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Keywords:

  • muscarinic receptor;
  • neural stem cells;
  • neuronal progenitor cells

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Development of the nervous system is accompanied by expansion and differentiation of the neuronal progenitors within the embryonic neuroepithelium. Although the role of growth factors in this process is well documented, there is increasing evidence for a role of neurotransmitters. Acetylcholine is known to exert many actions on developing neural cells, but its potential role in neurogenesis is unclear. Here, we show that the M1 muscarinic acetylcholine receptor is expressed in the neuroepithelium of the rat forebrain, where it is found on both nestin+ progenitor cells and TuJ1+ newly differentiated neurons. Furthermore, transcription is governed, at least in part, by regulatory cis elements that are also responsible for driving transcription in neuroblastoma cells. This represents the first demonstration of M1 receptors on neuronal progenitor cells and supports the notion that M1 muscarinic receptors may play a role in development of the nervous system prior to the onset of synaptogenesis and their subsequent role in neurotransmission.

Abbreviations used
ChAT

choline acetyltransferase

CP

cortical plate

DMEM

Dulbecco's modified Eagle's medium

IZ

intermediate zone

NGS

normal goat serum

PBS

phosphate-buffered saline

SVZ

subventricular zone

VZ

ventriclular zone

Early in development, the neuroepithelium of the central nervous system contains both neural stem cells and multipotential progenitor cells (Hockfield and McKay 1985; Davis and Temple 1994; Reid et al. 1995; Williams 1995; Williams and Price 1995; Alvarez-Buylla et al. 1998). Expansion and differentiation of these progenitors ultimately gives rise to the plethora of differentiated phenotypes that constitute the adult nervous system (Davis and Temple 1994; Kilpatrick and Bartlett 1995; Reid et al. 1995; Williams and Price 1995; Johe et al. 1996; Qian et al. 1997; Luskin 1998; He et al. 2001). It is this potential of neural stem cells and multipotential progenitor cells for repair of neurodegenerative damage that has stimulated a deal of interest in elucidating how individual phenotypes are established (Gaiano and Fishell 1998; Fricker et al. 1999; Wagner et al. 1999; Rubio et al. 2000; Toda et al. 2000; Philips et al. 2001; Price and Williams 2001; Veizovic et al. 2001). Although the specific molecular pathways that drive differentiation of neuronal stem cells and neuronal progenitors remain unclear, it is widely believed that these programmes of proliferation and differentiation require the interaction of extracellular and intrinsic signals (Edlund and Jessell 1999). While there is abundant evidence that points to the role of growth factors (Temple and Qian 1995; Gross et al. 1996; Johe et al. 1996; Qian et al. 1997; Williams et al. 1997; Park et al. 1999) in controlling neural differentiation, there is also increasingly persuasive evidence for a role for neurotransmitters. Many neurotransmitters are present in the brain prior to axonogenesis and synaptogenesis, raising the possibility that they may mediate non-classical signalling (Cameron et al. 1998; Nguyen et al. 2001). One transmitter that has been implicated in such signalling is acetylcholine, and studies have shown that activation of cholinergic muscarinic receptors can lead to DNA synthesis in astrocytes (Guizzetti et al. 1996) and oligodendrocytes (Cohen et al. 1996) and to an increase in proliferation of cortical neural progenitor cells (Ma et al. 2000; Li et al. 2001). Despite our extensive knowledge of the distribution of muscarinic receptor mRNA and protein in the adult murine brain (Buckley et al. 1988; Vilaro et al. 1990; Levey et al. 1995), we know very little of their expression in embryonic brain. Likewise, we have limited knowledge of the promoters and regulatory sequences responsible for driving cell-specific expression. Although, the promoters of the murine M1 and M4 genes have been characterised and some of the regulatory elements required for expression in cell lines identified (Wood et al. 1995; Mieda et al. 1996; Wood et al. 1996; Mieda et al. 1997; Wood et al. 1999; Garriga-Canut et al. 2001), we do not know if these same regulatory domains are responsible for driving expression in either embryonic or mature neuronal cells. This is important as there are numerous examples of discrete regulatory regions and elements that are used to direct neuronal transgene expression to different tissues (Vidal et al. 1990) or different regions of the nervous system (Hoyle et al. 1994; Severynse et al. 1995), or to control temporal changes in developmental expression (Desai et al. 2002). Against this background, we undertook to examine the expression of the M1 gene in the neocortical neuroepithelium, both in vivo and in culture, and to identify regulatory elements of the M1 gene that were responsible for driving expression in neuroepithelial cells. We report that the M1 gene is expressed extensively throughout the neuroepithelium of the E14 developing forebrain and that the M1 receptor is found on TuJ1-positive neurons and on nestin-positive progenitor cells, raising the possibility that acetylcholine could influence neuroepithelial development through activation of M1 muscarinic receptors. Furthermore, we have used reporter gene analysis to show that a region of the first exon of the M1 gene acts as an enhancer of M1 transcription in both neuroepithelial cells and neuroblastoma cell lines. We conclude that M1 receptors are expressed on neural progenitors and that transcription of the M1 gene in both neural progenitors and neuroblastoma cells is controlled by common cis regulatory elements.

