The gene for the helix-loop-helix protein, Id, is specifically expressed in neural precursors

DEVELOPMENTAL

BIOLOGY

154,

l-10

(1992)

The Gene for the Helix-Loop-Helix Is Specifically Expressed in Neural MELINDADUNCAN,*

EMANUEL M. DICICCO-BLOOM,~XINXIANG,*

Protein, Id, Precursors

ROBERTBENEZRA,$ANDKIRANCHADA*J

Departments of *Biochemistry and fNeuroscience and Cell Bi&gy/Pedia~trics, University of Medicine and Dentistry of New Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854; and *Department of CeU Biology, Mema‘al Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10021 Accepted

July

Jersey-

7, 1992

While mammalian neurogenesis has been characterized extensively, the molecules involved in regulating neural cell determination and differentiation remain ill-defined. There is accruing evidence that various members of the basic helix-loop-helix (bHLH) protein family critically regulate these biological processes in a number of tissues. Id, a negative regulator of bHLH proteins, was found to exhibit peak gene expression during mouse embryogenesis with a striking pattern in the central nervous system. Id transcripts were specifically localized to undifferentiated neural precursors of the ventricular zone and were not present in their differentiated derivatives. In addition, in the peripheral nervous system, dorsal root ganglia sensory precursors, known to be undifferentiated while dividing, also expressed Id mRNA. However, in the sympathetic nervous system and adrenal medulla, where differentiation and division occur simultaneously in precursors, Id was not expressed. Since Id transcript abundance inversely correlated with differentiation, this protein, similar to its Drosophila homolog, extramacrochaetae, may play a negative regulatory role in neural 0 1992 Academic Press. Inc. differentiation.

negatively regulated by the protein, extramacrochaetae (emc) (Alonso and Garcia-Bellido, 1988; Garrell and Modolell, 1990). Emc is a member of a protein family related to the bHLH protein family, the helix-loop-helix (HLH) proteins, and can complex with AC-Svia its HLH domain (Van Doren et al., 1991). However, since emc lacks a basic DNA binding domain, the DNA binding activity of the heterodimer is destabilized (Van Doren et al, 1991). Hence, the HLH proteins can act as negative regulators of bHLH-mediated transcriptional activation, leading to the speculation that differentiation arises from a balance between negative and positive regulators (Weintraub et ah, 1991; Van Doren et aZ., 1991). Id, the first mammalian HLH protein identified which did not possessa basic DNA binding domain, has been hypothesized to be the mammalian homolog of emc (Benezra et al, 1990). However, the majority of studies on Id have concentrated on its role in myogenesis (Weintraub et aZ., 1991; Benezra et ah, 1990) even though Id expression has been observed in a number of immortalized cell lines (Benezra et aZ., 1990; Sun et aL, 1991; Wilson et al, 1991). Therefore, to analyze the relationship of these studies to the function of Id in vivo, the tissue and temporal pattern of Id mRNA expression was investigated in the mouse.

INTRODUCTION

The mammalian central nervous system (CNS) develops by proceeding through a number of apparently discrete ontogenetic stages. This progression, from mitotic, undifferentiated precursor to migratory cell and finally terminally differentiated neuron, has been extensively characterized at the morphological level (Boulder Committee, 1970; McConnell, 1991). However, the molecular mechanisms involved in the maintenance of each stage and the transitions between stages are poorly understood. In Drosophila, many of the molecules involved in neural determination and differentiation have been characterized (Campos-Ortega and Knust, 1990). The genes of the achaetae-scute (AC-S) complex encode transcription factors involved in the determination of peripheral neurons in Drosophila (Campos-Ortega and Knust, 1990). These proteins are members of a family of regulatory molecules which exert positive control over cellular differentiation in a variety of developmental systems, from invertebrates (Campos-Ortega and Knust, 1990) to mammals (Weintraub et ah, 1991). This class of proteins possessesa basic DNA binding domain and a helix-loop-helix dimerization domain and is termed the basic helix-loop-helix (bHLH) proteins (Murre et al, 1989). The AC-S genes are known to be r To whom dressed.

