Id gene expression during development and molecular cloning of the human Id-1 gene

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 30 (1995) 312-326

Research report

Id gene expression during development and molecular cloning of the human Id-1 gene Weijia Zhu a John Dahmen a Alessandro Bulfone b, Muriel Rigolet a Maria-Clemencia Hernandez a, Wen-Lin Kuo c, Luis Puelles d, John L.R. Rubenstein b, Mark A. Israel a,, a Preuss Laboratory, Brain Tumor Research Center of the Department of Neurological Surgery, San Francisco, CA 94143, USA b Nina Ireland Laboratory of Developmental Neurobiology and the Center for Neurobiology and Psychiatry, San Francisco, CA 94143, USA c Department of Laboratory Medicine, School of Medicine, University of California, San Francisco, CA 94143, USA d Department of Morphology, University ofMurcia, Murcia, Spain

Accepted 17 January 1995

Abstract

Id genes encode helix-loop-helix proteins that inhibit transcription by forming inactive heterodimers with basic helix-loop-helix (bHLH) proteins, bHLH proteins normally form either homodimers or heterodimers with other bHLH proteins and bind to a DNA sequence element activating transcription. Id-containing heterodimers are inactive because Id proteins lack the basic amino acid region necessary to form a DNA-binding domain. We have examined the relative levels of Id-1 and Id-2 mRNA during normal development and in malignant tissues. In the course of these experiments we cloned and sequenced the human Id-1 cDNA. Two related cDNA molecules encoding human Id-1 mRNAs were identified. Id-la is a cDNA of 958 nucleotides and can encode a protein of 135 amino acids. Id-lb cDNA is 1145 nucleotides, can encode a protein of 149 amino acids, and appears to be a splice variant of Id-la. The amino acid sequence of human Id-1 is greater than 90% homologous to that of mouse Id-1. The patterns of Id-1 and Id-2 expression during mouse development vary widely, and we detected Id-1 expression in human fetal and adult tissues from lung, liver, and brain. High Id-1 mRNA expression was found in many human tumor cell lines, including those isolated from nervous system tumors. We mapped Id-2 to human chromosome 2p25.

Keywords: Id gene; bHLH protein; Gene expression 1. Introduction

H L H (helix-loop-helix) proteins are a large family of transcription factors that have been implicated in the developmental control of gene expression and cell differentiation [37,38]. One class of such transcription factors, the b H L H (basic helix-loop-helix) proteins, binds to a specific D N A enhancer element known as an E-box, C A N N T G , utilizing a region of basic amino acids immediately N-terminal to the H L H motif [6,38,39]. b H L H proteins always bind to D N A either as a homodimer or as a heterodimer formed by complexing with another b H L H protein [29]. E-box D N A sequences can bind ubiquitously expressed b H L H

* Corresponding author. Fax: (1) (415) 476-0388. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0169-328X(95)00017-8

molecules as well as tissue-specific b H L H factors that have been implicated in the regulation of tissue-specific gene expression [30,47,52]. b H L H proteins ubiquitously expressed in many tissues include the products of the E2A gene, E l 2 and E47 [37]; the family of myc-related genes; and other widely-expressed transcription factors such as USF [22], H E B [25], TFE3 [3], ITF2 [24], T F E B [48] and AP4 [26]. Other b H L H proteins are expressed in mammalian cells in a tissue-specific manner and found in muscle (MyoD, myogenin, Myf-5) [48], neuronal tissue (HEN1 and H E N 2 ) [7], and lymphoid tissue ( T A L l , TAL2, and LYL1) [32,36,53]. In mammalian ceils, the b H L H transcription factors that have been studied most thoroughly are expressed in a tissue-specific manner and control tissue-specific gene expression. MyoD, myogenin, and the Myf genes encode b H L H proteins that control muscle-specific genes in differentiating muscle cells. During muscle

w. Zhu et al. / Molecular Brain Research 30 (1995) 312-326

cell differentiation, the protein products of these genes form heterodimers with other E-box-binding proteins such as the products of the E2A gene or HEB [25,37]. This dimerization is mediated through the HLH domain and the dimers are capable of binding to regulatory elements that activate muscle-specific genes such as muscle creatinine kinase [15]. The finding of E-box sequences in the regulatory regions of other genes expressed in a tissue-specific manner, such as the immunoglobulin genes, is consistent with the tissuespecific expression of bHLH proteins in many different tissue types. Another class of HLH proteins, the Id proteins, provides a distinctive mechanism for modulating the enhanced transcription of tissue-specific genes mediated by bHLH proteins. Id proteins are HLH proteins that lack the basic amino acid region necessary to form a DNA-binding domain [4]. They inhibit transcriptional activation by bHLH proteins through the formation of inactive heterodimers [4,47]. A similar mechanism of dominant-negative transcriptional inhibition has been proposed for the Drosophila HLH gene, emc [17,21]. Emc inhibits the activity of the bHLH proteins encoded by the achete/scute and daughterless genes. T r a n s c r i p t i o n a l activation by d a u g h t e r l e s s achete/scute heterodimers is necessary for normal development of the PNS (peripheral nervous system), and in the absence of the emc gene product, too many PNS sense organs form [1,9]. The DNA-binding activity of these proteins is inhibited by their complexing with emc [50], providing important evidence for the proposed mechanism of action [17]. More recently a role for Id family gene expression in the regulation of cellular proliferation has been evaluated [2,23,27]. In several different cell types Id gene expression occurs following serum stimulation [[10], our unpublished data] and in some cell types, Id gene expression may be required for maximal proliferation [2,27]. One specific mechanism for regulating the effect of Id on proliferation is through its binding to the product of the retinoblastoma tumor suppressor gene, RB [27]. Conversely, antagonism of Id may also inhibit cell growth [14,46]. A role for Id gene expression in mediating cellular proliferation is consistent with its high level of expression in undifferentiated cell types which typically have a high proliferative potential. Several mammalian Id genes encoding HLH proteins that lack a basic DNA binding domain have now been identified. Four Id proteins, termed Id (Id-1) [4], Id-2 [47], HLH-462 (Id-3) [10], and Id-4 [44] have been cloned from rodents. Human homologs of ld-2 and HLH-462, named human Id-1 [23], Id-2 [5] and heir-1 [18], respectively, have also been identified. It has been proposed that the mammalian Id-1 and Id-2 proteins bind to proteins encoded by E2A and MyoD and inhibit the binding of these proteins to DNA [4,47].

