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Restricted expression of Slap-1 in the rodent cerebral cortex
Gene Expression Patterns 3 (2003) 437–440 www.elsevier.com/locate/modgep
Restricted expression of Slap-1 in the rodent cerebral cortex Pavlos Alifragisa,*, Zolta´n Molna´rb, John G Parnavelasa a Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
b
Received 28 January 2003; received in revised form 20 March 2003; accepted 20 March 2003
Abstract The deep layers of the mammalian cerebral cortex contain pyramidal neurons that project predominantly to subcortical targets. To understand the mechanisms that determine the identity of deeper layer neurons, a PCR based subtractive hybridisation was performed to isolate genes that are specifically expressed during the specification of these neurons. One of the genes we isolated was the rat homologue of the mouse Slap-1. SLAP-1 is an adaptor protein containing SH2-SH3 domains and it participates in the signalling of Receptor Tyrosine Kinases. In situ hybridisation studies have shown that Slap-1 is not substantially expressed before E17.At later stages, it is specifically and selectively expressed by deeper layer neurons and by neurons of layers II/III in the developing cortex. The specific timing and location of its expression, suggests that this gene may play a role in the differentiation of these neurons. q 2003 Elsevier Science B.V. All rights reserved. Keywords: telencephalon; piriform cortex; cerebral cortex; subplate; layer V; layer II/III; slap-1; SH2-SH3; adaptor protein
1. Results and discussion The cerebral cortex is the most prominent region of the dorsal telencephalon of the mammalian forebrain. In the mature brain, neurons of the neocortex are arranged in six layers, numbered I (most superficial) to VI. With the exception of layer I, all layers are formed in an inside-out pattern, in which the deep layers are laid down first followed by the addition of the more superficial layers (Berry and Rogers, 1965). Neurons within each layer share common properties including time of birth, sites of projection, physiological properties, and gene expression profiles (McConnell, 1995). Layer V/VI neurons, for instance, are the only neurons that project to subcortical areas (Koester and O’Leary, 1992; Kasper et al., 1994). In the rat, neurons destined for these layers are generated at the start of corticogenesis (E15-17) (Miller, 1988). In this study, we sought to isolate genes that are specifically expressed by these cells and, thus, might contribute to their morphological and functional identity. To narrow down our screen * Corresponding author. Present address: CSC Mammalian Neurogenesis Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK. Tel.: þ 44-20-8383-8298; fax: þ44-20-8383-8303. E-mail address: [email protected] (P. Alifragis).
on genes that are expressed by deeper layer neurons, cDNAs were prepared from tissue taken from embryonic cortex at the time of generation of these neurons, E15 and E17. A PCR based subtractive hybridisation was performed to isolate genes that are differentially expressed at E17. One of the differentially expressed genes isolated was the rat homologue of the mouse Slap-1 (GenBank accession number AY217759). Slap-1 was initially identified in a yeast two-hybrid screen using the cytoplasmic domain of the receptor tyrosine kinase Eph A2 as bait (Pandey et al., 1995). Eph’s constitute the largest known family of receptor tyrosine kinases and, together with their ligands, have been implicated as mediators of a variety of patterning events during embryonic development of the CNS (Flanagan and Vanderhaeghen, 1998). SLAP-1 is a molecule resembling the Src family of protein tyrosine kinases containing both SH2 and SH3 domains, but instead of the typical kinase domain that follows, it contains a unique COOH domain of 104 amino acids (Sosinowski et al., 2000). Slap-1 mRNA is predominantly expressed in lymphoid tissue, though low mRNA levels have been reported in the lung and brain (Sosinowski et al., 2000). Biochemical data suggest that SLAP-1 can inhibit PDGFs mitogenic response in NIH 3T3 fibroblasts antagonising Src kinase (Roche et al., 1998), while in the immune system it inhibits T-cell receptor
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(03)00090-5
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P. Alifragis et al. / Gene Expression Patterns 3 (2003) 437–440
(TCR) signalling during the process of negative/positive selection of CD4-CD8 thymocytes (Sosinowski et al., 2000, 2001). Here, we report the identification of Slap-1 as a gene that is differentially expressed by deeper layer neurons during cortical development, and provide a profile of its specific spatio-temporal expression pattern in the rat telencephalon. 1.1. Prenatal expression In order to verify the differential expression of Slap-1 in the rat telencephalon, In situ hybridisation experiments were carried out in embryonic rat brain sections. Consistent with our subtraction, Slap-1 transcripts were not detected in E15 rat brains (data not shown). By E17, the expression of Slap-1 was evident with strong signal intensities at deeper cortical layers and in the subplate (Fig. 1). Interestingly, Slap-1 transcripts were not detected in migrating neurons, but in neurons that had already reached their final destinations. Furthermore, expression of Slap-1 appeared throughout the rostro-caudal and dorso-ventral level with no apparent gradient. Lower levels of Slap-1 transcripts were also detected in the piriform cortex and in the developing hippocampus (Fig. 1A – C). However, we were unable to detect transcripts of Slap-1 in the ventral telencephalon, thalamus or hypothalamus (Fig. 1C). At later stages of development, the expression pattern became more refined.
