Expression pattern of the stem cell leukaemia gene in the CNS of the embryonic and adult mouse

Neuroscience 122 (2003) 421– 436

EXPRESSION PATTERN OF THE STEM CELL LEUKAEMIA GENE IN THE CNS OF THE EMBRYONIC AND ADULT MOUSE J. A. M. VAN EEKELEN,a* C. K. BRADLEY,a a ¨ J. R. GOTHERT, L. ROBB,b A. G. ELEFANTY,c a C. G. BEGLEY AND A. R. HARVEYd

different diencephalic, mesencephalic and metencephalic brain nuclei in adult CNS. Co-localisation of LacZ with the neuronal marker NeuN indicated expression in post-mitotic neurons in adulthood. LacZ expression by neurons was confirmed in tissue culture analysis. The nature of the pretectal, midbrain and hindbrain regions expressing LacZ suggest that SCL in adult CNS is potentially involved in processing of visual, auditory and pain related information. During embryogenesis, LacZ expression was similarly confined to thalamus, midbrain and hindbrain. LacZ staining was also evident in parts of the intermediate and marginal zone of the aqueduct and ventricular zone of the fourth ventricle at E12.5 and E14. These cells may represent progenitor stages of differentiating neural cells.Given the presence of SCL in both the developing brain and in post-mitotic neurons, it seems likely that the function of SCL in neuronal differentiation may differ from its function in maintaining the differentiated state of the mature neuron. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved.

a Centre for Child Health Research and WAIMR, University of Western Australia, at the Telethon Institute for Child Health Research, PO Box 855, West Perth WA 6872, Australia b The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Parkville VIC 3050, Australia c

Centre for Early Human Development, Monash Institute of Reproduction and Development, 27–31 Wright Street, Clayton VIC 3168, Australia

d School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia

Abstract—The basic helix–loop– helix (bHLH) transcription factor stem cell leukaemia (SCL) is a ‘master regulator’ of haematopoiesis, where SCL is pivotal in cell fate determination and differentiation. SCL has also been detected in CNS, where other members of the bHLH-family have been shown to be indispensable for neuronal development; however, no detailed expression pattern of SCL has so far been described. We have generated a map of SCL expression in the embryonic and adult mouse brain based on histochemical analysis of LacZ reporter gene expression in sequential sections of brain tissue derived from SCL-LacZ knockin mice. The expression of LacZ was confirmed to reflect SCL expression by in situ hybridisation. LacZ expression was found in a range of

Key words: SCL, tal-1 gene, bHLH-transcription factor, gene targeting, LacZ reporter gene, mouse embryogenesis.

The basic helix–loop– helix (bHLH) transcription factor SCL (stem cell leukaemia; tal-1, tcl-5) was first identified as a T-cell oncogene in the t(1;14) translocation in acute lymphoblastic leukaemia (reviewed by Begley and Green, 1999). The SCL gene is expressed in haematopoietic stem cells, progenitor cells and in committed erythroid, mast and megakaryocytic cells. It has a role in proliferation and self-renewal of multi-potent haematopoietic cells and their differentiation (Begley et al., 1991; Green and Begley, 1992; Green et al., 1991a, b, 1992; Visvader et al., 1991). Consistent with these findings, SCL-deficient embryos display absolute anaemia and absence of haematopoietic stem cells. They do not survive beyond embryonic day 10 (E10; Robb et al., 1995; Shivdasani et al., 1995). Thus, existing data indicate that SCL plays a pivotal role in establishing the transcriptional programme of haematopoietic stem cells. Although endogenous SCL gene expression has been described predominantly in the haematopoietic system, expression was also detected in embryonic brain, spinal cord and endothelium (Green et al., 1992b; Smith et al., 2002). To further study SCL expression in the mouse, the Escherichia coli LacZ reporter gene was introduced into the SCL locus. In these SCL-LacZ knockin mice, LacZ expression faithfully reproduced the pattern of SCL expression during haematopoietic differentiation (Elefanty et al., 1998). In addition, LacZ expression was demonstrated in the midbrain of whole mount stained heterozygous SCL-

Abbreviations: AChE, acetylcholinesterase; APT, anterior pretectal nucleus; AQ, aqueduct; bHLH, basic helix–loop– helix; CnF, cuneiform nucleus; DIG, digoxigenin; Dk, nucleus of Darkschewitsch; DLG, dorsal-lateral geniculate nucleus; DpMe, deep mesencephalic nucleus; DR, dorsal raphe; E, embryonic day; ECIC, external cortex of inferior colliculus; FBS, foetal bovine serum; GFAP, glial fibrillary acidic protein; IGL, intergeniculate leaflet; InC, interstitial nucleus of Cajal; IPC, caudal interpeduncular nucleus; IPL, lateral interpeduncular nucleus; IZ, intermediate zone; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; MGN, medial geniculate nucleus; MnR, median raphe nucleus; MZ, marginal zone; NeuN, nuclear neuronal protein; OPT, olivary pretectal nucleus; OT, nucleus of the optic tract; PAG, peri-aqueductal grey; PBG, parabigeminal nucleus; PBS, phosphatebuffered saline; PC, posterior commissure; PH, posterior hypothalamus; pnd, postnatal day; PrC, precommissural nucleus; PT, pretectum; RMC, magnocellular part of the red nucleus; RPF, retroparafascicular nucleus; RPO, rostral periolivary region; SC, superior colliculus; SCL, stem cell leukaemia; SGS, stratum griseum superficiale (superficial grey layer of superior colliculus); SN, substantia nigra; SO, stratum opticum (optic layer of superior colliculus); SP, substance P; SPO, superior paraolivary nucleus; SZ, stratum zonale (zonal layer of superior colliculus); T, tectum; Tg, tegmentum; VLG, ventral lateral geniculate nucleus; VLL, ventral nucleus of the lateral lemniscus; VTg, ventral tegmental nucleus; ZLI, zona limitans intrathalamica; 4V, fourth ventricle; 7N, facial nucleus. *Corresponding author. Tel: ⫹61-8-9489-7886; fax: ⫹61-8-94897700. E-mail address: [email protected] (J. A. M. van Eekelen).

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00571-2

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LacZ knockin embryos and adult brain tissue blocks (Elefanty et al., 1999). To more precisely define the expression pattern of SCL in the CNS, we have analysed the LacZ expression in sequential sections of adult and embryonic brain tissue from heterozygous SCL-LacZ knockin mice. A detailed map of SCL has been generated, defining the location and distribution of SCL expressing cells in the adult diencephalon, mesencephalon and metencephalon. SCL was not expressed in the telencephalon, including neural stem cell generating regions in the adult brain. During embryogenesis, SCL was detected in specific regions of the foetal brain known to differentiate into the range of regions that express SCL in adulthood. SCL was also expressed in parts of mesencephalic and metencephalic zones described as regions containing differentiating neural cells. This suggests a role for SCL in late neural differentiation in the developing CNS, which may be different from the role of SCL in post-mitotic neurons in the adult brain.

