SCG10 mRNA localization in the hippocampus: comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs)

BRAIN RESEARCH ELSEVIER

Brain Research 655 (1994) 177-185

Research report

SCG10 mRNA localization in the hippocampus: comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs) Toshiyuki Himi 1, Takashi Okazaki 2, Nozomu Mori * Dil,ision Of Neurogerontologv, Ethel Percy Andrus Gerontology Center, Unicersity of Southern California, Los Angeles, CA 90089-0191, USA

Accepted 24 May 1994

Abstract

SCG10 is a nerve growth factor (NGF)-inducible, neuron-specific protein whose expression is tightly correlated with axonal a n d / o r dendritic growth. We have recently shown that the mRNA encoding SCG10 is expressed at significant Icvels in certain subsets of neurons in the adult rat brain, while its expression is undetectable or negligible in other non-neuronal tissues. Here we show that regional SCG10 mRNA expression in the adult mouse brain is comparable to that in the rat, however, in the hippocampus its expression profile is distinct. In the mouse, SCG10 mRNA is expressed at high levels in pyramidal cells of CA3-CA4 sub-fields of Ammon's horn and at low levels in the C A I - C A 2 sub-fields, while it is found rather uniformly throughout the pyramidal cell layer of the rat hippocampus. SCG10 mRNA is not detectable in the dentate gyrus of the mouse hippocampus, although it is expressed in the rat dentate gyrus. Comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs) such as GAP-43, MAP2, al-tubulin and stathmin suggests that dentate granule cells express a different repertoire of neuronal growth-associated genes in mouse and rat. Key words: Gene expression; Growth-associated gene; GAP-43; Stathmin; pl9; MAP2; c~l-Tubulin: Plasticity; Brain; Neuron; In

situ hybridization

1. Introduction

N e u r o n a l growth-associated proteins ( n G A P s ) are a family of molecules whose expression is tightly correlated with ' n e u r o n a l growth' or 'neurite outgrowth'. Expression of these molecules is required for n e u r o n a l growth during development, but their expression persists into a d u l t h o o d where n e u r o n a l growth may correlate w i t h plasticity of the nervous system [14,30,38,41,42]. T h e most well-characterized n G A P is G A P - 4 3 (also designated as B-50, F1, p57 and neuromodulin) [6,14,18,38,41,47]. However, G A P - 4 3 is a G A P and it does not seem that only G A P - 4 3 is the master gene of the n e u r o n a l growth control. In this regard, m o r e studies of o t h e r n G A P s such as neurofilaments,

* Corresponding author. Fax: (1) (213) 740-8241. t Present address: Department of Biochemistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan. 2 Present address: Central Research Laboratories, Kyorin Pharmaceutical Co., Ltd., Mitarai, Nogi, Tochigi.329, Japan. 1.2The first two authors contributed equally to this work. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00648-V

microtubule-associated proteins (MAPs), a l - t u b u l i n , and SCG10 (see below) are also required for further u n d e r s t a n d i n g of the molecular basis of neuronal structural plasticity [21,25,30,48]. R e g u l a t e d expression of n G A P s forms a substantial basis for neuronal structural plasticity, i.e. regeneration and synaptic remodeling. It is therefore important to u n d e r s t a n d the mechanisms of n G A P gene expression in the brain, especially in the h i p p o c a m p u s where a significant a m o u n t of plasticity is known to take place [7,35]. SCG10 is a neural-specific and nerve growth factor (NGF)-inducible gene p r o d u c t that was initially isolated as a n e u r o n a l m a r k e r of the neural crest [5,46]. Expression of SCG10 m R N A is observed at high levels in developing n e u r o n s [46], and persists into adulthood at low but significant levels [20]. It is induced in PC12 cells and adrenal medullary chromaffin cells by N G F [5,45], and its induction is partially suppressed in the presence of glucocorticoids [3,45]. SCG10 is also upregulated during r e g e n e r a t i o n of injured postganglionic sympathetic nerve [3]. Thus SCG10 serves as a m a r k e r of neuronal plasticity and is a n o t h e r G A P [3,4,30]. In