Fig. 2. At E20, subplate (sp), layer V neurons, and cells in the piriform cortex (pc) continue to express Slap-1 (A–C). In addition, transcripts are detected in upper layer neurons (possibly even some layer II/III) at the dorso-medial cortex (white arrows in A and B). D is a high magnification of the boxed area in B. It can be clearly seen that slap-1 is restricted in the subplate and layer V. Scale bar: (A –C) 250 mm; (D) 50 mm.
At E20, strong expression was detected in the subplate and layer V neurons, and at the outer layers of the piriform cortex (Fig. 2). There was particularly strong expression at the ventral/lateral cortex, adjacent to the perirhinal cortex. In addition, Slap-1 transcripts were detected in a subset of upper layer (probably layers II/II) neurons, but only in the dorso-medial cortex (Fig. 2A,B white arrows). 1.2. Postnatal expression
Fig. 1. Slap-1 expression is first detected at E17 (A –C): The strongest expression is detected in the hippocampus (hp), subplate (sp), cortical plate, and in the piriform cortex (pc). It is expressed in a ventro-dorsal gradient matching the neurogenetic gradient of the cortex. Transcripts are detected throughout the rostro-caudal axis (A: rostral, B: medial, C: caudal). D: High magnification of the boxed area in B shows clearly labelled cells in subplate and layer V neurons. Scale bar: (A –C) 200 mm; (D) 50 mm.
After birth, Slap-1 expression appeared less intense and was downregulated with the exception of layer V in the neocortex and in areas of the piriform cortex. Thus, at P2, layer V neurons still expressed Slap-1 (Fig. 3), but the intensity of the signal has decreased. Furthermore, consistent with the prenatal expression pattern, Slap-1 was expressed in layers II/III in the dorso-medial cortex (Fig. 3A). Contrary to layer V neurons, layer II/III cells did not express Slap-1 uniformly. Our observations showed that the strongest signal was in layer II/III neurons in the middle regions along the rostro-caudal axis of the brain (at the level of the dentate gyrus), with a sharp boundary caudally (arrow, Fig. 3B) and a fading gradient dorsally. Furthermore, Slap-1 was down-regulated in subplate cells (Fig. 3). As the maturation of the cortex progressed, Slap-1 expression was still present at the outer layers of the piriform cortex at P7 (Fig. 4). However, Slap-1 was down- regulated
P. Alifragis et al. / Gene Expression Patterns 3 (2003) 437–440
439
axons towards their targets and whether the expression is restricted to a specific subset of layer V projection neurons.
2. Materials and methods
Fig. 3. Postnatally, the intensity of Slap-1 labelling decreases. (A) Expression of Slap-1 at P2 is detected in the subplate, and in neurons of layers V II/III. (B) Stronger signal intensities in upper cortical layers are detected above the hippocampus (hp) at the level of the dentate gyrus in a rostrocaudal gradient, with a sharp caudal boundary (arrowhead) and a medial high, rostral low gradient. Scale bars: 200 mm.