EXPERIMENTAL PROCEDURES Animals The SCLlacZ/w transgenic mice have been described previously (Elefanty et al., 1998) and carry the E. coli LacZ reporter gene introduced into the mouse SCL locus by homologous recombination. As a result, LacZ gene expression is under the control of SCL regulatory elements and simultaneously, the SCL coding sequence was deleted creating an SCL-null allele (SCL-LacZ knockin). These mice were back-crossed into C57B6/J mice for more than 10 generations. Six to 10 week old SCLlacZ/w and control SCLw/w wild type C57B6/J mice were used to determine the SCL expression pattern in the adult CNS. Timed pregnancies of crosses between SCLlacZ/w and SCLw/w mice yielded mice of the anticipated genotypes and in appropriate Mendelian ratios. Litters were harvested at E12, 14, 16, 18 and neonatal pups were killed on the morning of birth (postnatal day [pnd] 0) to determine the SCL expression pattern in the developing CNS. The animal experiments were approved by the Regional Animal Experimentation Ethics Committee.

Transgenic genotype analysis The genotypes of adult and neonatal mice were determined by PCR analysis of genomic DNA extracted from the tail tip. A small part of the lower embryonic body was used to extract DNA from unborn progeny. PCR amplification based on the use of a combination of three primers was applied to differentiate between the SCLw/w and SCLlacZ/w genotype. Forward primer A (5⬘ GTT TTG GTC TAG AGT TTG TGA GCC 3⬘) annealed to mouse SCL nucleotides 1416 –1440 in intron V (Begley et al., 1991) and is only present in the germline SCL allele. Forward primer B (5⬘ ATA TTG CTG AAG AGC TTG GCG GC 3⬘) annealed to the neomycin resistance gene included in the SCL-LacZ targeting vector and is only present in the SCL-LacZ knockin allele. A common reverse primer (5⬘ GCA TGC GTC TAG AGT TTG TGA GCC 3⬘) annealed in exon VI to mouse SCL nucleotides 1816 –1840. The PCR reaction included annealing at 65 °C and 35 cycles and the amplification products were analysed by gel electrophoresis.

Tissue preparation Adult and neonatal brain tissue was fixed by intra-cardial perfusion for 10 –15 min with ice cold 4% paraformaldehyde in phosphatebuffered saline (PBS), pH 7.2, followed by post-fixation of the

brain for 30 min at 4 °C. Embryonic brain tissue was fixed by immersion of the head for 1 h at 4 °C. If processed for whole mount staining, brain tissue was only rinsed repeatedly in PBS after fixation before further processing. If processed for cryosectioning, the fixed brain tissue was repeatedly rinsed in PBS, cryoprotected in 30% sucrose in PBS overnight, deep frozen in OCT and stored at ⫺80 °C until further analysis.

LacZ histochemistry Histochemical staining for ␤-galactosidase activity was performed on brain tissue blocks as well as cryosections (10 –100 ␮m thick) mounted on slides. For whole mount LacZ staining of adult brain tissue, brains were sectioned by hand either in the sagittal or coronal plane. In the sagittal plane, a mid-sagittal cut and an additional sagittal cut in parallel but approximately 1.5 mm more lateral to expose the lateral side of thalamus and midbrain (Paxinos et al., 2001), were made. In the coronal plane, a cut exposing the pretectal area and rostral part of the midbrain (at about Bregma ⫺2.5 mm) and an additional cut separating the cerebellum and brainstem from the midbrain (at about Bregma ⫺5.3 mm), were made. Brain tissue was then repeatedly rinsed in PBS before being washed three times 30 min. at room temperature in LacZ wash-buffer containing 5 mM EGTA, 0.01% deoxycholate, 0.02% NP-40, 2 mM MgCl2, 20 mM Tris buffer pH 8.0 and 100 mM PBS. Subsequently, the brain tissue was incubated overnight at 37 °C, protected from light in LacZ staining-buffer containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 and 1 mg/ml X-gal (Sigma) in addition to all wash-buffer ingredients. The tissue blocks were then washed in PBS, followed by three washes in PBS containing 3% DMSO, changed to 4% paraformaldehyde in PBS, pH 7.2, overnight and finally stored in PBS at 4 °C (Elefanty et al., 1999). Whole mount stained tissue was analysed using a Leica dissecting microscope. Some embryonic heads were embedded in paraffin, serial sectioned (7 ␮m), mounted and analysed by light microscopy (Leica). LacZ staining on cryosections was performed in a similar manner with slight modifications on the whole mount procedure described above. Cryosections (10 –100 ␮m thick) were prepared from brain tissue of all ages, cut in the sagittal and coronal plane and mounted on Superfrost Plus slides. Here, LacZ histochemistry included the use of 20 mM Tris buffer pH 7.3 and washing steps of 20 min each. After incubation in LacZ staining-buffer, the sections were washed in PBS, counterstained with Nuclear Fast Red, dehydrated and coverslipped. Tissue analysis was performed by light microscopy. As a negative control, adult brain tissue derived from wildtype C57B6/J mice (SCLw/w) was collected and processed as described, whereas during embryogenesis and at pnd0, SCLw/w and SCLlacZ/w littermates were compared. For the specific analysis of LacZ gene expression in adult brain that was used to compile the data represented in the schematic drawings (e.g. Fig. 4A), 10 ␮m cryosections were examined. For the photomicrographs presented in each of the Figs. 3–7 (e.g. Fig. 4B), 50 –100 ␮m cryosections stained for LacZ are presented. This is because the fine LacZ staining in thinner sections did not reproduce as well for photography compared with using the thicker sections.

Acetylcholinesterase (AChE) histochemistry Some sections (10 –100 ␮m) stained for ␤-galactosidase activity or sections adjacent to LacZ stained sections from SCLlacZ/w brain tissue, were stained for AChE activity. Mounted sections were washed in 0.1 M sodium maleate buffer, pH 6.0, followed by incubation for at least 2 h at RT in a staining solution containing 0.1 M sodium maleate buffer pH 6.0, 1.7 mM acetylthiocholine iodide (Sigma), 0.1 M sodium citrate, 30 mM CuSO4, 5 mM K3Fe(CN)6 and 10 mM Astra 1397 (reversible buterylcholinesterase inhibitor; Harvey and MacDonald, 1985). To stop the histo-

J. A. M. van Eekelen et al. / Neuroscience 122 (2003) 421– 436 chemical reaction, the sections were washes in distilled water, dehydrated, coverslipped and microscopically analysed.

Myelin/Nissl histochemistry Additional sets of adjacent sections from SCLlacZ/w brain tissue stained for ␤-galactosidase activity, were stained for myelin and Nissl (Bates et al., 2002). Briefly, mounted sections were immersed for at least 3 h at RT into a staining solution containing 0.2% Gold Chloride (hydrogen tetrachloroaurate; Sigma) and 20 mM PBS pH 7.2. The reaction was stopped in distilled water, the sections were fixed in sodium thiosulfate for 5 min, rinsed again in dH2O, Nissl-stained with Cresyl Violet acetate (Aldrich), dehydrated, coverslipped and microscopically analysed.