313

The DNA binding of other bHLH proteins, including c-myc and USF, is not affected by these Id molecules [47]. Consistent with their proposed function of inhibiting tissue-specific gene expression, steady state levels of Id-1, Id-2, or Id-3 mRNA have been observed to be decreased in association with the in vitro induced differentiation of several different cell types including skeletal muscle cells [4], lymphocytes [47,52], erythroleukemia cells [4,30], F9 embryonal carcinoma cells [4,47] and neuroblastoma cells [5]. Similarly, cells corresponding to increasing degrees of murine lymphoid differentiation express varying amounts of Id-1 and Id-2 mRNA [7]. Transfection of Id-1 into muscle precursor cells [4], myeloid precursor cell lines [30], or lymphoid cells [41] inhibits their differentiation. That inhibition is mediated, at least in part, by an activity of Id proteins inhibiting the activation of lineage-specific gene expression [4,5,13,18,28,41,47]. Several investigators have sought to examine the expression of Id genes during mouse development [16,19,40,51]. Id-1 is first expressed in the mouse around 7.5 days pc (post-coital), during gastrulation [51,40]. As differentiation progresses, expression of Id-1 becomes more restricted. Of particular interest is that its expression is not detected in all regions containing proliferative cells, although Id expression is typically higher in undifferentiated tissues compared to the levels obs e r v e d in c o r r e s p o n d i n g m a t u r e tissues [5,13,16,18,19,28,40,41]. Although Id-2 expression during development has not been as extensively studied, Id-2 is expressed in the central nervous system (CNS) and can be detected in both neural precursors and in fully-differentiated neurons [40]. In this report, we describe the DNA sequence of the human Id-1 gene, which contains an HLH domain that is identical to the HLH domain of the mouse Id-1 gene, and we compare the expression of Id-1 and Id-2 during development with a special emphasis on tissues of the developing nervous system. Because of the high levels of expression of these genes during development, we examined tumors, which are thought to arise in precursor cells of tissues that express Id genes during embryogenesis. We detected the expression of both Id-1 and Id-2 in a wide variety of tumors and tumor cell lines, suggesting a role for these genes in the regulation of expression of differentiated cellular features as well as those biologic activities that contribute to the malignant behavior of human tumors. 2. Materials and methods 2.1. Isolation, sequencing, and characterization o f hum a n Id-I

RACE-PCR was carried out as previously described and was used to synthesize and amplify cDNA corre-

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w. Zhu et al. /Molecular Brain Research 30 (1995) 312-326

sponding to the human ld-1 gene product [5,20]. First strand eDNA synthesis was accomplished using a poly(dT) primer covalently linked to a polylinker and total RNA extracted from the cultured glioblastoma cell line U251 [42]. This cDNA served as a template for PCR using the same poly(dT) primer and an Id-specific primer which had been designed by comparing the sequence of the Drosophila emc gene with that of mouse Id-1. For this primer, we selected a highly conserved region corresponding to the DNA sequence encoding amino acids 89 to 94 of the mouse Id protein. This primer had been used previously to amplify the human Id-2 gene from neuroblastoma mRNA [5]. The RACE-PCR cDNA was used to screen approximately 360,000 plaques of a human glioblastoma cell line U251 cDNA library (Clontech). Nineteen positive plaques were identified and six were subcloned into pBluescript SKII (Stratagene, La JoUa, CA). cDNAs were sequenced as double-stranded plasmids by the dideoxynucleotide chain-termination method using a protocol and sequenase from United States Biochemical (Cleveland, OH). DNA sequence analysis of GC rich regions of Id-1 were performed in parallel reactions using dITP and 7-deaza-dGTP in place of dGTP in the sequencing reactions. 2.2. Cell culture

The source of melanoma, lymphoma, lung cancer, colon cancer, medulloblastoma, neuroblastoma and glioblastoma cell lines other than the American Type Culture Collection, and their maintenance has been described [11,34]. SMS-KCNR was obtained from Dr. Patrick Reynolds (Los Angeles Childrens Hospital, Los Angeles, CA). SKNSH (F) was obtained from Dr. Neil Sidell (University of California, Los Angeles, CA), and H292 was obtained from Dr. Christopher Benz (University of California, San Francisco, CA). 2.3. R N A preparation and hybridization analysis

man Id-2 mRNA, an EcoRl fragment of the Id-2 cDNA encoding amino acids 75 to 116 was similarly radiolabeled and used as a hybridization probe. Blots were washed in 5 x SSC-0.1% SDS at room temperature and then in 1 X SSC-0.1% SDS at 65°C for 3(} min. As a control for the amount of RNA loaded, blots were stripped by heating in water at 100°C for 10 rain and rehybridized with a cDNA probe recognizing the constitutively expressed gene, glyceraldehyde 3' phosphate dehydrogenase (G3PDH). In experiments to semi-quantitatively assess relative levels of Id gene mRNA we used probes of approximately equivalent length and specific activity (approximately 5 x 10 7 cpm//~g): Id-1 (535 bp KpnI cDNA fragment from phId-lb DNA) and Id-2 (440 bp K p n I / H i n d I I I cDNA). In preliminary experiments we ascertained that these fragments recognize only the mRNA corresponding to the cDNA molecules from which they were derived (data not shown). 2.4. In situ hybridization analysis

Mouse embryos were obtained from timed pregnancies. The day of the vaginal plug was defined as embryonic day (E) 0.5. Animals were sacrificed and the embryos were prepared as previously described [8]. Cryostat sectioning and in situ RNA hybridization using 35S-labeled riboprobes was performed as previously described [43]. The Id-1 probe, p1002, was constructed from the mouse Id-1 cDNA clone previously described [4] and kindly provided by Dr. H. Weintraub, Seattle. DNA from this plasmid was restricted with EagI and the resulting fragment of DNA that was approximately 3400 bp in size was recircularized with T4 DNA ligase. This cloned DNA contains 3' untranslated sequences and approximately 40 bp of Id-1 coding sequence. The Id-2 probe used was a 235 bp stretch of DNA corresponding to 3' untranslated mRNA of Id-2 synthesized by PCR amplification of RACE PCR amplified cDNA as previously described [47]. 2.5. Chromosomal mapping of human Id-2

Total cellular RNA from cultured cells and mouse and human tissues was prepared using an RNAzol protocol (Tel-Test, Friendswood, TX). 20 Izg of total RNA was separated by electrophoresis on 1.2% agarose/formaldehyde gels that contained 1 Izg of ethidium bromide per ml to stain rRNA. RNA was transferred to nylon membranes (S & S, Keene, NH) in 10 X SSC buffer. Transferred membranes were ultraviolet cross-linked with 160,000 ~ J in a Stratagene UV Crosslinker. Blots were prehybridized overnight and hybridized for 24 h at 42°C in the presence of 50% formamide as previously described [5]. To detect human Id-1 mRNA, a 0.94 kb EcoR1 fragment consisting of a full-length Id-1 eDNA was labeled by random primer labeling (BRL, Bethesda, MD). To detect hu-

The chromosomal localization of human Id-2 was determined as described previously [32,33]. A genomic molecular clone of 9 kb encoding the 5' end of human Id-2, pgId-13, (our unpublished data) served as a probe for fluorescence in situ hybridization. Briefly, recombinant plasmid DNA was extracted, labeled with biotin14-dATP and hybridized to human metaphase spreads prepared from normal peripheral blood lymphocytes. Hybridized probes were detected using avidin conjugated with F1TC. Metaphase chromosomes were counterstained using propidium iodide and DAPI in an anti-fade solution. The DAPI banding pattern was used to determine the chromosomal band location at which the probe hybridized.