in the neocortex, and transcripts were only detected in a subset of layer V and II/III neurons in the dorsal and cingulate cortex as shown in Fig. 4. Finally, by P14, Slap-1 transcripts were detected only in the piriform cortex and in a few scattered cells in layers V and II/III of the dorso-medial cortex (data not shown). Taken together, these observations indicate that Slap-1 exhibits a dynamic expression pattern during cortical development. It is expressed predominantly by deeper layer neurons during a very restricted temporal window, when these cells reach and establish connections with their subcortical targets (Koester and O’Leary, 1992). Its expression in layer II/III neurons occurs at a time that these cells establish connections with other cortical areas (ipsilateral and contralateral). It is not known at present whether Slap-1 is involved in a process of guiding these
Sprague Dauley albino rats were used in this study. The morning the vaginal plug was found was defined as day 1. For the cDNA subtraction, mRNA was isolated using the Micro-Fast Track mRNA isolation kit from Invitrogen as per supplier’s instructions. Preparation of cDNA and the subtractive hybridisation was performed using the PCR-Select cDNA subtraction kit from Clontech according to manufacturers specifications. Briefly, cDNA was prepared from E15 and E17 brains. Both cDNA samples were digested with RsaI to create shorter blunt ended molecules. The digested E17 samples were then diluted and separated. Two different adaptors were ligated to the separated E17 samples. Subsequently, an excess of digested E15 cDNA was added to each E17 cDNA sample and they were heatdenatured and allowed to anneal. The annealed samples were then mixed together in the presence of fresh denatured E15 cDNA sample to allow the formation of differentially expressed E17 cDNAs with different adaptors on each end, and were subsequently PCR amplified. The E17 specific amplification products were cloned in T-easy vector (Promega) to create a library of E17 specific cDNAs. In situ hybridisation was carried out on 20 mm cryostat sections of fixed brains using DIG labelled antisense riboprobes. Hybridisation of the DIG riboprobe was carried out overnight at 55 8C. The hybridisation solution was 50% deionized formamide, 5 £ SSC, 10 mM b-mercaptoethanol, 10% dextran sulfate, 2 £ Denhards, 250 mg/ml(?) yeast t-RNA and 500 mg/ml Salmon Sperm DNA. For the detection, anti-digoxigenin Fabs coupled to alkaline phosphatase were used in combination with NBT/BCIP. Signal with the sense probe was not detected.
Acknowledgements This work has been supported by the European Community (Grant QLRT-1999-30158) and The Wellcome Trust (Grant 063974/B/01/Z) to JP and ZM.
References
Fig. 4. At P7, Slap-1 expression is maintained by some layer V and some layer II/III neurons in the dorsal, medial, and cingulate cortex. Expression is also maintained in some neurons in the outer layers of the piriform cortex (pc). Scale bar: 200 mm.
Berry, M., Rogers, A.W., 1965. The migration of neuroblasts in the developing cerebral cortex. J. Anat. 99, 691–709. Flanagan, J.G., Vanderhaeghen, P., 1998. The ephrins and Eph receptors in neural development. Annu. Rev. Neurosci. 21, 309–345. Kasper, E.M., Larkman, A.U., Lubke, J., Blakemore, C., 1994. Pyramidal neurons in layer 5 of the visual cortex. III. Differential maturation of
440
P. Alifragis et al. / Gene Expression Patterns 3 (2003) 437–440
axon targeting, dendritic morphology and electrophysiological properties. J. Comp. Neurol. 339, 495 –518. Koester, S.F., O’Leary, D.D.M., 1992. Functional classes of cortical projection neurons develop dendritic distinctions by classspecific sculpting of an early common pattern. J. Neurosci. 12, 1382–1393. McConnell, S.K., 1995. Constructing the cerebral cortex: neurogenesis and fate determination. Neuron 15, 761–768. Miller, M.W., 1988. Development of projection and local circuit neurons in neocortex. In: Peters, A., Jones, E.G. (Eds.), Cerebral Cortex, vol 7: Development and Maturation of Cerebral Cortex, Plenum Press, London, pp. 133 –175.
Pandey, A., Duan, H., Dixit, V.M., 1995. Characterization of a novel Srclike adapter protein that associates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270, 19201–19204. Roche, S., Alonso, G., Kazlauskas, A., Dixit, V.M., Courtneidge, S.A., Pandey, A., 1998. Src-like adaptor protein (Slap) is a negative regulator of mitogenesis. Curr. Biol. 8, 975 –978. Sosinowski, T., Pandey, A., Dixit, V.M., Weiss, A., 2000. Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling. J. Exp. Med. 191, 463–474. Sosinowski, T., Killeen, N., Weiss, A., 2001. The Src-like adaptor protein downregulates the T cell receptor on CD4 þ CD8 þ thymocytes and regulates positive selection. Immunity 15, 457–466.