Tissue culture Neural cultures were prepared from both the forebrains and midbrains of a litter of five neonatal (pnd2) SCL-LacZ knockin mice. Genotyping subsequently showed that two of these mice possessed the SCLlacZ/w genotype. The cerebral cortices and midbrains were removed, placed in L-15 (Liebovitz) medium (JRH Biosciences, USA), divested of meninges and chopped into small pieces. Mixed glial cultures were then prepared from each region using methods described previously (Chen et al., 1991; Harvey et al., 1992). In brief, tissue was dissociated enzymatically with 0.1% trypsin (Sigma) and 0.001% DNAase (Calbiochem, USA) for 30 min at 37 °C. The tissue was washed (3 times) with medium containing DNAase and resuspended with DMEM (high glucose; Gibco, Invitrogen, USA) containing 10% foetal bovine serum (FBS; JRH Biosciences). The suspension was then triturated with a fire-polished Pasteur pipette and with needles of decreasing size until a cloudy suspension was obtained. Most of the dissociated cortical and mesencephalic cells were pipetted into separate uncoated 75 cm2 flasks in DMEM plus 10% FBS. After 3 days, unattached cells (mostly neurons) were discarded and the purified mixed glial cell population was passaged into poly-L-lysine-coated (Sigma) chamber slides; fresh medium was placed on all cells. To obtain cultures with increased neuronal survival, a proportion of the dissociated suspension from each region was plated directly onto poly-L-lysine coated chamber slides. After 3 more days, cells were fixed with 4% paraformaldehyde and lacZ staining was performed as described. Cells were then washed with PBS containing 10% normal goat serum and double stained for glial fibrillary acidic protein (GFAP; polyclonal; Dako, Denmark) and ␤III-tubulin (TUJ1 clone; BabCO, Richmond, CA, USA). Cells were visualised with appropriate secondary antibodies labelled with FITC (TUJ1) and Cy3 (GFAP; both ICN, Aurora, OH, USA).

NeuN and GFAP peroxidase immunohistochemistry Some 10 ␮m sections stained for ␤-galactosidase activity, were co-stained for either a specific neuronal (nuclear neuronal protein [NeuN]; Mullen et al., 1992) or glial cell GFAP marker. The procedure described under LacZ histochemistry for sections on slides was performed up to the stage where the LacZ-stained sections would otherwise be counterstained. Here, the sections were immediately further processed for NeuN or GFAP immunohistochemistry starting with a 1 min fixation in 4% paraformaldehyde in PBS, pH 7.2, followed by a 20 min immersion in 0.6% H2O2 in PBS and an overnight incubation at 4 °C with each primary antibody. Monoclonal mouse NeuN antibody (MAB377; Chemicon) and monoclonal mouse GFAP antibody (Sigma) were diluted 1:100 and 1:400 respectively in PBS containing 1% BSA and 0.2% Triton X-100 (Ab-diluent). The sections were then incubated for 2 h at RT with biotinylated anti-mouse second antibody (Vectastain) diluted 1:800 in Abdiluent and for 1 h at RT with an avidin– biotin–peroxidase complex (Vectastain Elite ABC kit) at a dilution of 1:200 in PBS.

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3,3-Aminobenzidine tertrahydrochloride (Pierce) served as chromogen. In between all incubation steps the sections were washed three times in PBS, pH 7.2. Finally, the sections were dehydrated and coverslipped.

In situ hybridisation The SCL cDNA subclone 860 –1428 bp in pGem3Zf(⫹) (Begley et al., 1991) was linearised with EcoR1 and BamH1 to generate antisense and sense RNA probes respectively. The RNA probes were synthesised using SP6 and T7 RNA polymerase and were non-radioactively labelled with digoxigenin (DIG; Roche). DNA template was degraded by DNase-I and the RNA probes were purified by application of G-50 RNA purification columns (Roche). In situ hybridisation was performed on frozen 15–30 ␮m sagittal and coronal cryosections from SCLw/w mice fixed in 4% paraformaldehyde in PBS, pH 7.2, for 10 min at RT just prior to in situ hybridisation using DIG-labelled RNA probes as described by Schaeren-Wiemers and Gerfin-Moser (1993) with minor modifications. Our procedure included acetylation for 10 min at RT with 1.2% triethanolamine (Sigma) and 0.25% acetic anhydride (Fluka) in DEPC-treated distilled water. Prehybridisation was performed for 4 h at RT with 50% formamide, 5X SSC, 5X Denhardt’s, 250 ␮g/ml baker’s yeast RNA (Sigma) and 500 ␮g/ml herring sperm DNA (Sigma) in DEPC-treated dH2O. The sections were then incubated overnight at 70 °C with denatured antisense or sense DIG-RNA probes at a concentration of 200 ng/ml diluted in prehybridisation solution. Immunologic detection of hybridised DIG-labelled probes was accomplished by incubation with an anti-DIG antibody conjugated with alkaline phosphatase (Roche) and the use of NBT and BCIP as chromogens as described in detail by Schaeren-Wiemers et al. (1993). Slides were mounted with aqueous mounting medium, coverslipped and microscopically analysed.

RESULTS LacZ staining reflecting SCL expression Expression of ␤-galactosidase activity (LacZ staining) under control of the SCL regulatory elements was examined in the adult and embryonic CNS. In adult brain sections, positive staining was evident from the posterior hypothalamus (PH) to the lower brainstem. LacZ staining was most pronounced in a series of mesencephalic regions, including the superior colliculus (SC). Here, LacZ staining was detected throughout all seven layers of this tectal region (Fig. 1A), which were delineated and defined by staining for AChE (Fig. 1C; Edwards et al., 1986a) and myelin/Nissl (not shown) on adjacent sections. The density of LacZ stained cells was highest in the dorsal zonal (SZ) and superficial grey layer (SGS) of the SC, less dense in the optic layer (SO) of the SC, whilst the deeper layers of the SC showed the fewest LacZ stained cells (Figs. 1A, 7B). In control mice (SCLw/w C57B6/J), brain sections were devoid of LacZ staining (Fig. 1B), except for occasional nonspecific blue staining in the choroid plexus in the lateral ventricles (Fig. 5A) and/or fourth ventricle (4V). To confirm that the pattern of LacZ expression accurately reflected the pattern of SCL gene expression, we performed in situ hybridisation using anti-sense SCL probes. Brain sections from SCLw/w mice were incubated with DIG-labelled RNA probes and although the intensity of hybridisation signal was not as strong as in situ hybridisation using radioactive probes (Mori et al., 1999), similar staining patterns were