W. Zhu et al. / Molecular Brain Research30 (1995) 312-326

3. Results 3.1. Eualuation of Id -1 and Id-2 expression during deuelopment We examined the expression of Id-1 and Id-2 between embryonic days 12.5 and 16.5. The molecular probes that were used to detect Id-1 and Id-2 expression in this in situ analysis detect only Id-1 or Id-2 m R N A when used under the experimental conditions employed here (see Materials and methods). The expression of ld-1 outside of the CNS has been described previously [19,51]. In the course of this work, however, we noted at E12.5 expression of Id-1 in several structures not mentioned in previous publications. These tissues included kidney, olfactory epithelium, trigeminal ganglion, genital tubercle, and anterior pituitary (Table 2) (Figs. 1B and 2D,E,F). The expression of Id-2 (Fig. 1A) in extra-neural tissues has not been previously described. While there is considerable overlap in the expression patterns, Id-2 is expressed more extensively than Id-1 at E12.5 (Fig. 1A,B). We did not observe any non-CNS tissues where Id-1 was uniquely expressed. These genes were co-expressed in the genital tubercle, heart, large blood vessels, kidney, mandibular and maxillary mesenchyme (including in the regions of the tooth primordia), olfactory epithelium, Rathke's pouch, prevertebrae, otic vesicle and the trigeminal ganglion. Id-2 was expressed at much higher levels than Id-1 in the intestines (wall and lumen), liver, and lung epithelium. Within some of these structures, Id genes were differentially expressed

315

Table 1 List of anatomical abbreviations AQ, cerebral aqueduct A, atrium AOB, anterior olfactory CB. cerebellum nucleus C, neocortex CP, chorioid plexus CN, cerebellar nuclei DT, dorsal thalamus D, diencephalon GT, genital tubercle G, ganlionic emminence I, intestine HB, hindbrain L, liver K, kidney LGE, lateral ganglionic emminence LU, lung M, mammillaryarea LV, lateral ventricle IV, fourth ventricle MGE, medial ganglionic OB, olfactorybulb emminence OV. otic vesicle P, pituitary/Rathke's pouch and infundibulum TC, tela chorioidea of the PV, prevertebrae rhombencephalon PN, pontine nuclei sc, spinal cord S, septum OE, olfactoryepithelium T, tectum V, trigeminal ganglion TU, tongue VI, vibrissae VE, ventricle of heart III, third ventricle VT, ventral thalamus

in different tissues. For example, in the kidney, ld-1 was primarily expressed in the epithelium, whereas Id-2 was strongly expressed in both the mesenchyme and the epithelium. In the heart, Id-2 is expressed more extensively in the atrium. In the ventricle we were unable to detect Id-1 expression. These results are summarized in Table 2.

A

Fig. 1. Analysisof the expression of Id-2 and Id-I using in situ RNA hybridization at E12.5 of the mouse in parasagittal sections. A: ld-2; B: Id-1; C: Toluidine blue staining of panel A. Magnification bar in bottom of Panel A corresponds to 500 ~zm. Abbreviations are defined in Table 1.

W. Zhu et al. /Molecular Brain Research 30 (1995) 312-326

316

Table 2 Expression of Id-1 and Id-2 mRNA during murine development Tissue

Id-1

Id-2

Brain Genital tubercle Heart Intestine Kidney Liver

+ + + a.b +++ +++ + +++ ~ +

yes +++ +++ +++ c +++ e +++

Lung

+

f

Maxillary/mandibular mesenchyme Olfactory epithelium Pituitary (anterior) Pituitary (posterior) Submaxillarygland Teeth Tongue Ear Trigeminal ganglion Urogenital sinus Vertebral cartilage primordia

+ + + + + + + + + +

+ + + +

+ + + +

+ + + + + +

h

+ + +

j + + I +

+ + + + + + + + + + +

g

+ + + + + +

+ + + + + i

+ + + +

k + + m +

+ and - indicate the presence or absence of a detectable hydridization signal, b multiple pluses indicate relative levels of Id mRNA expression, c mucosa and external layer, d epithelium, e epithelium and mesenchyme, f interstitial cells, g epithelium, h dental lamina, i mesenchyme, i auditory vesicle, k epithelium of vesicle and VIII ganglia, l epithilium, m mesenchyme. a

3.2. Comparison o f Id-1 and Id-2 expression in the CNS during development 3.2.1. Expression o f ld-2 at E12.5 I n the spinal cord Id-2 is expressed t h r o u g h o u t the v e n t r i c u l a r zone a n d the m a n t l e in a dorso-ventral gradient, with strong expression in the roof plate (Fig.

2A,B,C). Two l o n g i t u d i n a l c o l u m n s of expressing cells that may flank the a l a r / b a s a l b o u n d a r y can also be observed (Fig. 2B). I n the h i n d b r a i n (HB), there are several distinct b o u n d a r i e s of expression n o t e d for the v e n t r i c u l a r zone ( V Z ) a n d the m a n t l e . Medial sections (Figs. 1A a n d 2A,B) show a V Z i n t e r - r h o m b o m e r i c expression b o u n d a r y at r l / r 2 (see arrow in 2A) a n d a m a n t l e b o u n d a r y at a b o u t r 4 / r 5 (see arrow in 2B). A m o r e lateral section (Fig. 2C) shows V Z expression e x t e n d i n g into r l a n d the c e r e b e l l a r area a n d m a n t l e expression t e r m i n a t i n g at r l / r 2 (see arrow). Expression is strong in the tela chorioidea of the r h o m b e n c e p h a l o n (TC), as well as in the i s t h m o c e r e b e l l a r plate (CB). I n this region t h e r e is expression in the isthmic area a n d r h o m b i c lip, the anlage of the cerebellar nuclei (Fig. 2C), b u t less or n o expression in the i n t e r p o s e d region. T h e entire Mar m i d b r a i n , t e c t u m (T), i n c l u d i n g the roof plate, expresses Id-2. T h e r e is less expression in the m i d b r a i n basal plate, except for a cluster of cells in the region of the p r i m o r d i u m of the s u b s t a n i a nigra (data not shown). W e have i n t e r p r e t e d the expression of Id genes in the d i e n c e p h a l o n a n d s e c o n d a r y p r o s e n c e p h a l o n based u p o n the p r o s o m e r i c m o d e l [8,43]. In this model, the f o r e b r a i n is subdivided into six transverse domains, or p r o s e n c e p h a l i c n e u r o m e r e s (prosomeres), that are named prosomere 1 through prosomere 6 (pl-p6). Expression in the V Z is strongest in the s u b c o m m i s sural part of p l . M a n t l e regions show weak expression, i n c l u d i n g parts of p l (PT) (Fig. 2A,B), p2 (DT) (Fig. 2B), a n d p3 (VT) (Fig. 2A,B). W i t h i n the secondary p r o s e n c e p h a l o n , expression in p4 extends from the

Fig. 2. Analysis of the expression of Id-2 and Id-1 in parasagittal sections of heads of E12.5 mice using in situ RNA hybridization. Id-2 expression is shown in Panels A, B and C; Id-1 expression is shown in panels D, E and F. The arrow in Panels A and D shows the VZ boundary at rl/r2: the arrow in Panel B shows the mantle boundary at r4/r5; the arrow in panel C shows the mantle boundary at rl/r2. Magnification bar in bottom of panel D corresponds to 250/~m. Abbreviations are defined in Table 1.