Restricted expression of Slap-1 in the rodent cerebral cortex Pavlos Alifragisa,*, Zolta´n Molna´rb, John G Parnavelasa a Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
b
Received 28 January 2003; received in revised form 20 March 2003; accepted 20 March 2003
Abstract The deep layers of the mammalian cerebral cortex contain pyramidal neurons that project predominantly to subcortical targets. To understand the mechanisms that determine the identity of deeper layer neurons, a PCR based subtractive hybridisation was performed to isolate genes that are specifically expressed during the specification of these neurons. One of the genes we isolated was the rat homologue of the mouse Slap-1. SLAP-1 is an adaptor protein containing SH2-SH3 domains and it participates in the signalling of Receptor Tyrosine Kinases. In situ hybridisation studies have shown that Slap-1 is not substantially expressed before E17.At later stages, it is specifically and selectively expressed by deeper layer neurons and by neurons of layers II/III in the developing cortex. The specific timing and location of its expression, suggests that this gene may play a role in the differentiation of these neurons. q 2003 Elsevier Science B.V. All rights reserved. Keywords: telencephalon; piriform cortex; cerebral cortex; subplate; layer V; layer II/III; slap-1; SH2-SH3; adaptor protein
1. Results and discussion The cerebral cortex is the most prominent region of the dorsal telencephalon of the mammalian forebrain. In the mature brain, neurons of the neocortex are arranged in six layers, numbered I (most superficial) to VI. With the exception of layer I, all layers are formed in an inside-out pattern, in which the deep layers are laid down first followed by the addition of the more superficial layers (Berry and Rogers, 1965). Neurons within each layer share common properties including time of birth, sites of projection, physiological properties, and gene expression profiles (McConnell, 1995). Layer V/VI neurons, for instance, are the only neurons that project to subcortical areas (Koester and O’Leary, 1992; Kasper et al., 1994). In the rat, neurons destined for these layers are generated at the start of corticogenesis (E15-17) (Miller, 1988). In this study, we sought to isolate genes that are specifically expressed by these cells and, thus, might contribute to their morphological and functional identity. To narrow down our screen * Corresponding author. Present address: CSC Mammalian Neurogenesis Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK. Tel.: þ 44-20-8383-8298; fax: þ44-20-8383-8303. E-mail address: [email protected] (P. Alifragis).
on genes that are expressed by deeper layer neurons, cDNAs were prepared from tissue taken from embryonic cortex at the time of generation of these neurons, E15 and E17. A PCR based subtractive hybridisation was performed to isolate genes that are differentially expressed at E17. One of the differentially expressed genes isolated was the rat homologue of the mouse Slap-1 (GenBank accession number AY217759). Slap-1 was initially identified in a yeast two-hybrid screen using the cytoplasmic domain of the receptor tyrosine kinase Eph A2 as bait (Pandey et al., 1995). Eph’s constitute the largest known family of receptor tyrosine kinases and, together with their ligands, have been implicated as mediators of a variety of patterning events during embryonic development of the CNS (Flanagan and Vanderhaeghen, 1998). SLAP-1 is a molecule resembling the Src family of protein tyrosine kinases containing both SH2 and SH3 domains, but instead of the typical kinase domain that follows, it contains a unique COOH domain of 104 amino acids (Sosinowski et al., 2000). Slap-1 mRNA is predominantly expressed in lymphoid tissue, though low mRNA levels have been reported in the lung and brain (Sosinowski et al., 2000). Biochemical data suggest that SLAP-1 can inhibit PDGFs mitogenic response in NIH 3T3 fibroblasts antagonising Src kinase (Roche et al., 1998), while in the immune system it inhibits T-cell receptor
1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-133X(03)00090-5
438
P. Alifragis et al. / Gene Expression Patterns 3 (2003) 437–440
(TCR) signalling during the process of negative/positive selection of CD4-CD8 thymocytes (Sosinowski et al., 2000, 2001). Here, we report the identification of Slap-1 as a gene that is differentially expressed by deeper layer neurons during cortical development, and provide a profile of its specific spatio-temporal expression pattern in the rat telencephalon. 1.1. Prenatal expression In order to verify the differential expression of Slap-1 in the rat telencephalon, In situ hybridisation experiments were carried out in embryonic rat brain sections. Consistent with our subtraction, Slap-1 transcripts were not detected in E15 rat brains (data not shown). By E17, the expression of Slap-1 was evident with strong signal intensities at deeper cortical layers and in the subplate (Fig. 1). Interestingly, Slap-1 transcripts were not detected in migrating neurons, but in neurons that had already reached their final destinations. Furthermore, expression of Slap-1 appeared throughout the rostro-caudal and dorso-ventral level with no apparent gradient. Lower levels of Slap-1 transcripts were also detected in the piriform cortex and in the developing hippocampus (Fig. 1A – C). However, we were unable to detect transcripts of Slap-1 in the ventral telencephalon, thalamus or hypothalamus (Fig. 1C). At later stages of development, the expression pattern became more refined.