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Cx IC SC

A

B

SZ

Cx

SGS

SZ

IC

SO SGI

SGS SAI

SGP

C

SAP

D

Fig. 1. SCL expression in the SC of the adult SCLlacZ/w mouse T. Photomicrographs present sagittal sections (rostral to the left) showing A: a high density of scattered LacZ stained neurons specifically in the dorsal layers of the SC (SZ, SGS and SO; 100⫻). B: The absence of LacZ staining in a midbrain section including the SC and IC of a SCLw/w control mouse (100⫻). C: AChE stained section showing the different layers of the laminated SC structure (100⫻). D: Alkaline phosphatase staining reflecting SCL mRNA in the SZ and SGS of a SCLw/w control mouse (200⫻; dotted line separates Cx from SZ). Nuclear Fast Red was used as a counterstain in A, B and D.

observed as compared with LacZ staining throughout the mesencephalon. Overlap in SCL and LacZ gene expres-

sion in the dorsal SZ and SGS layers of the SC is evident (Fig. 1A, D). In situ hybridisation with a DIG-labelled sense

Abbreviations used in the figures A9 –A10 AHi BIC Cb CG chp CIC Cx DCIC DLPAG DPO DT DTg Emi Fr IC IP IPR IS LR4V ME P PIL PF

dopaminergic nuclear complex amygdalohippocampal area nucleus brachium inferior colliculus cerebellum central grey choroid plexus cortex of the inferior colliculus cortex dorsal cortex of inferior colliculus dorsolateral periaqueductal grey dorsal periolivary region dorsal thalamus dorsal tegmental nucleus epimicrocellular nucleus fasciculus retroflexus inferior colliculus interpeduncular nucleus rostral interpeduncular nucleus isthmus lateral recess of fourth ventricle medulla pons posterior intralaminar thalamic nucleus parafascicular thalamic nucleus

PO PP PPT RMg Sag SAI SAP SCom SGI SGP SNC SNR SubB SubG VLPAG VZ WMS 3N

periolivary region peripeduncular nucleus posterior pretectal nucleus raphe magnus nucleus sagulum nucleus stratum album intermedium (intermediate white layer of superior colliculus) stratum album profundum (deep white layer of superior colliculus) subcommissural nucleus stratum griseum intermedium (intermediate grey layer of superior colliculus) stratum griseum profundum (deep grey layer of superior colliculus) compact part of substantia nigra reticular part of substantia nigra subbrachial nucleus subgeniculate nucleus ventrolateral periaqueductal grey ventricular zone whole mount stained oculomotor nucleus

J. A. M. van Eekelen et al. / Neuroscience 122 (2003) 421– 436

RNA probe as a negative control revealed no specific staining (not shown). Expression of SCL in neurons To characterise the cells which stained positively for LacZ, firstly, we co-stained cryosections for markers of either neurons or glial cells. Amongst immunoreactive neurons with distinct NeuN-stained cell nuclei, we found subsets of neurons in SCL expressing midbrain regions, which costained for LacZ (Fig. 2A). In contrast, GFAP immunopositive astrocytes scattered throughout SCL expressing regions did not co-stain for LacZ. Whilst positive staining for GFAP and LacZ was found in close proximity to each other, it did not coincide in the same cell bodies (Fig. 2B). Secondly, we analysed co-staining of LacZ and cell type specific markers in vitro in a series of CNS cultures. No LacZ expressing cells were seen in any of the cultures obtained from neonatal cerebral cortices (not shown). This is consistent with our in vivo analysis showing a lack of SCL expression in this region of the brain. We also saw no LacZ staining in the passaged mixed glial cell cultures derived from the midbrain. However, in the culture plated directly onto poly-L-lysine the occasional neuron immunopositive for ␤-III tubulin did express LacZ (Fig. 2C, D), whereas all glia and most neurons in these mesencephalic cultures did not stain for LacZ (Fig. 2E, F). Absence of LacZ staining in most neurons is consistent with our observation that in vivo only a subpopulation of midbrain neurons express SCL as well as the fact that these cultures were derived from a mixture of midbrain samples taken from SCLLacZ/w and SCLw/w pups. Expression of SCL in the adult CNS Detailed analysis of the expression pattern of LacZ in the CNS of the adult mouse was performed using microscopic analysis of parallel series of sections (10 ␮m) and stained for LacZ, AChE or myelin/Nissl. This multiple staining approach enabled us to orientate and distinguish important brain regions in the sagittal and coronal plane and facilitated comparison to the mouse brain atlas of Paxinos and Franklin (2001). This resulted in a precise description of the widespread localisation of LacZ expression throughout diencephalon, mesencephalon and metencephalon. The expression pattern of the LacZ is summarised in a combination of photomicrographs and schematic drawings, which correspond to the atlas of the mouse brain of Paxinos et al. (2001) (Figs. 3–7). In general, positive staining for LacZ was observed as blue foci in the cytoplasm of neuronal cell bodies (Fig. 2A). Within defined CNS regions, LacZ-stained neuronal cell bodies did not cluster together. Rather, they were scattered evenly over the different brain regions, indicating that only a subset of the neurons in each specific CNS region expressed LacZ. The staining intensity per neuron varied only marginally. Therefore, the staining pattern in specific CNS regions is described below as high density, moderate density or low density of scattered LacZ positive neurons in a specific region.

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Diencephalon. In the dorso-caudal part of the thalamus, the pretectal area contained several brain nuclei that expressed LacZ. A high density of scattered LacZ stained neurons was found in the precommissural nucleus (PrC; Fig. 3A, C), the anterior pretectal nucleus (APT; Fig. 4A, B), the olivary pretectal nucleus (OPT; Fig. 4A, B), the parafascicular thalamic nucleus, the retroparafascicular nucleus (RPF; Fig. 3C) and the subcommissural nucleus. A moderate density of scattered LacZ stained neurons was observed in the nucleus of the optic tract (OT; Fig. 4A, B) and in the posterior pretectal nucleus (Fig. 4A). More ventrally in the diencephalon, in rostro-caudal direction, a diagonal streak of LacZ positive neurons was observed in the PH (Fig. 3A, B). Here, a low density of LacZ stained neurons was detected in coronal sections from Bregma ⫺1.94 mm until Bregma ⫺2.46 mm. The most rostral tip of the streak (see arrow in Fig. 3B) was located just dorsolateral of the third ventricle and mid-lateral to the mamillothalamic tract. The most caudal end of the streak (see arrowheads in Fig. 3B) was found lateral to the ventral tip of the dorsal third ventricle. Along the lateral sides of the diencephalon, the density of LacZ stained neurons was high in the regions surrounding the medial geniculate nucleus (MGN) such as the posterior intralaminar thalamic nucleus, peripeduncular nucleus, subbrachial nucleus and nucleus brachium inferior colliculus (Fig. 5A, C and Fig. 6C, F), whereas the density of positively stained cells was only moderate in the dorso-lateral geniculate nucleus (DLG; Fig. 5E), the intergeniculate leaflet (IGL; Fig. 6B), the ventral lateral geniculate nucleus (VLG; Fig. 6B) and the subgeniculate nucleus. Mesencephalon/metencephalon. As described earlier, a high density of LacZ stained neurons was found in the tectum of the midbrain. Here, the two most dorsal layers of the SC, SZ and SGS (Edwards et al., 1986a), showed the highest density of LacZ positive neurons evenly scattered in rostro-caudal as well as midsagittal to a more lateral orientation. A high density of LacZ stained neurons was also found in the mid-caudal part of intermediate and deeper layers of the SC (Fig. 7A), which contrasted with a considerably less dense scattering of positive neurons in the lateral part of these deeper layers of the SC (Fig. 7B). A similar low density of LacZ stained neurons was also found in the (external and dorsal) cortex of the inferior colliculus (ECIC; Figs. 3A, 4A, C and 5A, C) and in the dorsolateral periaqueductal grey (Fig. 6E), whereas a moderate to high density of LacZ positive neurons was again detected in the ventrolateral periaqueductal grey (Fig. 6E) and more caudally in the cuneiform nucleus (CnF; Figs. 4A, C and 6G). Tegmental regions with a high density of scattered lacZ-stained neurons were the nucleus of Darkschewitsch (Dk; Fig. 3A, C), the laterodorsal tegmental nucleus (LDTg; Figs. 3A, C, E and 6H, I), the ventral tegmental nucleus (VTg; Fig. 3A), the caudal half (IPC; Fig. 3A, D) and the lateral (IPL) interpeduncular nucleus and the ventral nucleus of the lateral lemniscus (VLL; Fig. 5A, B). Other nuclei such as the dorsal raphe nucleus (DR; Fig. 3A), the substantia nigra (SN; Figs. 4A, 5A, B and 6D) and