W. Zhu et al. /Molecular Brain Research 30 (1995) 312-326

eminentia thalami (EMT) into the supraoptic paraventricular (SPV) area (Fig. 2A,B, see black arrowhead in 2B). Expression is also in the mammillary area (M) (Fig. 2A), and in the archicortex, where there is intense labeling at the site where the tela chorioidea attaches (Figs. 1A and 2A,B,C). Part of the adjoining primordia of the hippocampus is negative. In p5, there is strong expression in a rostro-caudal gradient along the neocortex (Figs. 1 and 2A,B,C) and more lateral piriform (paleo-) cortex (data not shown). Id-2 expression is preferentially found in the mantle zone, or preplate, compared to the VZ, p5 expression is also seen in the infundibulum (Fig. 2A). P6 labeling is found in the olfactory bulb (OB) and septal (S) primordia (Fig. 2A). 3.2.2. Expression o f ld-1 at E12.5

At E12.5, there is a strong similarity between the pattern of Id-1 and Id-2 expression. In the hindbrain and cerebellum. Id-1 expression is restricted largely to the VZ, where it has a similar, if not identical pattern to the Id-2 gene (Fig. 2D,E,F). Note, however, that the cerebellar nuclei do not express Id-1. In the alar plate

317

of the midbrain (tectum, T), Id-1 is expressed principally in the VZ (Fig. 2D,E,F). In the forebrain ld-1 expression is very limited and the pattern of expression diverges from that observed in Id-2. In the forebrain, Id-1 is strongly expressed in the chorioid plexus and infundibular floor region, and at a lower level in the VZ of the ganglionic eminences, septum and eminentia thalami (Figs. 1B and 2D,E,F). 3.2.3. Expression of ld-2 at E14.5

Expression of Id-2 in the hindbrain at E14.5 is largely limited to an alar domain extending across r8-r2, a region that gives rise to the cochlear and vestibular nuclei, and possibly part of the solitary tract nucleus and somatosensory column of the trigeminus (Fig. 3A). A subpial crescent of labeled cells is likely to represent the corpus ponto-bulbare (data not shown), which are migratory cells originating from the rhombic lip that give rise to the trapazoid body, and the pontine, olivary and lateral reticular nuclei (Fig. 3A). Although Id-2 is strongly expressed in these rhombic lip derivatives, it is not expressed in the rhombic lip-de-

Fig. 3. Analysis of the expressionof Id-2 and Id-1 in parasaginal sections of heads of E14.5 and E16.5 mice using in situ RNA hybridization.A: ld-2 expressionat E14.5. B: Id-1 expression at E14.5. C: Id-2 expressionat E16.5. D: Id-2 expression at E16.5. In panel B, the arrow shows Id-1 expression in the VZ of the rhombic lip and the arrowhead shows expression in the chorioid plexus of the telencephalon. In Panel C the discontinuity in neocortical expression is an artifact. Magnification bar in bottom of Panel D corresponds to 250/xm. Abbreviations are defined in Table 1.

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w. Zhu et al. / Molecular Brain Research 30 (1995) 312-326

rived external granule cells of the cerebellum. Within the cerebellum, Id-2 is expressed in the ventricular zone only in the midline areas (data not shown). More laterally, cerebellar expression is diffuse and is found in the primordia of the cerebellar nuclei and purkinje cells (Fig. 3A and data not shown). Finally, in the isthmus, expression is also seen in alar territory corresponding to the position of the parabigeminal and parabrachial nuclei (Fig. 3A). Expression of Id-2 in the midbrain is restricted mainly to the ventricular zone of caudal tectal plate (region of the inferior colliculus), although Id-2 mRNA is also found in the basal plate in the region of the developing substantia nigra. In the diencephalon there are scattered areas of weak expression in the epithalamus and in the ventral parts of pl and p2 (data not shown). The secondary prosencephalon has the highest levels of Id-2 expression as described previously [40], particularly in the neocortex and olfactory bulb primordia (see below). Elsewhere, there is expression in various prosomeric domains. In p4, expression is found in the mammillary area (MA) and eminentia thalami (EMT). Other p4 areas, including the CGE and ACx, may be negative. Expression in p5 appears in the H C C / A H region (data not shown). There is some expression in the infundibular region. The LGE and MGE are negative (Fig. 3A). The neocortex primordium expresses Id-2 at high relative levels in the rostral-caudal and latero-medial maturational gradients. In the neocortex, expression is in the mantle zone, with little or no expression in the proliferative zone. Expression in p6 is strongest in the olfactory bulb, which has the highest level in the brain.

layer and the deep cerebellar nuclei do not express Id-2 above background levels. There continues to be strong labeling of several rhombic lip derivatives, including the dorsally located corpus pontobulbare and the pontine primordium (Fig. 3D). In the midbrain, there is weak expression in the superficial stratum of the superior colliculus that contrasts with the negative inferior colliculus (data not shown). A faint patch of label is seen in the basal plate that appears to correspond with the substantia nigra. The diencephalon has expression in pl where the dorsomedial periventricular areas of the pretectum continue to express Id-2. In p2 the epithalamus is positive and low levels of expression are detectable in the dorsal thalamic populations. P3 is negative, as is much of p4, although there is some expression in the archicortex up to the apex of the intraventricular convex curve, which may correspond to the boundary between CA2 and CA3 (Fig. 3C). In p5 the neocortical and paleocortical plates, subplate and intermediate zones strongly express Id-2, while the VZ does not (Fig. 3C,D). The rostral boundary of the neocortical plate and subplate stops abruptly in the region of the anterior olfactory nucleus, whereas the intermediate zone expression is continuous with the olfactory bulb. Expression in p6 is strongest in the olfactory bulb, where it is found in the anlage of the glomerular layer, the deep OB layers, and the anterior olfactory nucleus (Fig. 3C,D). The medial and lateral septal nuclei also express Id-2 (Fig. 3D). 3.2.6. Expression of ld-1 at E16.5

Expression of Id-1 at E16.5 is only definitively seen in the chorioid plexuses and some periventricular areas, particularly in the subcommisural organs at the dorsal midline of pl (data not shown).

3.2.4. Expression o f Id-1 at E14.5

At this stage, there is considerable contrast between the pattern of expression of Id-2 and Id-1. Periventricular expression of Id-1 is seen widely throughout the brain except in the telencephalon, where only the chorioid plexus and its attachment taeniae are positive (Fig. 3B and data not shown). In the secondary prosencephalon, the ventricular zone in the region of the alar/basal boundary is positive (data not shown). Other positive VZ domains are found in the alar midbrain and hindbrain (see arrow in Fig. 3B). 3.2.5. Expression o f Id-2 at E16.5

As development proceeds the cerebellum shows irregular patches of Id-2 expression in groups of cells that are likely to be immature purkinje cells as they are forming characteristic cortical columns (Fig. 3C). Some sections show gaps of expression suggesting that some columns express Id-2, while others (particularly anterior and median areas) may not. The external granule

3.3. Isolation and DN.d sequence determination of a human Id-1 cDNA

Id-2 is known to be expressed in human fetal tissues and in some human tumors [5]. An important goal of our studies was to examine the expression of Id-1 in normal human tissues and in specimens of malignant tumors. At the time these studies were initiated the human homologue of Id-1 had not been cloned, and it was not possible to prepare a molecular probe that would reliably recognize the mRNA corresponding specifically to this member of the Id gene family. Using an ld-1 cDNA of approximately 600 bp, which was obtained by RACE-PCR as a probe, a cDNA library constructed from the human glioblastoma cell line U251 in lambda gtl0 vector (Clontech, Palo Alto, CA) was screened at high stringency. Six of 19 positive clones were subcloned into pBluescript SKII and their DNA sequence was determined. Recently the DNA se-

W. Zhu et al. / Molecular Brain Research 30 (1995) 312-326

quence of human cDNA homologues of the mouse Id-1 gene were reported [23]. The two types of clones isolated in these experiments were among those we isolated as well. We have named these Id-la and Id-lb, names similar to those originally chosen by Hara et al. [23] and numbered the DNA and amino acid sequence based on the proposed initiation site for mouse Id-1

ld l-a ldl-b

-62 bp -10

319

[4]. The two clones which were longest in size and representative of the full-length Id-la and Id-lb contained 958 bp and 1145 bp that includes both 5' and 3' untranslated regions (Fig. 4). We designated the plasmids containing these cDNA molecules as phld-la and phld-lb, respectively. As shown in Fig. 4, the DNA sequence and the

ttccgggcttccacctcatttttttcgctttcccattctgtttcagccagtcgccaagaatc gccaagaatc

Idl-a ldl-b

1 1

M ATG ATG M

K V AAA GTC A . A A GTC K V

A GCC GCC A

S AGT AGT S

G GGC GGC O

S AGC AGC S

T ACC ACC '/'