Fig. 2. At E20, subplate (sp), layer V neurons, and cells in the piriform cortex (pc) continue to express Slap-1 (A–C). In addition, transcripts are detected in upper layer neurons (possibly even some layer II/III) at the dorso-medial cortex (white arrows in A and B). D is a high magnification of the boxed area in B. It can be clearly seen that slap-1 is restricted in the subplate and layer V. Scale bar: (A –C) 250 mm; (D) 50 mm.
At E20, strong expression was detected in the subplate and layer V neurons, and at the outer layers of the piriform cortex (Fig. 2). There was particularly strong expression at the ventral/lateral cortex, adjacent to the perirhinal cortex. In addition, Slap-1 transcripts were detected in a subset of upper layer (probably layers II/II) neurons, but only in the dorso-medial cortex (Fig. 2A,B white arrows). 1.2. Postnatal expression
Fig. 1. Slap-1 expression is first detected at E17 (A –C): The strongest expression is detected in the hippocampus (hp), subplate (sp), cortical plate, and in the piriform cortex (pc). It is expressed in a ventro-dorsal gradient matching the neurogenetic gradient of the cortex. Transcripts are detected throughout the rostro-caudal axis (A: rostral, B: medial, C: caudal). D: High magnification of the boxed area in B shows clearly labelled cells in subplate and layer V neurons. Scale bar: (A –C) 200 mm; (D) 50 mm.
After birth, Slap-1 expression appeared less intense and was downregulated with the exception of layer V in the neocortex and in areas of the piriform cortex. Thus, at P2, layer V neurons still expressed Slap-1 (Fig. 3), but the intensity of the signal has decreased. Furthermore, consistent with the prenatal expression pattern, Slap-1 was expressed in layers II/III in the dorso-medial cortex (Fig. 3A). Contrary to layer V neurons, layer II/III cells did not express Slap-1 uniformly. Our observations showed that the strongest signal was in layer II/III neurons in the middle regions along the rostro-caudal axis of the brain (at the level of the dentate gyrus), with a sharp boundary caudally (arrow, Fig. 3B) and a fading gradient dorsally. Furthermore, Slap-1 was down-regulated in subplate cells (Fig. 3). As the maturation of the cortex progressed, Slap-1 expression was still present at the outer layers of the piriform cortex at P7 (Fig. 4). However, Slap-1 was down- regulated
P. Alifragis et al. / Gene Expression Patterns 3 (2003) 437–440
439
axons towards their targets and whether the expression is restricted to a specific subset of layer V projection neurons.
2. Materials and methods
Fig. 3. Postnatally, the intensity of Slap-1 labelling decreases. (A) Expression of Slap-1 at P2 is detected in the subplate, and in neurons of layers V II/III. (B) Stronger signal intensities in upper cortical layers are detected above the hippocampus (hp) at the level of the dentate gyrus in a rostrocaudal gradient, with a sharp caudal boundary (arrowhead) and a medial high, rostral low gradient. Scale bars: 200 mm.