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Fig. 2.

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the interstitial nucleus of Cajal (InC; Figs. 3C, 6C) and more caudally the sagulum nucleus (Fig. 5A) showed a moderate density of scattered LacZ-stained cells. A low density of scattered LacZ positive neurons was found in the median (MnR; Fig. 3D) and throughout the deep mesencephalic nucleus (DpMe; Figs. 4A, 7C). LacZ staining was rare in the parabigeminal nucleus (PBG; Fig. 6G), but a moderate density of scattered LacZ stained cells was found medial to the PBG as well as in the epimicrocellular nucleus immediately dorsal to the PBG and extending into the CnF and ECIC. In ventral metencephalon, a low density of stained neurons was found in the raphe magnus nucleus, the dorsal periolivary region (Fig. 4A), the rostral periolivary region (RPO; Figs. 4A, 7E) and in the superior paraolivary nucleus (SPO; Fig. 4A, D). Co-staining of adult brain sections for ␤-galactosidase and AChE activity revealed the presence of LacZ stained neurons in a variety of mesencephalic and metencephalic brain regions, which are known to stain intensely for AChE activity; e.g. the superficial layers of the SC, SN, IPC and IPL (Paxinos et al., 2001). LacZ staining did not, however, appear to be present in the medium to large AChE positive neurons found in many regions of the midbrain and hindbrain. For example, in the deep mesencephalic tegmentum, LacZstained cells were observed ventro-lateral to the magnocellular part of the red nucleus (RMC), but staining did not coincide with the medium to large AChE stained cells in that nucleus (Fig. 7C). Scattered LacZ-stained cells were observed in Dk, InC, DpMe and peri-aqueductal grey (PAG), but not in the large AChE-stained motorneurons of the adjacent oculomotor nucleus. Similarly, no LacZ staining was found in the facial nucleus (7N; Fig. 4D), nor in AChE stained neurons in locus coeruleus (LC), a nucleus that is close to a region (LDTg) containing a high density of scattered LacZ-stained cells (Fig. 7D). Finally, the medium to large AChE-stained neurons observed in RPO did not coincide with the LacZ expressing neurons in this region (Fig. 7E). Occasional LacZ-stained neurons were observed in the amygdalohippocampal area (Fig. 5D) in the ventrocaudal tip of the cortex. This was the only location outside the diencephalon, mesencephalon and metencephalon, where LacZ expressing neurons were found. No LacZ staining was detected in major brain structures such as the olfactory bulbs, basal ganglia, cerebral cortex, hippocampus and cerebellum. Expression of SCL in the developing CNS LacZ staining during early embryogenesis (E12.5, E14) was analysed in 7 ␮m thick serial sections derived from

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whole mount-stained embryos, which were paraffin embedded after LacZ staining. We considered the possibility that X-gal penetration would not be as effective with whole mount embryos at later ages and based analysis of LacZ expression at E16, E18 and pnd0 on LacZ staining of 30 ␮m cryosections. Here, the expression pattern of LacZ is summarised in a combination of photomicrographs and schematic drawings, which correspond to the atlas of the prenatal mouse brain by Schambra et al. (1992) (Fig. 8). E12.5. Distinct lacZ staining was detected in the intermediate zone (IZ) and marginal zone (MZ) of the tectum (T) and tegmentum (Tg; Fig. 8B, C). A welldefined spatially segregated subdivision of the neuroepithelium on the rostro-lateral sides of the aqueduct (AQ) stained positively for LacZ (Fig. 8C). This bilaterally symmetric subdivision of LacZ positive cells clearly outlined the border between the ventricular (proliferating) zone and the IZ of the T and Tg. Distinct LacZ staining was also observed in the region of the developing posterior commissure (PC; Fig. 8A, B), where most intense staining was found in the rostro-dorsal part of this fibre structure. In the sagittal plane, a wide band of LacZ stained cells in pretectum (PT; Fig. 8A, B) was evident. Furthermore, scattered LacZ staining was found in the developing A9 –10 dopaminergic nuclear complex, which gives rise to the SN and the VTg (Fig. 8B). Rostroventral to PT, LacZ staining was detected in the zona limitans intrathalamica (ZLI; Fig. 8B), which marks the boundary between the dorsal and ventral thalamus. In the mid- and hindbrain region of the developing CNS, a considerable number of positive LacZ stained cells were detected in the isthmus (Fig. 8B), which defines the border between the mesencephalon and metencephalon, and some positive LacZ-stained cells were found in the ventricular zone of the pons (Fig. 8B). A few patches of positive LacZ-stained cells were also found in the ventricular zone of the medulla (Fig. 8B). Although positive lacZ cells were observed in the ventricular zone of the 4V, they could not be detected in the 7–10 cell layers closest to the ventricle, which are possibly the proliferating cell layers of the neuroepithelium lining the 4V. E14. The expression pattern of SCL at E14 was very similar to the pattern found at E12.5, including the bilaterally symmetric subdivision of LacZ positive cells. A clear difference noted was intense staining in dorsal T at E14, a time when development of the upper regions of the SC is beginning (Fig. 8D–F; Edwards et al., 1986a). In addition, LacZ staining was identified in DR from this

Fig. 2. Characterisation of the SCL expressing cell type. A: Co-staining of LacZ with neuronal nuclear marker NeuN in neurons of the SGS in the tectal SC. Blue foci reflect ␤-galactosidase activity in the cytoplasm of neurons (arrowhead), which show brown NeuN immunostaining in the nuclear compartment (arrow; 10 ␮m cryosection, 1000⫻). B: Blue LacZ staining (arrowhead) was not found in GFAP stained astrocytes (arrow) in the PAG of the midbrain (10 ␮m cryosection, 1000⫻). C–F: Neonatal cell cultures from SCL-LacZ knockin mice stained for LacZ histochemistry and immunohistochemistry. Forty percent of the mice from this litter possessed a SCLlacZ/w genotype. The only cells found to express lacZ (blue spot in cell) were a small number of neurons derived from the mesencephalon (C, D). Note the neurites (arrows) that extend from the neuron in C. The vast majority of neurons that survived in the midbrain cultures were unlabelled for LacZ (F), as were the underlying glia (E). C, D and F immunostained for betaIII tubulin (green, FITC); E, same field as F, but with a different filter to reveal astrocytes immunostained for GFAP (red, Cy3). Scale in F for C–F⫽25 ␮m.

