both molecules may participate in similar extracellular interactions, but diverge concerning their signal transduction. Northern blot and in situ hybridizations demonstrated that punc was expressed during mid-gestation, but not at later stages. A band of 5.2 kb was detected in RNA from embryos at days E9.5, E10.5 and E11.5 (Fig. 1D). In situ hybridization (Fig. 2) showed that expression in the neuroectoderm precedes expression in the lateral plate mesoderm. Punc expression, e.g. in the neural tube, appears to be correlated with early cell proliferation. Associated with motor neuron differentiation, a drastic decrease of punc expression was observed. This could indicate that isl-1 may contribute to
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the downregulation of punc in motor neurons. However, after E11.5, punc mRNA was no longer detectable in any tissue, suggesting that transcriptional control of punc may
involve additional factors. Onset, spatial pattern, and sharp downregulation of punc expression are consistent with a role in cell differentiation in various tissues of the embryo.
Fig. 1. (A) E10.75, immunohistochemical staining for isl-1. (B) E10.75, in situ hybridization; punc expression is absent from motor neurons, in contrast to dorsal sensory neurons, ventral interneurons and cells of the ventral-medial zone; scale bars: 100 mm. Abbreviations: drg, dorsal root ganglion; fp, floor plate; mn, motor neuron; sc, spinal cord. (C) Domain structure of punc compared with DCC and NCAM. Red: identity above 40%. (D) Northern blot of total RNA from embryos at different stages (H, heads). (E) Predicted punc protein sequence (cDNA: Genbank accession number AF026465). Shaded area, signal peptide and transmembrane domain; boxes, immunoglobulin domains. (F) Alignment of individual lg domains of punc and DCC. Black, identity; gray, similarity; periods, gaps; asterisks, residues characteristic of lg domains.
J.M. Salbaum / Mechanisms of Development 71 (1998) 201–204
2. Method Differential display (manuscript in preparation), cDNA
203
screening of a E10.5 library (GIBCO) and DNA sequencing (Sambrook et al., 1989), and in situ hybridizations with digoxygenin-labeled riboprobes (Hogan et al., 1994) were
Fig. 2. Analysis of punc gene expression by in situ hybridization. Tissue: cryosections, 30 mm; scale bars: A–E,G,H,K,L: 100 mm; others: 500 mm. (A) Advanced primitive streak stage, E7.5, showing expression in the neuroectoderm. (B,C) Head fold stage, E8.5. Expression is restricted to the nervous system. (D,E) E9.5; punc staining is detected in the neural tube and lateral mesoderm. Ventral prosencephalon is negative. (F) Head, E10.5. Mesencephalon and dorsal diencephalon are labeled. Staining in the dorsal mesencephalon is decreased, ventral diencephalon is negative. Cranial mesenchyme and first branchial arch are positive. (G) Hind brain neural tube, E10.5; expression is evident in rhombomere 2, but absent from rhombomere 3, revealing a distinct transition. (H) Tail, E11.5. Dorsal root ganglia and neural tube, except motor neurons, are positive. (I–L) E10.5, series of transverse sections. Expression is detected throughout the neural tube, with regional restrictions in diencephalon (arrowhead) and caudal-medial telencephalon. Fore and hind limb buds and lateral plate mesoderm are strongly stained. (M–P) E11.5, sections approximately corresponding to I–L, illustrating a sharp decrease in expression compared with earlier stages. Telencephalon (M,N), cervical spinal cord (O), and limb buds (O,P) are negative, expression in diencephalon through rhombencephalon is restricted to the ventricular zone. Staining is still detectable in neural tube and dorsal root ganglia at lumbar levels (P). Motor neurons are already negative. Abbreviations: b1, first branchial arch; cm, cranial mesenchyme; di, diencephalon; ec, ectoderm; fl, fore limb; g, gut; h, heart; hl, hind limb; lpm, lateral plate mesoderm; m, mesoderm; mes, mesencephalon; nf, neural fold; nt, neural tube; pg. primitive groove; pm, paraxial mesoderm; pro, prosencephalon; r2, rhombomere 2; r3, rhombomere 3; rh, rhombencephalon; tel, telencephalon; VIII, third ventricle; VIV, fourth ventricle.
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J.M. Salbaum / Mechanisms of Development 71 (1998) 201–204
performed as described. A monoclonal antibody 39.4D5 (Tsuchida et al., 1994) against islet-1 was obtained from the Developmental Studies Hvbridoma Bank. Acknowledgements Expert technical assistance by Serge Golden is gratefully acknowledged. I wish to thank Drs. Gerald Edelman, Einar Gall, Fred Jones and Claudia Kappen for comments on the manuscript. This work was made possible by Neurosciences Research Foundation, which receives major support from Novartis. J.M.S. received support from the Richard Lounsbery Foundation. References Cunningham, B.A., Hemperly, J.J., Murray, B.A., Prediger, E.A., Brackenbury, R., Edelman, G.M., 1987. Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science 236, 799–806.