A GCC GCC A

T ACC ACC T

A GCC GCC A

A GCC GCC A

A GCG GCG A

G GGC GGC G

P CCC CCC P

S AGC AGC S

C TGC TGC C

17aa 17

Idl-a Idl-b

52 52

A GCG GVG A

L CTG CTG L

K AAG AAG K

A GCC GCC A

G GGC GGC G

K AAG AAG K

T ACA ACA T

A GCG GCG A

S AGC AGC S

G GGT GGT G

A G CJC'C; GGC GCG GGC A G

E GAG GAG E

V GTG GTG V

V GTG GTG V

R CGC CGC R

C TGT TGT C

34 M

ldl-a [dl-b

103 103

L CTG CTG L

S TCr TCT S

E GAG GAG E

Q CAG CAG Q

S AGC AGC S

V GTG GTG v

A GCC GCC A

I ATC ATC I

S TCG TCG S

R CGC CGC R

C TGC TGC C

A GCC GCC A

G GGG GGG G

G GGC GGC G

A GCC GCC A

G GGG GGG G

A GCG GCG A

51 51

[dl a Idl-b

154 154

R CGC CGC R

L CTG CTG L

P CCT CCT P

A GCC OCC A

L CTG CTG L

L CTG CTG L

D GAC GAC D

E GAG GAG E

Q CAG CAG Q

Q CAG CAG Q

V GTA GTA v

N AAC AAC N

V GTG GTG V

L CTG CTG L

L CTC CTC L

Y TAC TAC Y

D GAC GAC D

68 68;

205 205

M ATG ATG M

N AAC AAC N

G GGC GGC G

Y

Idl-a Idl-b

N

R

K

Idl-a Idl-b

256 256

Idl-a ld~-b

TOT C

C

TAC Y

S TCA TCA S

R CGC CGC R

L CTC CTC L

K AAG AAG K

E GAG GAG E

L CTG CI'G L

V GTG GTG V

P CCC CCC p

T ACC ACC T

L C'IG CTG L

P CCC CCC P

Q CAG CAG Q

85 85

V

K AAG AAG K

V GTG GTG V

E GAG GAG E

1 A'FI" ATI' I

L C'[~ CTC L

Q CAG CAG Q

II CAC CAC H

V GTC GTC V

I ATC ATC 1

D GAC GAC D

Y TAC TAC Y

I ATC ATC i

102 102

TGT TAC

AAC N

CGC R

AAG K

GTG V

S AGC AGC S

307 307

R AGO AGG R

D GAC GAC D

L C'IT CTT L

Q CAG CAG Q

L E TTG GAG " I I " G GAG L E

L CTG CTG L

N AAC AAC N

S TCG TCG S

E GAA GAA E

S TCC TCC S

E GAA GAA E

V G O'lff GGA G 2 r q ' GGA V G

'Y ACC ACC T

P CCC CCC I:'

G GGG GGG G

119 119

ldl-a Idl-b

358 358

G GGC GGC G

R CGA CGA R

G GGG GGG G

L CTG CTG L

P CCG CCG P

V GTC GTC V

R CGG CGG R

A GCT GCT A

P CCG CCG P

L CTC CIC I,

S AGC AGC S

T ACC ACC 'i"

L CTC CTC L

G GGC GGC G

E GAG GAG E

1 ATC ATC l

136 136

ldl-a ldl-b

409 409

S AGC AGC S

A GCC GCC A

L CTG CTG L

T ACG ACG T

A GCC GCC A

E GAG GAG E

................................................................................................... GTG AGA TCC AGA TCC GAC CAC TAG a t c a t c c t t a t a c c V R S R S I) tl AMB

Idl-a

427

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I,~2

Idt-h

465

g a c g g ggaaacggaggccagagagggcgtgggcgcttgcaccacttccgtccca•ccttgcgggtacctggctatgcgggggtgcctaaggagc

Id l-a Idl-b

427 559

..................................................................................................................................................... ctggaaaaagcgctcccccgtcgtgcttcctggggaagggggcgttcgctgcgctcggagcggcgtccctcccaacccgccggtctcatttcttctcg•

[dl-a ldl-b

427 659

................ tttcacag

ld l-a

473

ldl-b

712

Id l-a

569

)oi-~)

8~

AAC CGC AAG GTG

A GCG gcg

A GCA gca

C TGC tgc

V G'IT gtt

p CCT cct

A GCG gcg

D GAC gac

D (;AT gat

R CGC cgc

cctcccccagggaccggcggaccccagccatccagggggcaagaggaattacgtgctctgtgg

1 ATC atc

L TTG ttg

N AAC AAC N

C TGT tg t

n CGC cgc

OPA IGA tga

gtctcccccaacgcgcctcgccggatctg

142 1,:19

149

agcg

142 149

155 ~49

aggg

agaacaagaccgatcggcggccactgcgcccttaactgcatccagcc•ggggctgaggctgaggcactggcgaggagagggcgctcctctctgcac

Idl-a

666

t~ 1-b

905 acctactagtcaccagagactttagggggtgggattccactcgtgtgtttctattt~ttgaaaagcagacattttaaaaamggtcacgtttggtgcttctcaga

Idl-a

772

ldl-b

loll

tttctgaggaaattgctttgtattgtatattacaatgatcaccgactgaaaatattgttttacaatagttcttgtggggctgtttttttgttatt~aacaaataatttag

Idl-a Id I-b

881 1120

atggtgaaaaaaaaaaaa

F i g . 4. c D N A

sequence

and predicted

amino acid sequence

of two isoforms of human

Idl-a and ldl-b.

320

Zhu et aL / Mo&cu~r Brain R~earch 30 (1995) 312-326

Id-la

human

M K V A

S GS

Id-i

mouse

M K V A S G S - A A A A A G P

Id-la

human

SE

Q

SVA

I S R C A G G A G A R L P A L L

Id-I

mouse

SE

Q

S VA

I S R C A G -

Id-la

human

G C Y

S R L K E L V P T L P Q N R K V

Id-i

mouse

GCY

S R L K E

Id-la

human

Q L E

L N

S E

G T P G G R

Id-i

mouse

Q

L E

L N

S E S E V G T T G G R

Id-la

human

A

E A A

C V P A

D D R

I L C R

155

Id-i

mouse

A

E A

C V

D

I L

148

A

T A T A A A G P

LVP

D

R

G A G E V V R C L

35

- G E V V L G L

31

S C S L K A G R T A - -

- - T R L P

T LP

S E V

P A

S C A L K A G K T A S

DE

AL

LD

Q Q V N V L

L Y D M N

70

E Q Q V N V L L Y D M N

S K V E

63

I L Q H V

I D Y

I R D L

I L O H V

I D Y

I RD

G L P V R A P

L S T L N

G E

I S A

G L P V R A P

L S T L N

G E

O N R K V S

KVE

105

L

98

L T

140

I S A L A

133

C R

Fig. 5. Comparison of the predicted amino acid sequences for Id-la from human (Fig. 4) and mouse [20]. Dashes in the sequence have been inserted to optimize the alignment. The HLH domain of these proteins is underlined and extends from amino acid number 58 to 103 of the mouse ld molecule.