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Fig. 3. SCL expression pattern in adult SCLlacZ/w CNS, illustrated in the sagittal plane. A: Photomicrograph of WMS-brain cut at approximately 0.36 mm lateral to the mid-sagittal level. Blue LacZ staining is mostly confined to a series of midbrain and hindbrain regions. B: LacZ staining in diencephalic PH; the most rostral tip of the streak of blue staining is found around Bregma ⫺1.94 mm (arrow) and widens toward Bregma ⫺2.46 mm (arrowheads; 100⫻). C: LacZ staining in pretectal PrC and tegmental RPF, Dk, InC and LDTg (50⫻). D: LacZ staining in IP; note that the blue neurons are confined to the caudal part of IP (IPC). Although less densely scattered, blue neurons are also found all over the MnR region (100⫻). E: Higher power magnification of densely scattered LacZ staining in LDTg region closely bordering the 4V (100⫻). B–E represent relatively thick 60 ␮m cryosections stained for lacZ and AchE for illustrative purposes. In all views rostral is to the left.

age onwards. Occasional staining in the VZ of the rostral AQ and medial habenula, epithalamus and Rathke’s pouch (which will develop into pineal gland and pituitary respectively) was determined to be non-specific LacZ staining after observing similar staining in SCLw/w negative control embryos.

E16. Tectal LacZ staining was intense and clearly more confined to the most dorsal regions of the developing SC and IC (Fig. 8G, H). At this time in tectal development, retinal axons have started to invade the most superficial regions of the SC (Edwards et al., 1986b). Overall, however, the same range of pretectal, mesencephalic and

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Fig. 4. SCL expression pattern in adult SCLlacZ/w CNS, illustrated in the sagittal plane. A: Schematic presentation summarising the SCL expression pattern at approximately 0.96 mm lateral to the mid-sagittal level. The deep blue–light blue– grey colour gradient reflects a high, moderate or low density of scattered LacZ stained neurons in specific brain nuclei, respectively. B: High power photomicrograph of the pretectal region around the indicated lateral level from mid-sagittal, showing LacZ staining in APT, OPT and OT (100⫻). C: LacZ staining in tectal SC, IC and CnF. Note densely scattered LacZ stained neurons in specifically the dorsal layers of SC. The majority of LacZ stained cells in IC are found in its dorsal and external cortex (50⫻). D: LacZ stained cells are observed in the metencephalic SPO (arrows), but not in 7N (200⫻). B–D represent relatively thick 60 ␮m cryosections stained for lacZ and AchE (B, D) or 100 ␮m cryosection stained for LacZ without counterstain (C) for illustrative purposes. In all views rostral is to the left.

metencephalic regions showed SCL expression except for the thalamic ZLI (Fig. 8G–I). E18 and pnd0. From E18 onwards, defined LacZ staining in specific CNS regions such as thalamic DLG, pretectal nuclei, tegmental nuclei and metencephalic olivary nuclei could be distinguished (not shown). As the spatially restricted CNS regions in thalamus, midbrain and hindbrain became more apparent, the pattern of SCL expression more closely resembled the adult expression pattern of SCL. Like in the adult brain, no LacZ staining was found in any ventricular (proliferating) zone of the ventricular system in the CNS at these stages of brain development.

DISCUSSION Detailed histochemical analysis in this study demonstrated a much more widespread but specific expression of SCL in the embryonic and adult CNS than previously reported. In the rostro-caudal direction, SCL expression in discrete brain regions extended from the thalamus to the hindbrain and was almost completely absent in the forebrain. During the embry-

onic period of neuronal generation (E11–E16; Bayer and Altman, 1995), SCL expression was found in parts of the IZ of the mesencephalic neuroepithelium around the AQ and the ventricular zone of the 4V. SCL expression was not detected in neural stem cell generating and proliferating regions of the embryonic or adult brain. In mixed cell cultures from neonatal mice, we have shown that SCL expression was restricted to ␤-III tubulin immunopositive neurons. We have also shown that SCL expression in adult brain sections coincided with the specific neuronal marker NeuN (Mullen et al., 1992), indicating that SCL continued to be expressed in post-mitotic neurons in adulthood. SCL was expressed in post-mitotic migrating cells as well in the MZ of developing T and Tg, and in post-mitotic neurons in spatially restricted developing mesencephalic and metencephalic regions, likely to precede the specific pattern of SCL expression in the mature CNS. On the other hand, our data showing SCL expression in the intermediate and ventricular zone of the AQ and 4V respectively, support the idea that during early embryogenesis SCL was also expressed in zones of the neuroepithelium containing neural cells which have not yet differentiated into specific

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Fig. 5. SCL expression pattern in adult SCLlacZ/w CNS, illustrated in the sagittal plane. A: Photomicrograph of WMS-brain cut at approximately 1.68 mm lateral to the mid-sagittal level. B: Photomicrograph highlighting LacZ staining in the reticular part of SN, VLL and RPO (50⫻). C: LacZ staining observed more dorsally in the region surrounding the MGV at approximately 1.92 mm lateral to the mid-sagittal level (50⫻). D: At the same lateral level as in C, LacZ staining is observed in AHi (50⫻). E: LacZ staining shown in DLG at approx. 2.28 mm lateral to the mid-sagittal level (50⫻). B–E represent relatively thick 100 ␮m cryosections stained for lacZ with no counterstain for illustrative purposes. In all views rostral is to the left.

types of mesencephalic and metencephalic neurons. This interpretation of the data suggests a role for SCL in late neuronal differentiation in addition to a role for SCL in main-

tenance of mature neurons as described for SCL function in lineage determination in the haematopoietic system (reviewed by Begley and Green, 1999).