Ericson, J., Thor, S., Edlund, T., Jessell, T.M., Yamada, T., 1992. Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256, 1555–1560. Fearon, E.R., Cho, K.R., Nigro, J.M., Kern, S.E., Simons, J.W., Ruppert, J.M., Hamilton, S.R., Preisinger, A.C., Thomas, G., Kinzler, K.W., Vogelstein, B., 1990. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247, 49–56. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Pfaff, S.L., Mendelsohn, M., Stewart, C.L., Edlund, T., Jessell, T.M., 1996. Requirement for LIM homeobox gene lsl 1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84, 309–320. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Tanabe, Y., Jessell, T.M., 1996. Diversity and pattern in the developing spinal cord. Science 274, 1115–1123. Tsuchida, T., Ensini, M., Morton, S.B., Baldassare, M., Edlund, T., Jessell, T.M., Pfaff, S.L., 1994. Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79, 957– 970.
Gene expression pattern
Punc, a novel mouse gene of the immunoglobulin superfamily, is expressed predominantly in the developing nervous system J. Michael Salbaum* The Neurosciences Institute, 10640 John J. Hopkins Drive, San Diego, CA 92121, USA Received 25 November 1997; revised version received 12 December 1997; accepted 22 December 1997
Abstract A search for genes regulated by the LlM/homeodomain transcription factor islet-1 during development identified the novel mouse gene punc (putative neuronal cell adhesion molecule). Punc is a member of the immunoglobulin superfamily, exhibits a novel configuration of four immunoglobulin domains and two fibronectin-type III repeats, and resembles proteins involved in axon guidance. Punc expression in the embryo occurs in restricted patterns in both the nervous system and limb mesoderm. A sharp decrease in punc expression after embryonic day 11.5 suggests a function for punc early in mouse embryogenesis. 1998 Elsevier Science Ireland Ltd. Keywords: Axon guidance; Cell adhesion molecule; Cell differentiation; Cell proliferation; Cell surface; Development; Differential display; Downregulation; Embryo; Gene expression; Homeodomain; Immunoglobulin superfamily; Islet-1; Limb bud; Mesoderm; Midbrain; Motorneuron; Mouse; Neural plate; Neuroepithelium; Transcription factor; Transgenic mice
1. Introduction Transcription factors play key roles in orchestrating programs of gene expression as the basis for cell differentiation during development (Tanabe and Jessell, 1996). Understanding how such programs generate cellular phenotypes therefore requires elucidating the target genes that are regulated by particular transcription factors. Focusing on motor neuron differentiation, I have set up a screen to identify target genes for the LIM/homeodomain transcription factor isl-1 (Fig. 1A) (Ericson et al., 1992), which is essential for motor neuron development (Pfaff et al., 1996). Using overexpression of isl-1 in transgenic mice, mRNA differential display, and in situ hybridization, I have identified a novel mouse gene whose expression in the embryonic spinal cord was inversely correlated with the expression of isl-1 (Fig. 1B). Sequence analysis of cDNA clones predicted a protein (Fig. 1E) with a signal peptide, four immunoglobulin-like (lg) domains, two fibronectin-type III (FnIII) repeats, a transmembrane and a cytoplasmic domain. This organization (Fig. 1C) is reminiscent of cell adhesion mole* Tel.: +1 619 626 2162; fax: +1 619 626 2199; e-mail: [email protected]
0925-4773/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (98 )0 0005-7
cules such as NCAM (Cunningham et al., 1987), but constitutes a new subclass within the lg superfamily. Due to the predominantly neuronal expression, the new gene was designated punc, an acronym for putative neuronal cell adhesion molecule. Punc is most similar to the Deleted in Colorectal Cancer gene DCC (Fearon et al., 1990), with 41, 42, and 47% identity in the second, third and fourth lg domains, respectively (Fig. 1C,F). However, the FnIII repeats and the cytoplasmic regions differed significantly. This suggested that both molecules may participate in similar extracellular interactions, but diverge concerning their signal transduction. Northern blot and in situ hybridizations demonstrated that punc was expressed during mid-gestation, but not at later stages. A band of 5.2 kb was detected in RNA from embryos at days E9.5, E10.5 and E11.5 (Fig. 1D). In situ hybridization (Fig. 2) showed that expression in the neuroectoderm precedes expression in the lateral plate mesoderm. Punc expression, e.g. in the neural tube, appears to be correlated with early cell proliferation. Associated with motor neuron differentiation, a drastic decrease of punc expression was observed. This could indicate that isl-1 may contribute to
202
J.M. Salbaum / Mechanisms of Development 71 (1998) 201–204
the downregulation of punc in motor neurons. However, after E11.5, punc mRNA was no longer detectable in any tissue, suggesting that transcriptional control of punc may
involve additional factors. Onset, spatial pattern, and sharp downregulation of punc expression are consistent with a role in cell differentiation in various tissues of the embryo.