Comparison of Human Id-1, Id-2, and Id-3 Id-2

human

M K A F S

II Id-la human

. . . . . . . . . . . . . . .

II

I

I

M K A L S

Id-2

S D H S L G I S R

. . . . . . . . . . . . . . .

I Id-la

human

Id-2

I

II

Id-2

I

II ....

I I I I r I

III

40

II

I

II

70

Ill

K G P A - A E E - - P L S L L D D M ~

I I I

I I I I

45

I I I i

I I I I I I I P I I

75

I I

I

I

I I I I I

I I i I I

105

I I

I

f

I

Q L E L N S E

I

I

....

Id-2

S I L S L Q A

human

P

I

S A

L T A

I

I

J

il

ll0

111

I

S E V G T P G G R G L P V R A P L S T L N G E I

136

I

Q V V L A

human

80

Q I A L D S H P T I V S L H H Q R P G Q N Q A S R T P L T T L N T D I

Heir-I

Id-la

I

G C Y S R L K E L V P T L P Q N R K V S K V E I L Q H V I D Y I R D L

I human

Heir-i

17

H C Y S R L R E L V P G V P R G T O L S O V E I L O R V I D Y I L D L

human

Id-la

I I

I I I I I Heir-i

P V R G C - - - Y E A V C C L

D C Y S K L K E L V P S I P Q N K K V S K M E I L Q H V I D Y I L D L

I i i human

35

II

S K T P V - - D D - - P M S L L Y N M N

III

S E R S L A I A R G R G

human

Id-la

. . . . . .

I

S E Q S V A I S R C A G G A G A R L P A L L D E Q Q V N V L L Y D M N

II Heir-i

15

I

Heir-i

human

P V R S V - - - R K N S - - L

I

M K V A S G S T A T A A A G P S C A L K A G K T A S G A G E V V R C L

E P A P G P P

. . . . . . . . . . . . . . . . . . . .

S E F P S E L M

i

- S N D S K A

I

E A A

C V

P A

I . . . . . .

I

P H L P I Q T A E LAP

94

134

I I

D D R

I E L V

L C G

DG

I

I L C R

155

I

I S N D K R S F C H

119

Fig. 6. Comparison of the predicted amino acid sequences for human Id-2 [33], human Id-la (Fig. 4), and heir-1 [34]. Dashes have been inserted to optimize the alignment. Amino acids that are conserved among the proteins are indicated by vertical lines. The HLH domain has been underlined.

W. Zhu et al. / Molecular Brain Research 30 (1995) 312-326

amino acid sequence of the clones we isolated are somewhat different from those previously reported [23]. In both molecules reported in Fig. 4 we found that at codon 16, reported to encode serine, there was a A G C triplet encoding threonine. We also found three additional nucleotides not previously reported: we detected nucleotides GC at position 136-137 and G at position 141. These changes result in a deduced amino acid sequence of C A G G A (Fig. 4) instead of C R G A at position 45 to 49 [23]. Since our clones contain three additional bp compared to the previously reported Id-1 clones, the reading frame of the molecule is not changed distal to this region, but the amino acid sequence of aa 45-49 is different from that reported, and includes an additional amino acid. Fig. 4 also reports considerable untranslated D N A sequence both 5' to the presumed start site for translation and 3' to the termination codons, which has not been previously described. Both the cDNA molecules designated Id-la and Id-lb contain a single open reading frame. The open reading frame of p h l d - l a begins with an A T G codon 62 bp from the 5' end of the cloned molecule and ends with T G A at position 552. phld-lb includes 10 bp of 5' untranslated nucleotide sequence and ends with T A G at position 449 (Fig. 4). The open reading frame of p h l d - l a encodes a predicted protein of 155 amino acids containing a H L H motif. Isoforms of Id-1 corresponding to Idl-b in human have not yet been described in other species; however, comparison of the predicted amino acid sequence of human Id-la to that of mouse Id-1 indicates that they share greater than 90% amino acid homology (Fig. 5). As shown in Fig. 5, the H L H domains of human and mouse Id-1 correspond to amino acid 58 to 103 of the mouse homologue and are identical. Fig. 6 shows the human Id-1 protein sequence compared with human Id-2 [5] and heir-1 (human Id-3) [18], other human genes belonging to this family of transcription factors. There is a high degree of sequence identity between the H L H domains of Id-1, Id-2, and heir-1. Of the amino acid residues of the H L H domain, 80.9% were conserved between Id-1 and Id-2, whereas 69% were found conserved between Id-1 and heir-1. There is little sequence similarity between the three Id proteins outside the H L H domain, although short regions of similarity have been recognized at their 5' ends and 3' to the H L H region [23]. 3.4. Human Id-1 gene expression in normal tissue and in tumors

The predominant m R N A corresponding to Id-1 is approximately 0.9 bp, and in virtually all tissues examined, this 900 bp m R N A is the most prominent species (Fig. 7 and unpublished data). A second m R N A which

321

HUMAN BRAIN

LIVER

E72 A

El 22 A

LUNG E84 A

Id-1

G3PDH

Fig. 7. Examination of Id-1 m R N A expression in human fetal and . adult tissues. The age of the embryonic tissue from which total RNA was extracted is indicated above the corresponding lane. The Northern blot examined in this figure was re-examined in the lower panel with a probe corresponding to the human G3PDH gene as a control for RNA loading and transfer.

we believe corresponds to the Id-lb described in Fig. 4 is approximately 1.2 kb (data not shown). Based on comparisons with the expression of Id-1 in mouse tissues, we sought to determine whether there was a similar pattern of expression in selected human tissues. We had shown that, typically, there are higher levels of Id-2 gene expression in embryonic tissues than in tissues taken from the same organs in adults [5]. This pattern is recognizable in some tissues for other members of this gene family as well [51]. We detected low levels of Id-1 expression in both fetal liver and adult lung and liver tissues, and a high level of expression was detected in adult lung; this pattern of expression is consistent with the pattern observed by Duncan et al. [16] in mouse tissues. Moreover, we readily detected Id-1 gene expression in human fetal brain (72 days of gestation). Id-1 m R N A molecules of different sizes were detected in the fetal and adult tissue from liver and lung that we examined, although we have not yet detected a specific pattern of reproducible differences. The finding that Id-1 was expressed in a variety of fetal tissues [51], its role in the inhibition of gene expression in differentiated tissues [4], and the recent description of Id genes in enhancing cellular proliferation [2,27] led us to examine the expression of this gene in cell lines derived from a variety of human tumors. We examined medulloblastoma cell lines, D341 and

W. Zhu et al. / Molecular Brain Research 30 (1995) 312-326

322

D283; lung cancer cell lines, HUT69C and H292; colon cancer cell lines, Colo320 and Colo205; melanoma cell lines, WM373 and 3711T; and a lymphoma cell line, CA46 (Fig. 8 and Table 3). The highest levels of Id-1 expression were observed in the medulloblastoma line D341 and in the lung cancer cell lines studied. Because medulloblastomas are recognized as the most undifferentiated tumors of the CNS and because Id genes are developmentally regulated during brain maturation and implicated in the regulation of differentiation of several different tissues, we sought to determine if the Id-1 gene was expressed in other tumors arising in the nervous system. Neuroblastomas and neuroepitheliomas, tumors of the PNS that usually occur during childhood, are thought to arise in undifferentiated ceils originating in the embryonic neural crest [11,12,45,49]. Glioblastoma is a malignant brain tumor arising in the astrocytes of the CNS. We examined a series of cell lines from these tumors for the expression of Id-1 mRNA. We readily detected high levels of Id-1 m R N A in cell lines from each of these tumor types, although some cell lines did not express Id-1 that was detectable in the cultures we examined (Table 3). Id-1 m R N A was very abundant in glioblastoma cell lines and in the neuroblastoma cell line SK-N-AS. When Id-1 and Id-2 expression in these cell lines was compared, it was evident that many cell