Frozen sections

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

E14 and E17 embryonic brains were dissected out and fixed overnight in 4% paraformaldehyde in 0.1 m phosphate buffer at 4°C. They were then cryoprotected in 20% DEPC (Diethyl Pyrocarbonate, Sigma, St Louis, MO, USA) -sucrose in phosphate-buffered saline (PBS), embedded in optimum cutting temperature (OCT) embedding compound (Histequip Warwickshire, Uk) in moulds and snap frozen in liquid nitrogen. Frozen blocks were then stored at −80°C until required. Brains were sectioned coronally on a Bright cryostat at 16–20 μm. The sections were then collected on Superfrost Plus microscope slides (Merck), air-dried and stored at −80°C until required.

In situ hybridisation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Tissue sections from E14 and E17 rats were prepared as described above. In situ hybridisations using digoxygenin (DIG)-labelled riboprobes were performed on 16-mm frozen sections collected onto Superfrost Plus slides (Merck). Anti-sense and sense DIG-labelled riboprobes probes were synthesised from a PCR template amplified from an adult rat brain cDNA library. Linkers containing T7 and SP6 promotor sequences were directly ligated to the PCR products (261 bp: 980–1240 on GenBank M16404) by using Lig'nScrib (Ambion, Austin, TX, USA). Digoxigenin (DIG)-labelled M1 antisense and sense RNA probes were prepared by MAXIscript™in vitro transcription system (Ambion) using T7 and SP6 RNA polymerases, respectively.

All hybridisations were carried out at 65°C. Slides were mounted in Faramount aqueous mounting medium (Dako, Carpinteria, CA, USA) and photographed using a Nikon Eclipse microscope and processed with Lucia G and Adobe Photoshop imaging software.

Cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

The cerebral cortices of embryonic day 14 (E14) Sprague–Dawley rats were dissected in serum-free medium [Dulbecco's modified Eagle's medium (DMEM), Gibco Life Technologies, Rockville, MD, USA] supplemented with glucose, transferring, insulin, selenium, progesterone, thyroxine, tri-iodothyronine, putrescine and bovine serum albumin (Williams et al. 1997; Park et al. 1999) and dissociated into single-cell suspension by trituration through 21- and 23-gauge hypodermic needles. The cells were centrifuged and re-suspended in serum-free medium to give a final concentration of 4 × 106 cells per ml. Cells were then plated onto poly d-lysine (Sigma)-coated 13-mm coverslips, in 50 mL drops (2 × 105 cells). After leaving the cells to attach to the coverslips for 1 hour, an additional 0.5 mL of serum-free medium was added to each well. Half of the medium in each well was replaced with fresh medium every 2 days.

Immunohistochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

To determine whether M1 is expressed by progenitor cells, neurons or both cell types, coverslips were fixed in 4% paraformaldehyde in 0.1 m PBS on the day following plating and analysed by immunofluoresence. A rabbit polyclonal anti-M1 (1 : 200, Alamone Laboratories, Jerusalem, Israel; AMR-001) was used to identify M1 expressing cells. Neurons were identified using the anti-TuJ1 antibody (1 : 5000, Cambridge Bioscience, Cambridge, UK) that recognises a neuronal specific form of bIII tubulin and progenitor cells were identified using the anti-nestin antibody (Rat-401, hybridoma supernatant, 1 : 4, supernat, Developmental Studies Hybridoma Bank, Iowa). After fixation, cultures were incubated overnight at 4°C with the anti-M1 antibody and this antibody was visualized using a goat anti-rabbit Ig antibody coupled to fluorescein (GaRIg-Fl, 1 : 100, Southern Biotechnology, Birmingham, AL). Cells were then permeabilised by incubation for 5 min at room temperature (22°C) in 0.1% triton X-100 (Sigma) in PBS. Coverslips were then simultaneously incubated with the anti-Tuj1 and anti-nestin antibodies. TuJ1 staining was visualised using a goat anti-mouse immunoglobulin G2a coupled to Rhodamine (GαMIgG2a-Rh, 1 : 100, Southern Biotechnology) and nestin staining was visualised using a goat anti-mouse immunoglobulin G1 coupled to Biotin (GαM IgG1-Biotin, 1 : 100, Southern Biotechnology). The secondary antibodies coupled to biotin were then visualised by incubation with streptavidin-Alexa 350 (1 : 100, Molecular Probes, Eugene, OR, USA). Except where stated, all primary and secondary antibody incubations were carried out for 30 min at room temperature and the coverslips were washed three times in PBS after each incubation. All antibody dilutions were carried out in 10% normal goat serum (NGS, Sigma) in PBS. After the final wash, coverslips were mounted in Prolong anti-fade mounting medium (Molecular Probes) and analysed using a Nikon Eclipse fluorescence microscope using a 40 × or 60 × objective.

Reporter gene assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Luciferase constructs used in this study have been described previously (Wood et al. 1999). Transfections were carried out using Effectene (Qiagen, Valencia, CA, USA) according to manufacturer's instructions. Neuroepithelial cells were plated into 24-well plates (see above) and each well transfected with 200 ng reporter gene DNA and 1 ng pRL-CMV (Promega, Madison, WI, USA), 9.6 μL enhancer and 12 μL Effectene. Cells were harvested into 100 μL Reporter Passive Lysis Buffer (Promega). Luciferase measurements were carried out using the Promega dual luciferase assay system (DLR™) according to manufacturer's instructions using a Mediators PhL 1.8 96-well luminometer. Firefly luminescence was normalised to Renilla luminescence and the results expressed relative to normalised expression of empty vector, either pGL3 Basic (Promega) or pGL3 Inr (Wood et al. 1999).

The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Our first step was to examine the expression of the M1 gene in embryonic day 14 (E14) rat brain using in situ hybridisation. This age was selected because at E14 the dorsal forebrain (cortex) is almost entirely composed of a ventricular or germinal zone. This zone lines the lateral ventricles and contains rapidly dividing multipotential neurepithelial cells, neuronal precursor cells (neuroblasts) and newly differentiated neurons (McConnell 1991; McConnell and Kaznowski 1991). It is also the age at which dissociated cultures of neuronal progenitor cells can be established and maintained as a monolayer culture that is readily tractable to in vitro analyses (Williams and Price 1995; Williams et al. 1997; Park et al. 1999). This allows direct comparison of in vivo observations with in vitro manipulations.

Our in situ hybridisation studies show that M1 transcripts are widely expressed throughout the telencephalic ventricular zone at E14 (Figs 1a, b and c). In order to more clearly distinguish whether newly differentiated neurons express the M1 gene, we compared this distribution at E14 with that seen in sections of forebrain derived from E17 embryos, a developmental age at which many neurons have migrated out of the ventriclular zone (VZ) to form the cortical plate (Bayer and Altman 1991; Bayer et al. 1991). At E17, M1-expressing cells can be seen dispersed throughout both the VZ and the cortical plate (Fig. 1d). Labelled cells can also be seen throughout the intermediate zone (Fig. 1f). These latter cells probably represent migrating neurons. These observations clearly show that the M1 gene is expressed in both ventricular zone cells and the differentiating neurons of the cortical plate.

image

Figure 1. Expression of the M1 gene in the neuroepithelium and cortical plate of E14 and E17 forebrain. In situ hybridisation analysis of M1 muscarinic receptor gene expression in coronal sections through E14 (a, b, c) and E17 (d, e, f) forebrain. Tissue sections were processed as described in Materials and methods. Hybridisation signal can be seen throughout the neuroepithelium at E14 and in the ventricular zone (VZ), intermediate zone (IZ) and cortical plate (CP) at E17. LV, lateral ventricle. Signal obtained using sense probe can be seen in (e). (a, b, d, e) Scale bars represent 500 μm; (c, f) Scale bars represent 100 μm.