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situ hybridization studies of SCG10 m R N A in the adult rat brain revealed that it is expressed in certain subsets of neurons, including those in the olfactory bulb, piriform cortex, cerebellum, brain stem, and hippocampus [20]. Variously regulated expression profiles of SCG10, e.g. developmental regulation [5], NGF-inducibility [5,46], glucocorticoid-suppressibility [45], upregulation during axonal regeneration [3] and significant expression in subset neurons of the adult brain [20], are all reminiscent of GAP-43 [6,12,14,18,27,38,41,42,47]. However, there is no sequence similarity between SCG10 [46] and GAP-43 [22]. These two nGAPs may function in different aspects of neuronal growth during both development and adulthood. We have recently cloned and characterized the mouse SCG10 gene, and found that the mouse SCG10 protein sequence differs from the rat SCG10 only by one amino acid in 179 residues [36]. This strong amino acid sequence conservation seems to suggest that SCGI0 exerts an important, yet unknown, function in neurons. The study of the SCG10 gene has also indicated that the SCG10 gene was diverged from the stathmin gene by gene duplication [36]. Stathmin (also designated as p19, O p l 8 etc.) is a phosphoprotein that is more widely expressed in both neuronal and nonneuronal tissues [1,2,37,40,43,44] and its expression is developmentally regulated [1,2,23,37]. Interestingly, stathmin gene expression is apparently repressed in the adult liver, while it is rapidly induced during liver regeneration [23,37], suggesting that stathmin has a role in cellular proliferation. Phosphorylation of stathmin is rapidly induced in PC12 cells and striatal neurons by N G F and vasoactive intestinal peptide (VIP), respectively [9,13]. Thus stathmin phosphorylation seems to relate to neuronal growth a n d / o r stimulation. SCG10 may function in signalling processes at nerve terminals. Sobel and his colleagues proposed that the SCG10 homologue, stathmin, may be a signal transduction molecule that functions through rapid changes in its phosphorylation status thus transferring molecular information to other molecules [43,44]. Since amino acid sequences surrounding three serine residues as potential phosphorylation sites of stathmin are conserved in SCG10 [36,43] and SCG10 is a neural-specific gene [32,33,49], SCG10 seems to be a neural-specific phosphoprotein. The major structural difference between SCG10 and stathmin is that SCG10 contains a putative membrane-association domain, while stathmin lacks this domain [30,43]. Together with the observation that SCG10 mRNAs are expressed predominantly in neurons with long distance-projection axons a n d / o r extensive dendrites [20], the available evidence indicates that SCG10 may play a stathmin-like role at or near nerve terminals and relate to neuronal plasticity. More studies are needed, however, for further elucidation of SCG10 functions.

The present study shows the distribution of SCGI0 mRNAs in the mouse brain with special focus in lhc hippocampus. We compared the hippocampal expression of SCG10 mRNA with that of other neuronal growth-associated genes in mouse and rat. The results indicate that SCG10 mRNA is expressed in sub-structures of the hippocampus at different levels. The expression profile resembles in part some other nGAPs, though not identical, suggesting an unique role of this growth-associated protein.

2. Materials and methods 2.1. Animals, tissues and sections

C57BL mice (male, n = 7), Balb/c mice (female, n = 2) and Fischer 354 rats (male, n = 5) kept under stress-free conditions with a 12 h light/dark cycle, were anesthetized with pentobarhital (50-60 mg/kg, i.p.) and decapitated. For RNA extraction, the brain and other tissues were dissected, rinsed in phosphate-buffered saline, and processed immediately (see below). For in situ hybridization the brains were immediately removed and frozen at -30°C in a dry ice-isopentanol bath. Coronal, sagittal and horizontal cryosections were cut into 13 ~zm on a cryostat and thaw-mounted onto gelatinecoated glass slides. The sections were stored at -80°C until use. 2.2. Cultured cells

Mouse NIH-3T3 fibroblasts were maintained in Dulbecco's modified Eagle's minimum essential medium (D-MEM) supplemented with 10% fetal calf serum. Mouse C1310 neuroblastoma (NB4IA3, American Type Culture Collection #CCL147) were kindly provided by I. Rozovsky and maintained in Ham's F-10 supplemented with 15% horse serum and 2.5% fetal calf serum. 2.3. Plasmids

Mouse SCG10 genomic subclones, pSG313 and pSG501, were used for RNase protection and in situ hybridization assays, respectively. Characterization of these clones had been described previously [36]. Control probes for RNase protection assay included the constitutively expressed metabolic enzyme, phosphoglycerate kinase (PGK) [31] and cytoskeletal protein, y-actin [t9]. Complementary DNA clones of rat growth-associated genes were pSCG10-8.6 [45], pSch2b-1 [20], pGAP-ECO [22], pMAP2-19a [15], and pECL-24 [39]. Each plasmid corresponds to SCG10, stathmin, GAP-43, MAP-2a, and al-tubulin, respectively. 2.4. RNase protection assay