-~

N

~-

o

-6

=

N

<

Id-1

G3PDH

Fig. 8. E x a m i n a t i o n o f Id-1 m R N A e x p r e s s i o n in h u m a n t u m o r cell lines. T h e n a m e o f t h e cell line f r o m w h i c h t o t a l R N A w a s e x t r a c t e d is i n d i c a t e d a b o v e t h e c o r r e s p o n d i n g l a n e . T h e N o r t h e r n b l o t e x a m i n e d in this f i g u r e w a s r e - e x a m i n e d in t h e l o w e r p a n e l w i t h a p r o b e c o r r e s p o n d i n g to t h e h u m a n G 3 P D H g e n e as a c o n t r o l f o r R N A loading and transfer.

Table 3 Id g e n e e x p r e s s i o n in h u m a n t u m o r cell lines Cell line Neuroblastoma SHF SK-N-SH SK-N-AS SK-N-DZ Glioblastoma U251 SF295 U87 Ul18 U343 Neuroepithelioma SKNMC TC32 Medulloblastoma D341 D283 Melanoma WM373 3711T Lung HUT69C H292 Colon Colo320 Colo205 Lymphoma CA46

Id-1 a

Id-2

+ + + + + + + +

+ + + + + + + -

+ + + + -

+ + + + + -

+ + + + + + +

+ + + + +

+ + + + + +

+ + + + +

-

-

-

+ + + + + +

+ + -

+

-

-

+

a Id-1 a n d Id-2 e x p r e s s i o n w a s e x a m i n e d b y N o r t h e r n blot h y b r i d i z a t i o n a n a l y s i s as i n d i c a t e d in M a t e r i a l s a n d M e t h o d s . D a s h e s ( - ) i n d i c a t e t h a t e x p r e s s i o n w a s n o t d e t e c t a b l e . Pluses ( + ) i n d i c a t e t h e relative level o f g e n e e x p r e s s i o n d e t e c t e d in t h e s e d i f f e r e n t cell lines f o l l o w i n g c o r r e c t i o n f o r d i f f e r e n c e s in R N A l o a d i n g as d e t e c t e d by examination of the blots for G3PDH expression.

lines express both genes, although, in general, Id-2 levels are lower than Id-1 levels in glioblastomas (Table 3 and see below). Cell lines from several other human tumor types, including the commonly occurring carcinomas of lung and colon, had readily detectable levels of Id-1 and Id-2 m R N A (Table 3). Examination of the pattern of Id gene expression in Table 3 indicates that with few exceptions those cell lines in which Id-1 is highly expressed are also characterized by relatively high levels of Id-2 expression, and cell lines in which the level of Id-1 expression is low also have relatively low levels of Id-2 expression. In an effort to look more critically at the possibility that the regulated level of expression of different members of the Id gene family may be similar in the different cell lines we examined, we designed an experiment to eva[uate the relative levels of Id gene expression in a series of different human tumor cell lines (Fig. 9). We chose to examine cell lines originating in glial tumors because of our interest in Id expression in the CNS and these tumor cell lines were among those in which our survey revealed the highest levels (Table 3). For this examina-

HI,, Zhu et al. / Molecular Brain Research 30 (1995) 312-326

1

2

3 4 5

6

323

Z

"ld-1

Id-2

G3PDH

Fig. 9. Examination of Id gene mRNA in glioblastoma cell lines. Similar amounts of mRNA (approximately 20/~g per lane) from glioma tumor cell lines was examined with comparably probes which uniquely recognize either Id-1 or Id-2 mRNA as described in the Materials and methods section, The cell lines examined were Y87 (lane 1), SF767 (lane 2), SK-MG-13(lane 3),SK-MG-12(lane 4), SK-MG-2(lane 5), U178 (lane 6), U87 (lane 7), SNBI9 (lane 8). Hybrid.ization to a probe for G3PDH provided a control for DNA loading and transfer.

tion we loaded total R N A from 8 different glioma tumor cell lines onto 2 different blots, and prepared equivalent probes that recognized uniquely Id-1 or Id-2 m R N A (see Methods). As seen in Fig. 9, glioma tumor cell lines that expressed high levels of Id-1 tended to express high levels of Id-2 and a corresponding pattern was seen in cell lines expressing relatively lower levels of these two related genes. In other experiments we have found that Id-3 m R N A expression also reflects closely the relative level of expression of these other Id family m e m b e r s (data not shown). Id gene expression in human tumor tissues and the biologic activity of these genes in inhibiting differentiation and enhancing growth calls attention to their potential to contribute to the malignant transformation of normal cells. Because such genes might even act as oncogenes their chromosomal location can provide important clues regarding the tumor types in which they might be of pathologic importance. Id-3 has been m a p p e d to h u m a n chromosome lp36, a region frequently deleted in many tumor types [18], and Id-1 has been m a p p e d to human chromosome 20 (R. Benezra and R. Chiganti, personal communication). We used fluorescence in situ hybridization of labeled Id-2 DNA,

pgld-13, to normal human chromosomes, and we m a p p e d Id-2 to Ch 2p25.

4. Discussion Understanding the molecular pathways over which tissue-specific gene expression is mediated is of central importance for the study of cellular differentiation and the disorders that affect it. Ultimately the signals and clocks that control these processes must interface with the regulatory elements of individual genes that encode the unique characteristics of different types of differentiated cells. The emerging picture of tissue-specific transcription factors that can either positively or negatively regulate the expression of such genes suggests we may find that the molecular mechanisms mediating differentiation are involved in influencing the precise balance of expression required for normal development. Transcriptional regulators belonging to the Id family of genes efficiently antagonize the gene-activating effects of the several different b H L H transcription factors.

324

W. Zhu et al. /Molecular Brain Research 30 (1995) 312-326

There are now four members of the Id group of genes recognized in mammalian tissues [4,10,44,47]. In humans they are know as Id-1 [23], Id-2 [5], and Id-3 (heir 1) [18]. The fourth family member, Id-4, has been identified in mouse tissues [44], and we have detected the expression of this gene in some human tissues (data not show). Our characterization of the human Id-1 homologue extends this family of genes and our understanding of their possible functions. Our finding that the human Id-1 shares a high degree of homology within its HLH region with other family members of this group of proteins is compatible with the finding that the different mouse Id proteins dimerize with a similar spectrum of bHLH proteins [47]. Human Id-1 is highly divergent from the other human Id genes both 5' and 3' to this domain (Fig. 3). The coexpression of Id-1 and Id-2 in many different cell types (Tables 2 and 3) and the observation that, within a single cell type, there are apparently multiple different bHLH proteins expressed that can form heterodimers with one another and with the Id proteins suggest that, eventually, regions outside the HLH domain will be found to impart considerable specificity to the actions of the Id proteins. The divergent regions of the different Id molecules may be sites for interaction with other proteins, and they may enhance the precision of lineagespecific gene expression, which is clearly important for the proper functioning of highly specialized cells. Our evaluation of the expression of Id-1 and Id-2 during development does not allow identification of the molecular mechanisms that regulate the expression of these genes. Previously the expression of Id-1 and Id-2 were examined individually [16,19,40,51], whereas in this study we compared the expression of these genes in adjacent sections of mouse embryos. This approach reveals interesting relationships between these genes. Several tissues co-expressed these genes, whereas others expressed only one. There were also some tissues w h e r e t h e r e was reciprocal epithelial/mesenchymal expression of these genes. Tissues that express high levels of both genes include the meseneephalon, rhombeneephalon, spinal cord, prevertebrae, genital eminence, PNS, pituitary, olfactory epithelium, and craniofacial mesenchyme. In some tissues, Id-1 and Id-2 appear to be co-expressed in the same cells, although the resolution of the in situ technique does not allow one to know this with certainty. These tissues would include trigeminal ganglion, pituitary, olfactory epithelium, genital eminence, prevertebrae and the ventricular zones of the mesencephalon, rhombencephalon, and spinal cord. While Id-1 and Id-2 are co-expressed in apparently the same proliferating, undifferentiated cells, Id-2 continues to be expressed in differentiated descendants of these precursor cells in the mesencephalon, rhombencephalon, and spinal cord.