correspondence

and

reprint

requests

should

MATERIALS

AND

METHODS

(CBA/J x C5’7B1/6J)Fl female mice were placed with males of the same genotype. The morning on which a

be ad-

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$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

VOLUME 154, I992 Adult

Embryo

Liver

Femur Muscle

I-

I

Kidney I

Id

Head

Brain

FIG. 1. Northern blot analysis of Id transcripts in embryonic and adult mouse tissues. Equal amounts of total RNA (10 pg) for each tissue and 4 fig of total 9.5-dpc head RNA as a control were applied to gels. RNA blots were sequentially hybridized with the murine Id cDNA (Benezra et al, 1990) and an oligonucleotide complementary to murine 2% ribosomal RNA (Barbu and Dautry, 1989). An Id transcript of 1.3 kb was detected in all tissues analyzed. Abbreviation used: dpc, day postcoitum.

copulatory plug was present, indicating ing, was designated 0.5 dpc2

Northern

successful mat-

Blot H&ridizaticm

RNA was prepared using the guanidine thiocyanate/ cesium chloride centrifugation method (Chirgwin et aL, 1979), electrophoresed in 1.2% agarose/formaldehyde gels, transferred to Duralon UV membranes (Stratagene, La Jolla, CA) and crosslinked by ultraviolet light. Membranes were prehybridized for 2-4 hr at 42°C in a solution containing 50% formamide, 5~ SSC, 10x Den-

2 Abbreviations yuridine.

used: dpc, day postcoitum; BUdR, 5-bromo-Z-deox-

hardt’s, 0.1 mg/ml sonicated, denatured salmon sperm DNA, 0.001 M EDTA, pH 8.0, 0.2% SDS, and 0.05 M phosphate buffer, pH 6.8. Membranes were then hybridized overnight at 42°C in hybridization buffer (50% formamide, 5~ SSC, 1 X Denhardt’s, 0.02 M phosphate buffer, pH 6.8, 0.1 mg/ml sonicated, denatured salmon sperm DNA, 0.001 MEDTA, pH 8.0,0.2% SDS, and 10% dextran sulfate) with the murine Id cDNA (Benezra et aL, 1990) as the radiolabeled probe (Maniatis et aL, 1982). Unhybridized probe was removed with two room temperature washes (2~ SSC, 0.05% sodium pyrophosphate, 0.01 M phosphate buffer, pH 6.8,O.OOl M EDTA, 0.5% SDS) followed by one 45-min wash at 65°C in the same buffer. Blots were then rehybridized to an oligonucleotide complementary to 28s ribosomal RNA as a control for gel loading and transfer. The sequence of the 28s ribosomal oligonucleotide was AACGATGAGAG-

DUNCANETAL.

Id Expression

in Neural

FIG. 2. Spatial localization of Id mRNA in 12.5-dpc mouse embryos. Photomicrographs mouse embryo hybridized to the antisense (A) or sense (B) strand of Id mRNA or stained an index for ongoing DNA synthesis (Miller and Nowakowski, 1988). In the central neuroepithelium (arrowheads, C) of the hindbrain (myl, met) and midbrain (mes) but is patterns were observed in embryos with or without BUdR pretreatment. Abbreviations cephalon; met, metencephalon; myl, myelencephalon; tel, telencephalon; Md, mandible;

TAGTGGTATTTCA (Hassouna et aZ., 1984) and the hybridization conditions were as described (Barbu and Dautry, 1989).