Total cellular and tissue RNAs were extracted by the procedures of Cathala et al. [8] and Chomczynski and Sacchi [10], respectively. The RNA samples, 10 /zg each, were hybridized with 32P-labeled antisense probes corresponding to the mouse SCG10 gene containing the third exon (pSG313) [36], the mouse phosphoglycerate kinase (PGK) cDNA [31], and the human y-actin eDNA [19] in 25 tzl of a single reaction. These plasmids were digested with EcoRI, TaqI and Pst I, respectively, and then transcribed using SP6 RNA polymerase. The sizes of SCG10, PGK and ~,-actin probes were about 370, 220, and 150 nucleotides long, respectively, and the protected fragments were about 160, 200, and 140 nucleotides long after digestion in a mixture of RNase A and T1. Molecular size markers were 35S-labeled

7". Himi et al. / B r a i n Research 655 (1994) 177-185 pBR322 digested with E c o R l and Hinfl. For quantification of autoradiograms, intensities of the protected fragments were quantified using a densitometer (Bio-Rad, model GS-670). Values are averages of two separate protection assay of two independent animals and two scanning of the RNase protection data. Comparisons were performed in a linear range of hybridization (as to R N A a m o u n t ) and of film (as to exposure time). Detailed conditions of RNase protection assays employed are essentially as described previously [20]. 2.5. In situ hybridization The regional expression of SCG10 transcripts was examined in detail by in situ hybridization of both young (l-month-old) and

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mature (3- to 6-month-old) adult mouse brains. For comparison. young adult rat (3-month-old) brains were also used. Sections were fixed in 4 ~ paraformaldehyde and acetylated in a solution containing 0.1 M triethanolamine, 0.9% NaCI, 0.5% acetic anhydride at pH 8.0. Following dehydration in a graded series of ethanol, sections were hybridized in situ using 35S-labeled antisense or sense riboprobes of the mouse genomic clone, pSG501 (containing the fifth exon of the mouse SCGI0 gene [36]) or the rat SCGIII c D N A clone (SCG10 8) [46]. Hybridizations using other n G A P probes (all from rat clones) were processed in parallel using coronal sections containing mouse and rat hippocampi, The GAP-43 plasmid (pGAP-ECO) [22] was linearized by B a m H I restriction digest and was transcribed using SP6 R N A polymerase. The MAP-2 plasmid (clone 19a) [15] was linearized by B a m H l and was transcribed using T3 polymerasc.

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Fig. 1. Expression of SCG10 m R N A in mouse tissues and cells. Relative m R N A levels were determined by R N a s e protection assay using SCG 10 plasmid and two control plasmids, i.e. phosphoglycerate kinase (PGK) and -,/-actin. The plasmid pSG313 containing a part of exon 3 was used as a template to make a 370 nt probe for SCG10 (lane 1). Control probes were the mouse phosphoglycerate kinase (PGK) c R N A of 220 nt (.lane 2), and the h u m a n 7-actin c R N A of 150 nt (lane 3). T h e hybridization of each of these probes with brain RNA, followed by digestion with ribonuclease resulted in the protection of 160 nt, 200 nt, and 140 nt bands, respectively (lane 4-6). RNase protection experiments were carried out with carrier t R N A only (lane 8), or R N A s from cells and tissues: C1310 neuroblastoma (lane 9), NIH-3T3 fibroblast (lane 10), testis (lane 11 and 18), liver (lane 12 and 19), kidney (lane 13 and 20), spleen (lane 14 and 21), heart (lane 15 and 22), and brain (lane 16 and 23). R N A s in lanes 11-16 and 18-23 were from different animals. Molecular size markers are 35S-labeled pBR322 digested with EcoRI and Hinfl. Positions of probes and protected fragments are indicated by arrowheads on the left and right margins, respectively.

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The al-tubulin plasmid (pECL-24) [39] was linearized by ,4l,all and was transcribed using T7 polymerase. Methods of hybridization and analyses of film autoradiography and emulsion-dipped slides were essentially the same as described previously [20,34].