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Fig. 6. SCL expression pattern in adult SCLlacZ/w CNS, illustrated in the coronal plane. From rostral to caudal, A: photomicrograph of WMS-brain cut at approximately Bregma ⫺2.46 mm. B: High power photomicrograph taken around the same level as in A, highlighting LacZ staining in IGL and VLG (100⫻). C: schematic presentation summarising the SCL expression pattern at approximately Bregma ⫺2.92 mm. The deep blue–light blue– grey colour gradient reflects a high, moderate or low density of scattered LacZ stained neurons in specific brain nuclei, respectively. D: LacZ staining observed in both the compact (SNC) and the reticular part (SNR) of the SN around the same level as in C (100⫻). E: LacZ staining found in the immediate region around the AQ around Bregma ⫺3.40 mm (100⫻). F: LacZ staining surrounding the MGN, whereas the MGN itself seemed devoid of blue cells (100⫻; Bregma ⫺3.40 mm). G: LacZ staining found immediately medial and dorsal to PBG, extending into CnF and ECIC (100⫻; Bregma ⫺4.48 mm). H: Photomicrograph of WMS-brain cut at approximately Bregma ⫺5.34 mm. I: LacZ staining observed at the same level as in H in the region immediately ventral to 4V (100⫻). B, D–G and I represent relatively thick 60 ␮m cryosections stained for lacZ and AchE for illustrative purposes.

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Fig. 7. Photomicrographs show a combination of LacZ staining and AChE staining in coronal sections of the mesencephalon and metencephalon. A: LacZ staining in the dorsal SC. Only in the deeper layers of the mid-caudal part of SC, a high density of LacZ stained neurons was observed in (200⫻; 10 ␮m). B: In contrast to A, more (rostro)-lateral in the SC, a high density of scattered LacZ-stained neurons was confined to the three dorsal layers of SC (200⫻; 10 ␮m). C: LacZ staining observed in DpMe around RMC at Bregma ⫺3.80 mm (100⫻; 60 ␮m). D: As in RMC (C), relatively large AChE positive neurons in LC are devoid of lacZ staining (100⫻; 60 ␮m). E: LacZ staining in the RPO of the ventral hindbrain. Note that the blue foci do not co-localise with medium to large AChE-stained neurons (arrow; 200⫻; 10 ␮m).

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Fig. 8. The expression pattern of SCL in embryonic SCLlacZ/w CNS. Schematic presentations and photomicrographs summarise the SCL expression pattern at three different embryonic ages in the sagittal plane just lateral to a mid-sagittal cut (A, B, D, E, G–I), or horizontal plane. (C, F) A: LacZ staining found in the rostro-dorsal part of PC and in the pretectal region of CNS at E12.5 (200⫻, 7 ␮m). B: schematic presentation displaying LacZ staining at E12.5. The deep blue–light blue– grey colour gradient reflects a high, moderate or low density of scattered LacZ stained neurons in specific brain regions, respectively. C: LacZ staining is detected in a bilaterally symmetric subdivision of the IZ and the MZ of Tg at E12.5 (200⫻; 7 ␮m; level indicated by line in B). D: LacZ staining in the dorsal SC at E14 (200⫻; 7 ␮m). E: schematic presentation displaying LacZ staining at E14 as in B. F: Dorsal view of LacZ staining in upper SC at E14 (50⫻; 7 ␮m; level indicated by line in E). G: LacZ staining in the dorsal SC at E16 (100⫻; 30 ␮m). H: Schematic presentation displaying LacZ staining at E16 as in B. I: LacZ staining in caudal Tg at E16 (200⫻; 30 ␮m). All photomicrographs show Nuclear Fast Red as counterstain. The anterior–posterior orientation of the heads equals the left–right orientation in the sagittal sections and up– down orientation in the horizontal sections.

The pattern of SCL expression was based on the expression pattern of the reporter gene LacZ under transcriptional control of the SCL regulatory sequences and coincided with earlier reports based on in situ hybridisation to detect SCL mRNA (Green et al., 1992b; Mori et al., 1999). The results in this study are also consistent with previous reports examining LacZ expression in knockin and transgenic models (Elefanty et al., 1999; Sinclair et al., 1999). However, those studies did not define SCL expression with the precision described here. In one study focussed on regulatory sequences in the SCL gene, the significance of enhancer elements in the 5⬘ region of the SCL gene was assessed in transgenic mice, in which LacZ expression was under direct control of these isolated SCL enhancer elements. A specific element, the 5⬘ SCL neural promoter, stretching from 0.9 kb upstream of the SCL coding region to exon 3, appeared responsible for directing SCL expression to midbrain, hindbrain and spinal cord as shown in whole mount-stained E12.5 embryos (Sinclair et al., 1999).

The involvement of various classes of transcription factors in the process of neuronal differentiation is well established. Amongst them, bHLH factors have been demonstrated to play an essential role in neuronal determination and differentiation during embryogenesis (Anderson, 1997; Kageyama et al., 1997; Lee, 1997; Guillemot, 1999). Tissue-specific determination of cell fate by bHLH factors is generally achieved through dimerisation and DNA binding to activate downstream genes (Murre et al., 1994) and activation cascades of bHLH factors to control neuronal development have been demonstrated (Anderson, 1997). Comparison between the spatial pattern of expression of SCL and other bHLH factors showed a high degree of overlap between SCL and tal-2 (Mori et al., 1999). Both bHLH factors are associated with T cell acute lymphoblastic leukaemia upon activation by chromosomal translocations (Baer 1993; Begley and Green, 1999). Under normal conditions, tal-2 is also expressed in the developing mouse brain and null mutations of tal-2 result in distinct dysgenesis of the midbrain T (Bucher et al., 2000). Although

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embryonic expression of tal-2 is slightly more extended than SCL, it was very similarly restricted to the developing dorsal diencephalon, mesencephalon and metencephalon, including the IZ of the AQ (Mori et al., 1999). Another bHLH factor of interest is the mammalian achaete-scute homolog one (Mash1), which may itself be regulated by SCL (M. A. Hall and C. G. Begley, unpublished observation). Expression of Mash1 in region restricted and well-defined zones in the rat mesencephalon, metencephalon and myelencephalon appears to parallel the expression of SCL in the developing mouse brain at E12.5. Overall, however, expression was much more widespread than SCL, extending from the telencephalon to the spinal cord (Lo et al., 1991). Interestingly, no overlap in expression was found between SCL and neuronal bHLH factors NSCL-1 and 2, originally cloned as a result of their high sequence homology within the bHLH domain of SCL (Begley et al., 1992). Involvement of SCL and related transcription factors in brain development requires their expression during the period of neuronal generation (E10 –E16 in rodent) after neurulation has been achieved. Similar to SCL, Mash1 expression in the developing brain appeared at E10.5 when neurogenesis begins (Lo et al., 1991; Sinclair et al., 1999; Bayer and Altman, 1995). Temporal differences in expression of transcription factors, but more likely cell differentiation stagespecific differences in expression could dictate their specific role in neuronal generation (Lee, 1997). In principle, during neurogenesis neuronal precursors proliferate in the ventricular zone and migrate through the IZ to the MZ, where the cells terminally differentiate (Bayer and Altman, 1995). We have shown SCL expression in the intermediate and MZ of the mesencephalic neuroepithelium. SCL was also expressed in the ventricular zone of the 4V, where an IZ is not defined, but where SCL was consistently absent in the first seven to 10 cell layers lining this brain vesicle. Likewise, neurogenin 1 has been observed in the IZ and MZ (Ma et al., 1997) and NeuroD genes were found expressed in migrating and/or differentiating cells as well as terminally differentiated neurons (McCormick et al., 1996). Mash1 and tal-2, were also expressed in the ventricular zone of neuroepithelium (Lo et al., 1991; Ma et al., 1997; Mori et al., 1999). Moreover, Mash1 was only transiently expressed, preceding the expression of terminal differentiation markers (Lo et al., 1991). Taken together, our data therefore clearly imply a previously unrecognised role for SCL in neuronal development. However, whether SCL will be regarded as a neuronal determination factor, similar to neurogenin (Sommer et al., 1996) and Mash1 (Guillemot et al., 1993) involved in early neuro-developmental events, or as a neuronal differentiation factor, such as NeuroD (Lee, 1997) involved in terminal steps of neurogenesis, remains undetermined. Our findings of persistent expression of SCL during late embryogenesis into postnatal life and distinct expression of SCL in border regions such as the ZLI in the dorsal diencephalon and the isthmus also argue for a potential role of SCL in regionalisation of the developing brain. In this context, several parallels can be drawn between SCL and the paired-box containing family of transcription factors (Pax-genes). Similar to SCL, Pax-genes in general are expressed throughout