Pretreatment

of Tissue for in Situ Hybridization

Pregnant female mice were injected intraperitoneally with 50 pug/g body weight of 5-bromo-2’-deoxyuridine (BUdR; Sigma, St. Louis, MO) and were sacrificed by cervical dislocation 30 min later (Miller and Nowakowski, 1988). Embryos were dissected and tissues fixed overnight in a freshly made solution of 4% paraformaldehyde in phosphate-buffered saline. The following day, they were dehydrated through graded ethanols and then were cleared with 1:l toluene:ethanol and finally 100% toluene. The tissue was infiltrated with Paraplast plus (Oxford, St. Louis, MO) with 4 X 30 min changes. The tissue was then oriented in paraffin embedding molds and was allowed to cool. After the paraffin was set, the blocks were placed in the cold room where they could be stored indefinitely. The g-pm sections were prepared, floated out in boiled, distilled water, and were mounted on 3-aminopropyl-triethoxysilane (Sigma)-treated slides (Rentrop et al., 1986). The mounted slides were baked at 37°C overnight and then were stored dessicated at 4°C.

Preparation

3

Precursors

of adjacent, parasagittal sections through a 12.5-dpc immunohistochemically for BUdR (C) incorporation as nervous system, Id mRNA is localized to the mitotic absent from the forebrain (di, tel). Identical expression used: cp, choroid plexus; di, diencephalon; mes, mesenMx, maxilla; H, heart; L, liver; G, gut.

of Single-Stranded

Id mRNA

Probes

The full-length cDNA clone used for transcription has been previously described (Benezra et ak, 1990). The insert of 928 nucleotides was cloned into the SmaI and EcoRI site of Bluescript SK- (Stratagene) and largescale plasmid recovery was performed. To prepare the template to transcribe antisense mRNA, the plasmid was cut with BamHI, gel purified, and the DNA was recovered from the agarose utilizing GeneClean, (BiolOl, La Jolla, CA). The sense strand template was prepared similarly except that the Id containing plasmid was cut with EcoRI. Two hundred and fifty microcuries of [35S]UTP (NEN, Boston, MA, 1320 Ci/mmole) was evaporated to dryness in a Speed vat concentrator and transcription was initiated by adding 1 wg of template DNA, 8 11 of a solution containing (2.5 mlMeach of rATP, rCTP, and rGTP), 4 ~1 buffer (BRL, Gaithersburg, of 5~ T3/T7 transcription MD), 2 ~1 of 0.1 M dithiothreitol, 40 units of RNasin (Promega, Madison, WI), 25 units of T7 RNA polymerase (Promega, Madison, WI) for antisense strand preparation or 25 units of T3 RNA polymerase (BRL, Gaithersburg, MD), and diethylpyrocarbonate (DEPC)treated water to 20 ~1. The reaction was placed at 37°C for 1 hr, and then 20 units of RNasin was added along with 50 units of RNAase-free DNAase (Boehringer-

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DEVELOPMENTALBIOLOGY

VOLUME154,1992

FIG. 3. Spatial and temporal localization of Id transcripts in the developing mouse brain. Photomicrographs are paired adjacent sections hybridized to the antisense strand of Id (A,C,E,G,I) or stained immunohistochemically (B,D,F,H,J) for BUdR-incorporated DNA. (A) A sagittal section of a 9.5-dpc embryo showing major Id mRNA expression localized to neuroepithelium of the spinal cord, rhombencephalon, and mesencephalon in addition to the branchial arch, developing gut, tail, and somites. (C) Transverse section of a 10.5-dpc embryonic brain showing Id expression localized to the dorsal neuroepithelium of the myelencephalon. (E) Transverse section of a 12.5-dpc embryonic head showing Id expression in neuroepithelium of the spinal cord and neural retina, the anterior choroid plexus, and the mesenchyme of the head. The bright ring surrounding the neural retina is an artifactual signal from melanin in the eye. (G) Transverse section of a 14.5-dpc fetal head showing Id mRNA localized to the neuroepithelium lining the fourth ventricle, the medial wall of the lateral ventricle, and the posterior and anterior choroid plexus. (I) Transverse section of a 16.5-dpc fetal head showing that little Id mRNA expression remains in the brain except in the hippocampus. The neural retina (tip of arrow) continues to proliferate and express Id. Abbreviations used: rho, rhombencephalon; mes, mesencephalon; pro, prosencephalon; ba, branchial arch; H, heart; SC,spinal cord; myl, myelencephalon; di, diencephalon; tel, telencephalon; cp, choroid plexus; met, metencephalon; IV, fourth ventricle; LtR, lateral recess of the fourth ventricle; hi, hippocampus.