Table 1 Quantitation of SCG10 mRNA levels in mouse tissues SCG10 mRNA levels were determined by quantifying intensity of the protected bands corresponding to the SCGI0 mRNA in RNase protection assay (see Fig. 1). The values are presented relative to the expression level in brain as 100

3. Results

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3.1. SCGIO m R N A expression in mouse tissues and cells

We first examined SCG10 mRNA levels in various tissues of adult mice by RNase protection assay using the recently isolated mouse SCG10 genomic clones. A massive amount of SCG10 mRNA was detected in the brain, while other tissues showed undetectable or negligible levels of expression (Fig. 1). Quantification of mRNA bands on the RNase protection blot showed that the heart had a significant level of expression (Table l). The relative intensity of the signal in the heart was 2.4% of that in brain RNA. It was confirmed by separate experiments that the detection of the

SCG10 band in the heart is not due to leakage from the adjacent sample containing brain RNA (data not shown). A longer exposure of the autoradiogram (not shown) revealed that very low levels of SCGIO mRNA were detectable in the testis, while no SCG10 mRNA was detected in other tissues such as the liver, kidney

Fig. 2. SCO10 m R N A distribution in the mouse brain. In situ hybridization was performed using brain sections from 1-month-old mice. X-ray film images of parasagittal (A), horizontal (B), and coronal (C,D) sections are shown. Abbreviations of neuronal structures are as follows: 7, facial nucleus; BS, brain stem; Cb, cerebellum; Cx, cortex; Hp, hippocampus; LL, lateral lemniseus; Ob, olfactory bulb; Pit, piriform cortex; St, striatum; Th, thalamus. Bar = 2 mm.

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Fig. 3. Expression of n G A P m R N A s in the rat and mouse hippocampi. A series of in situ hybridization was employed using rat n G A P probes against hippocampal coronal sections from three month-old rat and mouse. Shown in the top row of the panel are a schematic drawing of neuronal structures in the rat coronal section including the hippocampus (a), bright-field views of the rat and mouse hippocampi at a large magnification ( x 100) (b,c), and a background image as a result of hybridization using an SCG10 sense probe (d). Abbreviations of neuronal structures are as follows: ca1, Cajal's area 1; ca3, Cajal's area 3; dg, dentate granule cell layer; cing, cingulate; ml, molecular layer of the dentate gyrus; slm, stratum l a c u n o s u m / m o l e c u l a r e of the cal region; sub, suhiculum. In the second to the bottom rows, in situ hybridization images using SCG10 (e-h), stathmin (i-l), GAP-43 ( m - p ) , M A P 2 (q-t), and a l - t u b u l i n (u-x) are presented, respectively. The left two (e,f,i,j,m,n,q,r,u,v) and the right two (g,h,k,l,o,p,s,t,w,x) photographs are of the rat and mouse brains, respectively. Arrowheads in panel g and n indicate the boundary between the CA2 and CA3 fields. Scale bars shown at the bottom of the panel are from the left to the right 2 ram, 0.4 mm, 0.4 m m and 2 mm, respectively.

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71 Himi et aL / Brain Research 655 (1994) 177-1~5

and spleen. SCG10 m R N A was also detected in uninduced mouse neuroblastoma C1310 cells at modest levels, but not in the NIH-3T3 fibroblasts (Fig. 1).

3.2. SCGIO mRNA expression in the mouse brain The overall expression profile of SCG10 m R N A in the adult mouse brain is essentially the same as that observed in the rat [20], including high expression in brain stem nuclei, the piriform cortex, granule cells of the cerebellum, and mitral cells of the olfactory bulb (Fig. 2). However, the pyramidal ceils of the mouse hippocampal sub-fields CA3 to CA4 were heavily labelled with the SCG10 antisense probe, whereas SCG10 m R N A signals were undetectable in the granule cell layer of the mouse dentate gyrus (Fig. 2C, see also Fig. 3g). It was also noted that the signal intensity of the SCG10 m R N A changed drastically between the CA2 and CA3 regions, and therefore the boundary of the CA2 and CA3 regions was clearly marked in the mouse (Figs. 2C and 3g,h), while the expression continued at a similar level through the C A 3 / 4 and C A 1 / 2 regions in the rat (Fig. 3e,f). Such a distinction at the C A 2 / C A 3 boundary was not observed in the rat [20] (see also Fig. 3e,f). These expression profiles, i.e. clear and high expression in the CA3 field and no detectable expression in the dentate gyrus, were distinct from the SCG10 m R N A distribution in the rat hippocampus [20], but were strikingly similar to that of GAP-43 in the rat brain [12,27].