In other tissues, these genes were clearly differentially expressed. The most prominent of these included the forebrain, lung, teeth, urogenital sinus, kidneys and intestines. In the telencephalon, Id-1 and Id-2 have reciprocal expression patterns. Id-2 is expressed at high levels in several domains (the neocortex, paleocortex, archicortex, and olfactory bulb) where Àd-1 expression cannot be distinguished from background levels. On the other hand, Id-1 is expressed in basal telencephalic primordia (MGE, LGE) (Fig. 1), where Id-2 is expressed at background levels. In some tissues it is possible to hypothesize the reciprocal expression of Id-1 and Id-2 in epithelial and mesenchymal cells within a developing structure (teeth, urogenital sinus, lung) (Table 2). For instance, at E14.5 in the teeth, Id-1 is expressed in the epithelial dental lamina whereas ld-2 is expressed in the mesenchyme. At E14.5 in the urogenital sinus, Id-1 is expressed in the epithelium and Id-2 is expressed in the mesenchyme. At E12.5 in the lung, Id-1 is expressed at low levels in mesenchymal tissue and Id-2 is expressed in the epithelium. More complex patterns of Id gene expression may also exist in some organs. In the hindbrain, Id-1 and Id-2 are expressed in similar anteroposterior and dorsoventral regions, but differ in their expression along the mediolateral axis (radial dimension). Here, Id-1 is restricted in its expression to the VZ, whereas Id-2 is expressed both in the VZ and in the postmitotic mantle layer. This is also true in the rhombic lip and its derivatives. Id-1 is expressed in the proliferative zone, whereas Id-2 is expressed in a subset of rhombic lip derivatives (e.g. pontine nuclei). A similar pattern is also seen in the spinal cord and in the tectum. This suggests that in these domains Id-1 may play a role in proliferative, undifferentiated cells, whereas Id-2 may also function in regulating the differentiation of the postmitotic migrating cells in the mantle. Also, Id-2 is expressed in postmitotic differentiating cells of the cerebral cortex, olfactory bulb and the cerebellum. This is compatible with the finding of others that during gestation and in adult mice, Id-2 continues to be expressed in specific subsets of neurons in these structures, demonstrating that Id-2 expression can be found in postmitotic, fully-differentiated cells [40]. Id genes inhibit differentiation and enhance cellular proliferation - two key characteristics of malignant tissues. Although Id-2 mRNA expression has been observed in tumors of the human nervous system [5] and Id-3 is expressed in cell lines from embryonal neuroblastoma [18], Id-1 gene expression has not been systematically studied in tumors of any type. We found that both Id-1 and Id-2 were widely expressed in cell lines derived from a variety of different tumors including such commonly occurring carcinomas as those arising in lung and colon. These genes were also expressed in many types of primary nervous system tumors. Both

w. Zhu et al. / Molecular Brain Research 30 (1995) 312-326

Id-1 a n d Id-2 w e r e e x p r e s s e d in astrocytic t u m o r s as well as in t u m o r s arising in cells with e v i d e n c e o f neuronal differentiation, including both neuroblastoma and neuroepithelioma. Medulloblastoma, a primitive CNS tumor of early childhood, expressed readily det e c t a b l e levels of Id-1, a l t h o u g h we w e r e u n a b l e to d e t e c t Id-2 g e n e expression. It is likely t h a t h u m a n t u m o r cell lines can p r o v i d e i m p o r t a n t r e a g e n t s for the i d e n t i f i c a t i o n of tissue-specific b i n d i n g p a r t n e r s of t h e I d g e n e p r o d u c t s . O n g o i n g e x p e r i m e n t s in o u r labs suggest that, while s o m e p r o t e i n s t h a t b i n d t h e s e g e n e s can b e i d e n t i f i e d in m a n y d i f f e r e n t tissues, m a n y tissues have e v i d e n c e of novel b i n d i n g p a r t n e r s (our u n p u b l i s h e d data). T h e c y t o g e n e t i c l o c a l i z a t i o n o f g e n e s i m p o r t a n t for m a l i g n a n t t r a n s f o r m a t i o n has f r e q u e n t l y p r o v i d e d a clue to t h e t u m o r types in which specific g e n e s play a role of p a t h o l o g i c i m p o r t a n c e . O u r m a p ping o f Id-2 to C h 2p25 p l a c e s it c y t o g e n e t i c a l l y in t h e r e g i o n o f c h r o m o s o m a l r e a r r a n g e m e n t s p r e v i o u s l y reco g n i z e d to o c c u r in a s s o c i a t i o n with H o d g k i n ' s d i s e a s e [31,36,45] a n d a l e i o m y o m a [35]. G i v e n the k n o w n activities of I d g e n e s to inhibit d i f f e r e n t i a t i o n a n d e n h a n c e p r o l i f e r a t i o n , Id s e e m s a s t r o n g c a n d i d a t e for c o n t r i b u t i n g to t h e m a l i g n a n t p r o p e r t i e s o f t h e s e tumors.

Acknowledgements This r e s e a r c h was p a r t i a l l y s u p p o r t e d by g r a n t s to M.A.I. f r o m t h e N a t i o n a l I n s t i t u t e for N e u r o l o g i c a l Disease and Stroke (NS#31076), Theodora Betz Found a t i o n , a n d P r e u s s F o u n d a t i o n as well as by g r a n t s to J . L . R . R . f r o m the M a r c h o f D i m e s , N A R S A D , t h e J o h n M e r c k F u n d , P f i z e r P h a r m a c e u t i c a l s , a n d the N a t i o n a l I n s t i t u t e o f M e n t a l H e a l t h R O 1 MH49428-01 a n d K O 2 MH01046-01. G e n e m a p p i n g studies w e r e s u p p o r t e d by U S D O E c o n t r a c t s D E A C 0 3 7 6 S F 0 0 0 9 8 . F o r fellowship s u p p o r t , we wish to t h a n k t h e C a n c e r R e s e a r c h F o u n d a t i o n of A m e r i c a (J.R.B.). W e t h a n k N h u n g H u y n h for p r o v i d i n g e x c e l l e n t t e c h n i c a l assistance. W e a p p r e c i a t e the a s s i s t a n c e of Lucy de la C a l z a d a a n d Cheryl C h r i s t e n s e n in the p r e p a r a t i o n of this m a n u s c r i p t a n d t h a n k S u s a n E a s t w o o d E L S ( D ) for e d i t o r i a l advice.

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