Mannheim, Indianapolis, IN) and was incubated at 37°C for another 15 min. The mixture was phenol/chloroform extracted and then was precipitated by adding 0.8 ~1 of 1 M dithiothreitol, 2 ~1 of 3 M sodium acetate, pH 5.2, and 50 ~1 of 100% ethanol and placing at -80°C for 1 hr. The RNA was pelleted in a microcentrifuge and then was resuspended in 40 ~1 of DEPC-treated water and 10 ~1 of 5 M ammonium acetate. The sample was then reprecipitated at -80°C for 1 hr (Titus, 1991). The RNA was again pelletted and redisolved in 50 ~1 of DEPC-treated water. One microliter of this solution was reserved to

check transcript length on a denaturing acrylamide gel. Fifty microliters of 0.2 M sodium carbonate, pH 10.2, was then added to the RNA and the sample was incubated at 60°C for 55 min in order to hydrolyze the transcripts to an average fragment length of 150 base pairs. This reaction was precipitated by adding 3 ~1 of 3 M sodium acetate, pH 6.0,5 ~1 of 10% acetic acid, and 300 ~1 of ethanol and was placed at -80°C for 1 hr. The RNA was then pelletted, dried, and dissolved in 100 ~1 of 10 mM Tris, 1 mM EDTA, pH 8.0, with 2 ~1 of 1 M dithiothreitol added (Angerer et a& 1987). The microgram

DUNCAN

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Id Expression

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FIG. 4. Photomicrograph of adjacent, transverse sections through the neck of a 12.5-dpc embryo hybridized with the antisense strand of Id (A) or stained with hematoxylin and eosin (B). The dorsal root ganglia, which contain mitotic, undifferentiated precursors (Rohrer and Thoenen, 1987; Lawson and Biscoe, 1979) expressed high levels of Id mRNA. Abbreviations used: drg, dorsal root ganglia; SC, ventricular zone of the spinal cord.

yield of RNA was calculated from the specific activity of the radiolabeled nucleotides used in the transcription and the cpm’s of the incorporated probe. The hydrolyzed probe length was determined by running 1~1 of the final solution on a denaturing acrylamide gel. In Situ Hybridization

Slides with attached sections were deparaflinized and hydrated to 0.85% saline. They were then equilibrated in IX PBS for 5 min followed by postfixation in 4% paraformaldehyde for 20 min. After removal of the paraformaldehyde with two 5-min PBS washes, the slides were treated for 8 min with 20 pg/ml proteinase K in 50 m&i Tris-HCl, 5 mM EDTA, pH 7.5. The slides were again postfixed in 4% paraformaldehyde. In order to reduce background, the slides were acylated for 10 min with 625 ~1 acetic anhydride in 350 ml of 0.1 M triethanolamine, pH 8. The salts were removed from the sections with successive washes of PBS, 0.85% saline, and DEPCtreated water. The slides were then dehydrated to 100% ethanol and allowed to air dry (Copp and Cockcroft, 1990). For prehybridization, the sections were ringed with Duco cement (DuPont) and then 1 ml of prehybridation solution (4x SET, 0.02% Ficoll-400, 0.02% polyvinylpyrrolidone, 0.1% Pentex BSA (ICN, Costa Mesa, CA), 500 pg/ml sheared and denatured salmon sperm DNA, 600 pg/ml yeast total RNA, and 50% deionized formamide) was carefully placed over the sections and was allowed to sit undisturbed in a humid chamber for 3 hr. For hybridization, 35 ng of probe was mixed with 350 ~1 of deionized formamide and was incubated at 80°C for 1 min. After the samples were quenched on ice, 350 ~1 of 2~ hybridization buffer (SX SET, 0.04% Ficoll-400,