3.3. Comparison of the expression of mRNAs encoding neuronal growth-associated proteins (nGAPs) in the rat and mouse hippocampus Inspired by our findings that hippocampal SCG10 m R N A expression is significantly different between the rat and mouse, we hypothesized that SCG10 and probably other neuronal growth-associated genes also may be expressed in slightly different manners among different species. To test this hypothesis we examined m R N A expression of other nGAPs, including GAP-43, MAP2, al-tubulin, and stathmin, by in situ hybridization. These nGAPs sequences are highly conserved during evolution: for example, the mouse and rat SCG10 proteins differ only by a single amino acid in 179 residues; stathmin by a single amino acid in 149 residues; and GAP-43 by three amino acids in 226 residues. High degrees of amino acid sequence conservation are also reflected at the nucleic acid sequence levels. Therefore, rat nGAP clones were used as probes for hybridization of both rat and mouse brain sections. The rat nGAP antisense cRNA probes detected specific signals in both rat and mouse sections in our standard in situ hybridization conditions (see Materials and methods for details).

Fig. 3 shows a panel of representative hybridization signals of various nGAPs in the rat and mouse coronal sections containing the hippocampus. It was noted that there are many similarities and dissimilarities in the expression profiles of these nGAP mRNAs. In the rat, for example, SCG10 m R N A expression resembled that of al-tubulin in that these two mRNAs arc expressed at high levels in hippocampal neurons and at low levels in the cortex and thalamus (Fig. 3e.u). In the hippocampus, SCG10 and al-tubulin mRNAs were expressed in both the pyramidal cell layer as well as the dentate granule cell layer (Fig. 3f.v). While MAP-2 m R N A is also expressed in both layers of the hippocampal neurons, its m R N A expression is unique in migrating into dendritic structures (Fig. 3q,r) as has been pointed out by Matus and his colleagues [16]. In contrast, GAP-43 m R N A is not expressed in the dentate gyrus, whereas high levels of expression are observed most prominently in the CA3 pyramidal cells and ceils in the hilus and at moderate levels in CA1 sub-field (Fig. 3n) as well as various neuronal structures throughout the cortex and thalamus (Fig. 3m). Note that the boundary of the CA2 and CA3 fields is clearly distinguished by a striking difference in the GAP-43 m R N A expression level (Fig. 3n. arrowhead). In the mouse, on the other hand, the distinction of GAP-43 m R N A expression at the C A 2 / C A 3 boundary is not obvious (Fig. 3o). In addition, GAP-43 mRNA is not expressed in dentate granule cells in either rat or mouse (see Fig. 3m-p). Expression of MAP-2, a ltubulin and stathmin looks similar between the rat and mouse hippocampus (Fig. 3r,s,v,w,j,k). Interestingly, GAP-43 m R N A expression in the rat hippocampus was strikingly similar to S C G t 0 m R N A expression in the mouse hippocampus (compare Fig. 3 panels n and g). In both cases, m R N A was expressed at high levels in the pyramidal cells of the CA3 field, but only marginal levels in the CA2 and CA1 fields, with a clear boundary between the CA3 and CA2 sub-fields. In both cases, m R N A was not expressed at detectable levels in the dentate granule cells. Thus, among the five nGAPs tested here, SCG10 and GAP-43 were the only examples that showed distinction in their m R N A expression profiles in the rat and mouse.

4. Discussion Our results examining SCG10 m R N A expression in mouse tissues and cells are consistent with the fact that SCG10 gene expression is specific to neurons [32,33,49], However, slight levels of the mRNA were detected in the heart, and faint levels were detected in the testis. The detection of the messages in the heart may be due to innervating neurons present in this tissue or partly due to contamination of a portion of the aorta contain-