brain development in restricted regions and in adulthood in subsets of post-mitotic cells. This suggests a role in commitment of precursor cells to different neuronal cell fates as well as in maintenance of specific brain cell subtypes (Stoykova and Gruss, 1994; Kawakami et al., 1997). Expression of specific members of the Pax-gene family has been demonstrated in morphological boundaries underlying brain segmentation, such as Pax6 in the PC marking the boundary between forebrain and midbrain (Schwarz et al., 1999), Pax2 and 5 in the isthmus marking the junction between mid and hindbrain (Schwarz et al., 1997, 1999) and Pax7 in embryonic T preceding development of the laminae of the SC (Kawakami et al., 1997; Matsunaga et al., 2001). These Pax-gene expression domains are considered organising regions for brain structure development. We have shown that these regions also express SCL in the same time frame of brain development, raising the possibility that SCL might also function as an organiser protein. Previous studies on the regulation of SCL have shown regulation of the SCL gene by the zinc-finger transcription factors of the GATA family: GATA2 in the haematopoietic system (Go¨ttgens et al., 2002) and most likely GATA3 in neural tissue (Sinclair et al., 1999). Co-expression of GATA3 and SCL in a subpopulation of GATA3 expressing cells of the ventral spinal cord during embryogenesis supports this finding (Smith et al., 2002). In addition, our results indicate a considerable overlap in expression patterns of SCL and GATA3 in the mesencephalon and metencephalon during development as well as in the adult brain (Oosterwegel et al., 1992; van Doorninck et al., 1999). Alternatively, these observations might suggest the possibility that SCL and GATA3 are co-localised in neural tissue and exert their function on downstream genes through formation of a multi-protein complex. Such an interaction of transcription factors to compose a multiprotein complex, including SCL and GATA1 amongst others, is found in erythroid cells to regulate gene expression (Valge-Archer et al., 1994; Wadman et al., 1997). The widespread but specific distribution of SCL in the adult CNS is striking. Until the present study, adult expression of SCL was only reported in midbrain T and in ventral hindbrain, expression domains visible from the outside of complete whole mount-stained mouse brains (Elefanty et al., 1999). Our analysis of sectioned brain tissue goes beyond that study to describe a detailed map of SCL throughout the CNS in sagittal and coronal orientation. Our findings imply that SCL in the adult brain potentially plays a role in different functional circuits, based on known functions of CNS regions that contain SCL expressing neurons. Firstly, SCL may function in the processing of visual information based on a relatively dense subpopulation of SCL expressing neurons in the dorsal ‘visual’ layers of the SC, specifically in the SGS layer of SC known to be targeted by retinal axons, and in the thalamic dorsal and ventral LGN and IGL (Sefton et al., 2003). Furthermore, associated functions of this circuit, such as pupillary light reflex and coordination of motor function related to vision have been shown to involve a series of mesencephalic regions, which include several SCL expressing pre-tectal nuclei, PAG, DK

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and the caudal reticular part of SN (Klooster and Vrensen, 1998; Onodera and Hicks, 1998). Secondly, involvement of SCL in the processing of auditory information is suggested by its expression in IC, VNLL and nuclei of the olivary complex in the ventral medulla oblongata (Webster, 1995). Thirdly, SCL may play a role in the mediation of sensation of pain. This last assumption is partly based on SCL expression in neurons of brain regions known to take part in the pain circuit, such as PAG and DR (Willis et al., 1995). Furthermore, there is some overlap between the expression pattern of SCL and the neuropeptide substance P (SP), which has been extensively reported to contribute to pain perception (Halliday et al., 1995; Willis et al., 1995; Mogil et al., 2000; Commons and Valentino, 2002). Interestingly, expression of preprotachykinin (the precursor for SP) in neurons of the rat dorsal visual layers of the SC and the dorsal cortex of the IC parallels to some extent the expression of SCL in neurons in the equivalent mouse brain regions, although these tectal areas are not considered part of the pain circuit. In particular, SCL expression in neurons of the deeper layers of the medial SC, which project to the SCL expressing CN, resembled SP expression (Wynne et al., 1995; Harvey et al., 2001). Based on the pattern of expression of SCL throughout embryogenesis and in adulthood, different roles for SCL are likely within the brain. The question remains how SCL contributes to the function of specific post-mitotic neurons in the adult brain and whether this mechanism of action differs significantly from the way SCL exerts its role in neuronal development. It is accepted that the function of SCL relies on formation of multi-protein complexes. In each specific cell type, heterodimerisation partners of SCL and therefore downstream target genes may vary (Begley and Green, 1999). Moreover, DNA binding via the transactivation domain of SCL is not always required for function. In leukaemogenesis and during early haematopoiesis, DNA binding of SCL is not essential (Aplan et al., 1997; Porcher et al., 1999; O’Neil et al., 2001). Here, SCL may sequester other (DNA binding) proteins, whereas maintenance of mature erythrocytes is dependent on DNA binding of SCL (Aplan et al., 1992). In CNS, temporal and spatial differences in the cellular phenotype of SCL expressing progenitors and/or neurons may underlie different functions of SCL. Further co-expression analysis as well as SCL deletion studies, for example in conditional transgenic knock out mice, will provide new means to investigate the role of SCL in neural development and in differentiated neural cells. Acknowledgements—We thank K. Becher for the PCR genotype analysis of the transgenic mice, M. Pollett for the tissue culture preparations, N. Symons for technical assistance and K. Smith for critical reading of the manuscript. The work was supported by the Australian National Health and Medical Research Council.

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(Accepted 21 July 2003)