0.04% polyvinylpyrrolidone, 0.2% Pentex BSA, 200 pg/ ml sheared, denatured salmon sperm DNA, 200 kg/ml yeast total RNA; 20% dextran sulfate), 3.5 ~1 of 20% SDS, and 7 bl of 1 M dithiothreitol were added. Fifty microliters of this mixture was overlaid over each section and then the slides were coverslipped and placed on Whatman paper soaked with 4~ SSC/50% formamide. The chamber was sealed with plastic wrap and then was placed at 50°C for 16-18 hr (Copp and Cockcroft, 1990). To wash off any unhybridized probe, the slides were soaked in formamide wash buffer (50% formamide, 1X SSC, 0.01 Mdithiothreitol) until the coverslip floated off and then were moved into formamide wash buffer at 50°C for 30 min. To remove the formamide, the slides were washed in 0.5~ SSC for 30 min. To remove any unhybridized, single-stranded probe, the slides were then treated with 5 units/ml RNAase Tl, 100 fig/ml RNAase A in 3.5~ SSC for 30 min at 37°C. The RNAase was removed with two lo-min washes with 3.5~ SSC and the remainder of unhybridized probe was removed with a 2-hr wash in 0.1~ SSC. The sections were then dehydrated through an increasing ethanol/O.3 Msodium acetate, pH 5.5, series and air dried (Copp and Cockcroft, 1990). The sections were exposed to /3-max x-ray film (Amersham, Arlington Heights, IL) for 2 days (Chun et al, 1991) and then were dipped in NTB-2 x-ray emulsion (Kodak, Rochester, NY). The slides were exposed for a time based on the autoradiogram intensity and then were developed in D-19 developer (Kodak, Rochester, NY) and fixed in Kodak fixer. The excess fixer was washed from the emulsion with a 20-min wash in running dH,O and then the slides were dehydrated to xylene and mounted with Pro-Texx mounting medium (ASP, McGaw Park, IL). The hybridization was visual-

DUNCAN ET AL.

Id Expression

in Neural

Precursors

7

FIG. 5. Photomicrographs are paired adjacent sections of embryos hybridized with the antisense strand of Id (A,C), either stained with thionin (B) or stained immunohistochemically with an anti-tyrosine hydroxylase antibody (D). (A,B) Transverse sections through the upper thorax of a 14.5-dpc embryo. Sympathetic ganglia exhibited no Id expression (A) but demonstrated tyrosine hydroxylase immunoreactivity (data not shown). The blood cells seen in the common cardinal vein and pulmonary artery are artifactually detected by dark-field optics. The figure was intentionally overexposed to detect possible silver grains in sympathetic ganglia. Thus, there appears to be a more intense hybridization signal in the drg at 14.5 dpc than at 12.5 dpc (see Fig. 4). However, in the original material, the hybridization signal in DRG is stronger at 12.5 dpc. (C,D) Sagittal sections of a 14.5-dpc fetal adrenal gland showing that the adrenal medullary islands lacked Id mRNA expression while exhibiting positive tyrosine hydroxylase immunoreactivity. At this stage adrenal medullary cells exhibited BUdR nuclear staining (data not shown), indicating ongoing mitotic activity (Miller and Nowakowski, 1988). Abbreviations used: L, liver; med, adrenal medullary islands; car, adrenal cortex; K, kidney; SC,spinal cord; drg, dorsal root ganglia; R, rib, V, vertebra; sg, sympathetic ganglion; pa, pulmonary artery; ccv, common cardinal vein; E, esophagus; B, bronchus; A atrium.