T. Himi et al. /Brain Research 655 (1994) 177-185

ing sensory neurons which send the feedback signal controlling blood pressure to the autonomic nervous system. The significance of the detection of SCG10 m R N A in testis is not understood at present. To further examine the expression of SCG10 m R N A in the testis, we performed in situ hybridization experiments using SCG10 antisense probes of mouse and rat testis sections. We, however, did not observe any significant numbers of grains over background (N. Mori et al., unpublished observations). The fact that SCG10 m R N A is not expressed in mouse dentate granule cells suggests that the mouse may be a suitable animal to test whether SCG10 is induced in the dentate gyrus in response to electrical activation, such as seizure and long-term potentiation (LTP), or to various lesions that lead to synaptic remodelling. It would be of interest to examine whether SCG10 m R N A is induced following seizures, by which GAP-43 gene expression is induced [26]. Again, by analogy to GAP-43 [11,17], it is interesting to examine whether SCG10 could be modulated at phosphorylation levels and could be changed following LTP. Differential expression of SCG10 m R N A in the mouse and rat hippocampi may suggest that granule cells of the dentate gyrus have different potentials as to growth response between the two rodents: for example, mouse dentate gyrus granule cells may be more plastic than those neurons in other species. However, this may suggest that SCG10 expression in the rat dentate granule cells is not biologically important, since such expression of SCG10 m R N A in this cell population seems apparently unnecessary in the mouse. Our preliminary in situ hybridization experiments showed that SCG10 m R N A is also expressed at moderate levels in the dentate granule cells of the rabbit and in humans (N. Mori et al., unpublished). Therefore, the mouse case may be an exception, but further comparative studies using more species are necessary to conclude this issue. It is also important to examine the expression of SCG10 protein in the hippocampus in order to determine whether SCG10 is located in nerve terminals as predicted by the structural model [46]. Preliminary studies by immunohistochemistry and immunoelectron microscopy indicate that SCG10 protein is expressed in the dentate granule cells in the rat and is present along dendrites and axons, suggesting that SCG10 is transported toward nerve terminals (Y. Sugiura et at., manuscript in preparation). At present, it is not clear whether SCG10 is a presynaptic or postsynaptic molecule, although SCG10 is found in axons and growth cones of developing neurons [46]. Despite many similarities in the regulation of SCG10 and GAP-43 gene expression, there is little evidence thus far as to the functional implication of co-regulation of the two growthTassociated molecules. Since both SCG10 and GAP-43 are co-regulated temporally

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and spatially, expressed in subset neurons of the adult hippocampus, and accumulated in growth cones of growing neurons, SCG10 and GAP-43 may play synergistic or supplemental roles in neuronal growth and plasticity. In this regard, it is of interest to point out the results of our recent collaborative studies that have demonstrated that SCG10 and GAP-43 mRNAs are independently induced (or not induced) in different types of sprouting response following neuronal deafferentation in the cortico-striatal pathway, suggesting that SCG10 plays a role in paraterminal sprouting, while GAP-43 in collateral axonal sprouting (H.-W. Cheng et al., manuscript in preparation; see also Cheng et al., Soc. Neurosci. Abstr., 17 (1991) 305.3). Other brain lesion experiments in the hippocampus, e.g. timbria-fornix lesion and entorhinal cortex lesion also showed that SCG10 and GAP-43 mRNAs differentially responded during synaptic remodelling (E. Schauwecker et al., manuscript in preparation). Thus, accumulating evidence is increasingly suggestive that both SCG10 and GAP-43 have certain roles in the adult hippocampus, which is separate from their roles in axonal elongation during development. Therefore, it is extremely important to clarify their functions in mature neurons for better understanding of the roles of SCG10 and GAP-43 in the functioning and plasticity of the mature brain. In addition, more studies of other nGAPs are required for further understanding of the molecular basis of neuronal structural plasticity, and our current study conforms an effort toward this direction.

Acknowledgements The authors thank the following individuals for providing plasmids: Drs. M. Fishman, A. Matus, and C.E. Finch for GAP-43, MAP2, and al-tubulin, respectively. We also thank Elyse Schauwecker for critical reading of the manuscript. Excellent technical assistances by Minghua Cao and Huang Guo are greatly acknowledged. This work was supported in part by grants from NIH (AG07909) and the Max Factor Foundation.

References [1] Amat, J.A., Fields, K.L. and Schubart, U.K., Stage-specific expression of phosphoprotein p19 during spermatogenesis in the rat, Mol. Reprod. Det,., 26 (1990) 383-390. [2] Amat, J.A., Fields, K,L. and Schubart, U.K., Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia, Det,. Brain Res., 60 (1991) 205-218. [3] Anderson, DJ., Development and plasticity of a neural crest derived neuroendocrine sublineage. In: The Assembly of the Nercous System, Alan R. Liss, 1989, pp. 17-36.

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