Fig. 1. (A) E10.75, immunohistochemical staining for isl-1. (B) E10.75, in situ hybridization; punc expression is absent from motor neurons, in contrast to dorsal sensory neurons, ventral interneurons and cells of the ventral-medial zone; scale bars: 100 mm. Abbreviations: drg, dorsal root ganglion; fp, floor plate; mn, motor neuron; sc, spinal cord. (C) Domain structure of punc compared with DCC and NCAM. Red: identity above 40%. (D) Northern blot of total RNA from embryos at different stages (H, heads). (E) Predicted punc protein sequence (cDNA: Genbank accession number AF026465). Shaded area, signal peptide and transmembrane domain; boxes, immunoglobulin domains. (F) Alignment of individual lg domains of punc and DCC. Black, identity; gray, similarity; periods, gaps; asterisks, residues characteristic of lg domains.
J.M. Salbaum / Mechanisms of Development 71 (1998) 201–204
2. Method Differential display (manuscript in preparation), cDNA
203
screening of a E10.5 library (GIBCO) and DNA sequencing (Sambrook et al., 1989), and in situ hybridizations with digoxygenin-labeled riboprobes (Hogan et al., 1994) were
Fig. 2. Analysis of punc gene expression by in situ hybridization. Tissue: cryosections, 30 mm; scale bars: A–E,G,H,K,L: 100 mm; others: 500 mm. (A) Advanced primitive streak stage, E7.5, showing expression in the neuroectoderm. (B,C) Head fold stage, E8.5. Expression is restricted to the nervous system. (D,E) E9.5; punc staining is detected in the neural tube and lateral mesoderm. Ventral prosencephalon is negative. (F) Head, E10.5. Mesencephalon and dorsal diencephalon are labeled. Staining in the dorsal mesencephalon is decreased, ventral diencephalon is negative. Cranial mesenchyme and first branchial arch are positive. (G) Hind brain neural tube, E10.5; expression is evident in rhombomere 2, but absent from rhombomere 3, revealing a distinct transition. (H) Tail, E11.5. Dorsal root ganglia and neural tube, except motor neurons, are positive. (I–L) E10.5, series of transverse sections. Expression is detected throughout the neural tube, with regional restrictions in diencephalon (arrowhead) and caudal-medial telencephalon. Fore and hind limb buds and lateral plate mesoderm are strongly stained. (M–P) E11.5, sections approximately corresponding to I–L, illustrating a sharp decrease in expression compared with earlier stages. Telencephalon (M,N), cervical spinal cord (O), and limb buds (O,P) are negative, expression in diencephalon through rhombencephalon is restricted to the ventricular zone. Staining is still detectable in neural tube and dorsal root ganglia at lumbar levels (P). Motor neurons are already negative. Abbreviations: b1, first branchial arch; cm, cranial mesenchyme; di, diencephalon; ec, ectoderm; fl, fore limb; g, gut; h, heart; hl, hind limb; lpm, lateral plate mesoderm; m, mesoderm; mes, mesencephalon; nf, neural fold; nt, neural tube; pg. primitive groove; pm, paraxial mesoderm; pro, prosencephalon; r2, rhombomere 2; r3, rhombomere 3; rh, rhombencephalon; tel, telencephalon; VIII, third ventricle; VIV, fourth ventricle.
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J.M. Salbaum / Mechanisms of Development 71 (1998) 201–204
performed as described. A monoclonal antibody 39.4D5 (Tsuchida et al., 1994) against islet-1 was obtained from the Developmental Studies Hvbridoma Bank. Acknowledgements Expert technical assistance by Serge Golden is gratefully acknowledged. I wish to thank Drs. Gerald Edelman, Einar Gall, Fred Jones and Claudia Kappen for comments on the manuscript. This work was made possible by Neurosciences Research Foundation, which receives major support from Novartis. J.M.S. received support from the Richard Lounsbery Foundation. References Cunningham, B.A., Hemperly, J.J., Murray, B.A., Prediger, E.A., Brackenbury, R., Edelman, G.M., 1987. Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science 236, 799–806.
Ericson, J., Thor, S., Edlund, T., Jessell, T.M., Yamada, T., 1992. Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1. Science 256, 1555–1560. Fearon, E.R., Cho, K.R., Nigro, J.M., Kern, S.E., Simons, J.W., Ruppert, J.M., Hamilton, S.R., Preisinger, A.C., Thomas, G., Kinzler, K.W., Vogelstein, B., 1990. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247, 49–56. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Pfaff, S.L., Mendelsohn, M., Stewart, C.L., Edlund, T., Jessell, T.M., 1996. Requirement for LIM homeobox gene lsl 1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84, 309–320. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Tanabe, Y., Jessell, T.M., 1996. Diversity and pattern in the developing spinal cord. Science 274, 1115–1123. Tsuchida, T., Ensini, M., Morton, S.B., Baldassare, M., Edlund, T., Jessell, T.M., Pfaff, S.L., 1994. Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79, 957– 970.