ized with a Wild Leitz Ortholux dark-field condenser.

microscope fitted with a

BUdR Immunohistochemistry Sections adjacent to those used for in situ hybridization were exposed to 0.1% trypsin for 30 min, 37°C 2 N

HCL for 30 min, and were incubated with of anti-BUdR antibody (Becton-Dickinson) (Miller and Nowakowski, 1988). The bound detected by the Vectastain ABC Elite Kit peroxidase method using nickel/cobalt (Miller and Nowakowski, 1988). Specific

a 1:50 dilution for 30 min antibody was (Vector Labs) enhancement staining was

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DEVELOPMENTALBIOLOGY

not observed in sections from control embryos which did not receive BUdR injection. Tyrosine Hydroxylase

Detection

Tyrosine hydroxylase immunohistochemistry was performed using the Vectastain ABC peroxidase method as previously described (DiCicco-Bloom et ak, 1990). Thionin staining was by standard methods (Miller and Nowakowski, 1988). RESULTS

In order to gain insight into the biological role of HLH proteins in mammals, the temporal and site-specific expression pattern of Id was defined in the mouse. In all tissues analyzed by Northern blot hybridization, a single transcript of 1.3 kb was detected. Interestingly, the peak of Id mRNA expression occurred during early ontogeny in most tissues (Fig. l), although Id transcripts were found at variable levels in all major adult organs (Fig. 1, top). To further characterize the embryonic role of Id at the cellular level, in situ hybridization studies were performed on the 12.5-day postcoitum (dpc) embryo since this is the earliest time point that most organs are readily discernable. A uniform Id mRNA distribution was detected in a number of developing tissues including the mandible, maxilla, somites, lungbud, and gut mesenchyme (Figs. 2A and 2B). The most striking observation, however, was the specific localization of Id expression to the ventricular zone, the proliferative neuroepithelial layer of the central nervous system (CNS) which gives rise to diverse neuronal and glial cell populations (Boulder Committee, 1970). Following precursor proliferation in the ventricular zone, newly born cells migrate distally and undergo differentiation. While Id mRNA was detected in the ventricular zone of the hindbrain and midbrain, it was absent from the forebrain and postmitotic, differentiated neural populations (Figs. 2A-2C). To explore this differential expression of Id mRNA, a spatial and temporal analysis was performed during neurogenesis. At 9.5 and 10.5 dpc, Id mRNA was detected in the ventricular zone of the spinal cord, rhombencephalon, and mesencephalon (Figs. 3A-3D). However, Id exhibited a dorsoventral gradient of gene expression (Figs. 3A and 3C) suggesting that precursor heterogeneity may be one mechanism by which different mature neural populations are produced (McConnell, 1991; Roelink and Nusse, 1991; Gavin et aL, 1990; Lo et aL, 1991). By 12.5 dpc, the expression pattern was more homogeneous and was detected in both the dorsal and ventral ventricular zones of the midbrain, hindbrain (Fig. 2A), and spinal cord (Fig. 3E). By 16.5 dpc,