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The M1 receptor is expressed on both neurons and neuroepithelial cells in culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Although in situ hybridisation studies on sections of embryonic brain allow us to conclude that the M1 gene is expressed throughout the neuroepithelium at both E14 and E17, they cannot resolve whether M1 expression is restricted to progenitor cells, differentiating neurons or to both. In order to address this issue, we prepared dissociated cell cultures from the developing cerebral cortex of E14 rat embryos. Our previous studies have shown that the majority of cells in these cultures at E14 are multipotential neuroepithelial cells that express the intermediate filament protein nestin, but do not express markers that distinguish differentiated cell-types (Williams 1995; Williams et al. 1997; Park et al. 1999). The remaining cells in these cultures are neurons characterized here by their expression of β-3 tubulin, a specific neuronal marker. Cells isolated from E14 cortex were plated on poly-D-lysine-coated coverslips in defined medium. After 24 h, cultures were triple stained with antibodies against Tojl nestin and M1. The M1 receptor was expressed by 27% of the total cells in the culture, including both TuJ1 immunopositive neurons and nestin immunopositive NE cells (Fig. 2). In accordance with previous observations (Williams et al. 1997; Park et al. 1999), around 20% of the cells expressed TuJ1, of which approximately half (10.7% of total cell population) also expressed the M1 gene. As a percentage of the total cell population, 5.2% of cells were (nestin/TuJ1+/M1+) and 5.5% of cells were (nestin+/TuJ1+/M1+). We define both of these cell populations as neurons as, in the rat, nestin expression is retained in young neurons although it is lost as these cells mature. Accordingly, some newly differentiated neurons transiently express both nestin and TuJ1. Of the total cell population, 15.4% were nestin+/TuJ1/M1+, indicating that a population of progenitor (or neurepithelial) cells express M1 receptors. Ninety-five per cent of all nestin-positive cells expressed the M1 gene while a small number of cells (0.5%) expressed the M1 gene but were neither TuJ1 nor nestin immunopositive (nestin/TuJ1/M1+).

image

Figure 2. Expression of the M1 gene in high-density dissociated cell cultures of E14 dorsal forebrain. Cell culture and immunostaining was carried out as described in Materials and methods. M1, Tuj1 and nestin immunoreactivity are shown as green (a, b), red (c, d) and blue (e, f), respectively. Both Tuj1 immunopositive/nestin immunonegative neurons (arrows) and nestin-immunopositive/Tuj1 immunonegative progenitors (arrow heads) show M1 immunoreactivity. Scale bars represent 10 μm.

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Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Next, we sought to identify those regulatory regions of the M1 gene that may be responsible for directing transcription in neuroepithelial cells. In previous studies, we have characterised the gene structure and promoter of the rat M1 gene and have identified regulatory domains within the first exon that are required for cell-specific expression of reporter gene constructs in IMR32 neuroblastoma cells (Pepitoni et al. 1997; Wood et al. 1999). We took this series of reporter gene constructs and transfected them into high-density cultures of dissociated E14 neuroepithelium. In accordance with these earlier studies, constructs containing the first exon plus as little as 372 bp of 5′ flanking sequence (pGL3-372/+420) were sufficient to drive reporter gene expression and further deletion of 5′ flanking sequence had little effect on reporter gene expression. However, deletion of a 436-bp fragment from within the first exon abolished expression. A more discrete deletion consisting of a 182-bp sequence containing two putative control regions, a polypyrimidine tract and a conserved region, also led to a greatly reduced reporter gene expression (Fig. 3a). These same deletions also abolished or severely attenuated reporter gene expression in neuroblastoma cell lines (Wood et al. 1999). We next used reporter gene constructs containing these two regions cloned upstream of initiator of the adenovirus major late promoter to see if they could behave as classical enhancers in neuroepithelial cells. Both constructs drove activated expression, but the construct containing only the putative regulatory regions was markedly more effective and drove 32-fold expression over empty vector (Fig. 3b). This is qualitatively similar to results obtained on cells lines, but the relative strength of the two enhancers is more marked in neuroepithelial cells (Wood et al. 1999). These data clearly indicate that common regulatory elements regulate M1 expression in both neural cell lines and neuroepithelial cells.