in the neocortex, and transcripts were only detected in a subset of layer V and II/III neurons in the dorsal and cingulate cortex as shown in Fig. 4. Finally, by P14, Slap-1 transcripts were detected only in the piriform cortex and in a few scattered cells in layers V and II/III of the dorso-medial cortex (data not shown). Taken together, these observations indicate that Slap-1 exhibits a dynamic expression pattern during cortical development. It is expressed predominantly by deeper layer neurons during a very restricted temporal window, when these cells reach and establish connections with their subcortical targets (Koester and O’Leary, 1992). Its expression in layer II/III neurons occurs at a time that these cells establish connections with other cortical areas (ipsilateral and contralateral). It is not known at present whether Slap-1 is involved in a process of guiding these
Sprague Dauley albino rats were used in this study. The morning the vaginal plug was found was defined as day 1. For the cDNA subtraction, mRNA was isolated using the Micro-Fast Track mRNA isolation kit from Invitrogen as per supplier’s instructions. Preparation of cDNA and the subtractive hybridisation was performed using the PCR-Select cDNA subtraction kit from Clontech according to manufacturers specifications. Briefly, cDNA was prepared from E15 and E17 brains. Both cDNA samples were digested with RsaI to create shorter blunt ended molecules. The digested E17 samples were then diluted and separated. Two different adaptors were ligated to the separated E17 samples. Subsequently, an excess of digested E15 cDNA was added to each E17 cDNA sample and they were heatdenatured and allowed to anneal. The annealed samples were then mixed together in the presence of fresh denatured E15 cDNA sample to allow the formation of differentially expressed E17 cDNAs with different adaptors on each end, and were subsequently PCR amplified. The E17 specific amplification products were cloned in T-easy vector (Promega) to create a library of E17 specific cDNAs. In situ hybridisation was carried out on 20 mm cryostat sections of fixed brains using DIG labelled antisense riboprobes. Hybridisation of the DIG riboprobe was carried out overnight at 55 8C. The hybridisation solution was 50% deionized formamide, 5 £ SSC, 10 mM b-mercaptoethanol, 10% dextran sulfate, 2 £ Denhards, 250 mg/ml(?) yeast t-RNA and 500 mg/ml Salmon Sperm DNA. For the detection, anti-digoxigenin Fabs coupled to alkaline phosphatase were used in combination with NBT/BCIP. Signal with the sense probe was not detected.
Acknowledgements This work has been supported by the European Community (Grant QLRT-1999-30158) and The Wellcome Trust (Grant 063974/B/01/Z) to JP and ZM.
References
Fig. 4. At P7, Slap-1 expression is maintained by some layer V and some layer II/III neurons in the dorsal, medial, and cingulate cortex. Expression is also maintained in some neurons in the outer layers of the piriform cortex (pc). Scale bar: 200 mm.
Berry, M., Rogers, A.W., 1965. The migration of neuroblasts in the developing cerebral cortex. J. Anat. 99, 691–709. Flanagan, J.G., Vanderhaeghen, P., 1998. The ephrins and Eph receptors in neural development. Annu. Rev. Neurosci. 21, 309–345. Kasper, E.M., Larkman, A.U., Lubke, J., Blakemore, C., 1994. Pyramidal neurons in layer 5 of the visual cortex. III. Differential maturation of
440
P. Alifragis et al. / Gene Expression Patterns 3 (2003) 437–440
axon targeting, dendritic morphology and electrophysiological properties. J. Comp. Neurol. 339, 495 –518. Koester, S.F., O’Leary, D.D.M., 1992. Functional classes of cortical projection neurons develop dendritic distinctions by classspecific sculpting of an early common pattern. J. Neurosci. 12, 1382–1393. McConnell, S.K., 1995. Constructing the cerebral cortex: neurogenesis and fate determination. Neuron 15, 761–768. Miller, M.W., 1988. Development of projection and local circuit neurons in neocortex. In: Peters, A., Jones, E.G. (Eds.), Cerebral Cortex, vol 7: Development and Maturation of Cerebral Cortex, Plenum Press, London, pp. 133 –175.
Pandey, A., Duan, H., Dixit, V.M., 1995. Characterization of a novel Srclike adapter protein that associates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270, 19201–19204. Roche, S., Alonso, G., Kazlauskas, A., Dixit, V.M., Courtneidge, S.A., Pandey, A., 1998. Src-like adaptor protein (Slap) is a negative regulator of mitogenesis. Curr. Biol. 8, 975 –978. Sosinowski, T., Pandey, A., Dixit, V.M., Weiss, A., 2000. Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling. J. Exp. Med. 191, 463–474. Sosinowski, T., Killeen, N., Weiss, A., 2001. The Src-like adaptor protein downregulates the T cell receptor on CD4 þ CD8 þ thymocytes and regulates positive selection. Immunity 15, 457–466.