VOLUME1%$1992

there was little proliferation in the ventricular zone of the midbrain (Fig. 35) and Id mRNA levels were correspondingly decreased (Fig. 31). Therefore, as cells of the ventricular zone cease dividing and undergo migration and differentiation, Id mRNA expression diminishes. In contrast to the mid- and hindbrain, virtually no Id mRNA expression was detected in the forebrain between 9.5 and 12.5 dpc (Figs. 2A, 3A, 3C, and 3E). At 14.5 dpc, expression appeared along the full extent of the medial telencephalic wall and was especially intense in the hippocampus (Fig. 3G). By 16.5 dpc, the remaining Id expression and mitotic activity in the forebrain were localized only to the hippocampus (Figs. 31 and 35). These observations suggest that neural precursors of the midbrain and hindbrain differ from those of the forebrain during neurogenesis, a contention supported by retroviral analysis of neural lineage (McConnell, 1991) and gene expression patterns during CNS development (McConnell, 1991; Lo et aL, 1991; Kessell and Gruss, 1990). The expression of Id mRNA in undifferentiated, proliferating neural precursors suggested that Id was associated with the mitotic and/or the undifferentiated state of the cell. To further explore this issue, cells of the neural crest-derived peripheral nervous system (PNS), including the sensory (Rohrer and Thoenen, 1987) and sympathoadrenal lineages (Anderson and Axel, 1986), were examined since the relationships of cell division to phenotypic differentiation have been characterized in detail (DiCicco-Bloom et ab, 1990; Rohrer and Thoenen, 1987; Anderson and Axel, 1986; Rothman et ah, 1980). At 12.5 dpc, mitotic precursors were undifferentiated in sensory ganglia (Rohrer and Thoenen, 1987) and exhibited Id expression (Fig. 4) similar to the cells of the CNS ventricular zone. In contrast, precursors of sympathetic ganglia and the adrenal medulla exhibit differentiated traits while dividing (DiCicco-Bloom et ah, 1990; Rohrer and Thoenen, 1987; Anderson and Axel, 1986; Rothman et aL, 1980). Id mRNA expression was not detected in either cell type at 12.5 dpc (data not shown) or 14.5 dpc (Fig. 5) indicating that Id is not related to mitosis per se in precursor cells. It should be noted that in Fig. 5, the expression of Id mRNA in the DRG is artifactually high (see Fig. 5 legend). Residual expression in the dorsal root ganglia at 14.5 dpc may represent mRNA in postmitotic (Lawson and Biscoe, 1979), but undifferentiated neurons in sensory ganglia. Alternatively, Id expression may be localized to other neural precursors, that is, the glia. However, our preliminary results have found Id protein expression in the small, recently generated neurons but not the glia in dorsal root ganglion cell cultures generated from 12.5-dpc embryos (data not shown).

DUNCANETAL.

Id Expression

These data in the peripheral nervous system suggest that Id expression does not correspond to mitotically active cells in the nervous system. Rather, the inverse correlation of Id expression levels with differentiated phenotypic traits suggests that Id plays a role in regulating the onset of neural differentiation. DISCUSSION

This study describes the spatial and temporal expression of the gene for the helix-loop-helix (HLH) protein, Id, in the developing mouse nervous system. It should be noted that the following conclusions regarding Id function are based on the likely assumption that Id mRNA levels reflect the amount of Id protein translated in the cell. Indeed, recent tissue culture experiments indicate that neuronal precursors express Id protein as detected by immunocytochemistry using polyclonal antibodies (data not shown). These observations revealed that Id mRNA was regionally expressed in embryonic precursor populations of the CNS and the signal was most intense in phylogenetically older structures (Gans and Northcutt, 1983) (spinal cord, hindbrain, and midbrain). Even in the recently evolved forebrain, Id expression was restricted to the most primitive region, the hippocampus (Pearson and Pearson, 1976). Therefore, it appears that a number of highly conserved molecules [Id (Benezra et aZ., 1990), Dlx (Price et aZ., 1991), and HOX family members (Kessell and Gruss, 1990)] are involved in the development of the nervous system in organisms as diverse as Drosophila and mammals. In contrast, the newly evolved and more complex forebrain which is responsible for higher cognitive functions in mammals, may well use novel sets of molecules during neurogenesis. The studies of neural precursors in the PNS and CNS suggests that Id plays an important inhibitory role in neural differentiation. Id mRNA levels were negatively correlated with differentiation in dividing precursors, in the nervous system and the adrenal but not the mitotic state. It is possible that the Id protein inhibits positive bHLH regulators in mitotic neural precursors. Subsequently, differentiation may depend on reduced levels of the negative regulator Id, a model implied from in vitro experiments (Weintraub et al., 1991; Benezra et al., 1990). This is also consistent with the genetic analysis of the Drosophila homolog of Id, emc (Alonso and GarciaBellido, 1988) which negatively regulates neural development. Further, known interactions of emc with bHLH achaetae-schute complex proteins in Drosophila neurogenesis (Van Doren et al., 1991) raise the possibility that Id interacts with bHLH proteins during mammalian development. While recent studies of the mammalian achaetae-scute homolog (MASH 1) (Lo et al., 1991) sug-

in Neural

9

Precursors

gest that bHLH proteins are involved in neural ontogeny, specific interactions remain to be defined. More generally, these studies indicate a remarkable functional conservation of HLH-bHLH protein interactions in mammalian nervous system development. We thank members of the Chada laboratory, M. Roth, I. B. Black, and D. Reinberg for critical reading of the manuscript and R. Nowakowski and J. Lee for technical assistance. This work was supported by the Dysautonomia Foundation Inc. (E.M.D-B.) and the NIH (KC.).

REFERENCES Alonso, L. A. G., and Garcia-Bellido, A. (1988). Extramncrochuetae, a tram-acting gene of the achuete-scute complex of Drosophila involved in cell communication. Wilhelm Roux’s Arch Dev. Biol. 197, 328-338. Anderson, D. J., and Axel, R. (1986). A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. Cell 47, 1079-1090. Angerer, L. M., Cox, K. H., and Angerer, R. C. (1987). Demonstration of tissue specific gene expression by in situ hybridization. Methods Enzymol. 152,649-661. Barbu, V., and Dautry, F. (1989). Northern blot normalization with a 28s oligonucleotide probe. Nucleic Acids. Res. 17,7115. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990). The protein Id: A negative regulator of helix-loop-helix binding proteins. Cell 61,49-59. Boulder Committee (1970). Embryonic vertebrate central nervous system. Anat. Rec. 166,257-261. Campos-Ortega, J. A., and Knust, E. (1990). Genetics of early neurogenesis in Drosophila melarwgaster. Annu. Rev. Genet. 24,387-407. Chirgwin, J. M., Przbyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18,5294-5299. Chun, J. J. M., Schatz, D. G., Oettinger, M. A., Jaenisch, R., and Baltimore, D. (1991). The recombination activating gene-l (RAG-l) is present in the murine central nervous system. Cell 64,189-200. Copp, A. J., and Cockroft, D. L. (1990). “Postimplantation Mammalian Embryos: A Practical Approach”. IRL, Oxford, England. DiCicco-Bloom, E., Townes-Anderson, E., and Black, I. B. (1990). Neuroblast mitosis in dissociated culture: Regulation and relationship to differentiation. J. Cell Biol. 110,2071-2085. Gans, C., and Northcutt, R. G. (1983). Neural crest and the origin of vertebrates: A new head. Science 220,268-274. Garrell, J., and Modolell, J. (1990). The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell 61,39-49. Gavin, B. J., McMahon, J. A., and McMahon, A. P. (1990). Expression of multiple novel Wnt-l/int-1 related genes during fetal and adult mouse development. Genes Dev. 4,2319-2332. Hassouna, N., Michot, B., and Bachellerie, J. P. (1984). The complete nucleotide sequence of mouse 28s rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes. Nucleic Acids Res. 12,3563-3599. Kessel, M., and Gruss, P. (1990). Murine developmental control genes. Science 249,374-379. Lawson, S. N., and Biscoe, T. J. (1979). Development of mouse dorsal root ganglia: An autoradiographic and quantitative study.J. Neurocytol. 8,265-274. Lo, L. C., Johnson, J. E., Wuenschell, C. W., Saito, T., and Anderson, D. J. (1991). Mammalian achaetae-scute homolog-1 is transiently ex-

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