image

Figure 3. M1 reporter gene expression in high-density dissociated cell cultures of E14 dorsal forebrain. Reporter constructs have been described previously (Wood et al. 1999) and transfection protocol and luciferase measurements are described in Materials and methods. All data are normalised to Renilla luciferase expression driven by co-transfected pRL-CMV (Promega) and promoter activity is expressed as fold-over luciferase activity obtained with empty vector, pGL3 basic (a) or pGL3Inr (b). Data represent the mean of at least three independent experiments, each performed in triplicate, and the error bars represent the SEM.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References

Although the role of growth factors in regulating the generation and fate of neural progenitors has received a great deal of attention, the potential role of neurotransmitters in regulating these activities has only recently fallen under this spotlight (Cameron et al. 1998; Nguyen et al. 2001). This disparity is due, in part, to the relative death of knowledge concerning expression of neurotransmitter receptors on neural progenitor cells. In this study we have focused upon the expression of the M1 muscarinic acetylcholine receptor in cells derived from the forebrain neuroepitheliuim of the E14 rat. At this developmental age, the ventricular zone consists mainly of neural stem cells, multipotential progenitor cells, neuronal precursor cells and newly formed neurons (Bayer and Altman 1991; Bayer et al. 1991). Some neurons are also migrating out of the VZ towards the pial surface to form the cortical plate.

In situ hybridisation showed that, at E14, M1 expression was evident within cells of the ventricular zone, whilst at E17, expression could be seen in VZ cells as well as in the intermediate zone (IZ) and in the developing cortical plate. Although the IZ and developing cortical plate contains post-mitotic neurons, the VZ contains a mixture of dividing progenitor cells and newly differentiated neurons. Therefore, in order to determine whether M1 was expressed by both neurons and progenitor cells, we analysed the phenotype of M1 expressing cells in culture using the Tuj1 antibody to identify young neurons and an anti-nestin antibody to identify progenitor cells. These studies indicated that M1 is expressed on both nestin-positive/TuJ1-negative cells and on TuJ1-positive cells, clearly indicating that M1 is expressed on both progenitor cells and on neurons. As not all progenitor cells express the M1 receptor, it is possible that M1 expression is restricted to progenitor cells that are already committed to generating neurons that may or may not continue to express the M1 receptor.

Although usually associated with classical synaptic transmission, muscarinic receptor activation can also lead to a diverse array of responses in non-innervated developing tissue, including folding of embryonic retina (Yamashita and Fukuda 1993), elevation of Cainline image in retinal ventricular zone cells (Wong 1995), blockade of apoptosis in cerebellar granule cells (Yan et al. 1995), inhibition of neurite outgrowth in sympathetic neurones (Small et al. 1995) and stimulation or inhibition of mitosis (Conklin et al. 1988; Ashkenazi et al. 1989). However, there are few studies that have addressed the expression or function of muscarinic receptors in neuroepithelial cells. In an earlier study, Ma et al. (2000) reported muscarinic receptor gene expression in mitogen-expanded cultures derived from rat dorsal telencephalon where they detected M2, M3, M4 and M5, but not M1, transcripts. The cultures used in the present study are not mitogen expanded and represent a primary culture, indicating that mitogen expansion might selectively amplify specific subpopulations of neural progenitors, or that these culture conditions suppress expression of the M1 gene. Nevertheless, carbachol activation of muscarinic receptors in these mitogen-expanded cultures increased both proliferation of precursor cells (mainly TuJ1) and the number of TuJ1+ cells, indicating that acetycholine has the potential to alter number and/or fate of progenitor cells. Although this action is clearly attributable to activation of muscainic receptors, use of carbachol does not allow identification of the receptor subtype responsible. Neuroepithelial cells derived and expanded from E10.5 neural tube also show an increase in intracellular Ca2+ in response to acetylcholine, although the receptor type mediating the response was not determined (Cai et al. 2002). However, all these studies rely upon agonist challenge, and for these ideas to have any foundation requires that acetylcholine is also present in the neuroepithelium. Neurogenesis of the cholinergic neurones of the basal forebrain that gives rise to the pyramidal projection to the cortex, hippocampus and hindbrain, occurs between E12 and E17 (Armstrong et al. 1987; Semba and Fibiger 1988; Brady et al. 1989) and choline acetyltransferase (ChAT) immunoreactivity is not detectable until E17 (Armstrong et al. 1987), whilst ChAT immunoreactive fibres are not detectable in the cortex or hippocampus until the first three post-natal weeks (Dinopouolos et al. 1989; Gould et al. 1991; Thai et al. 1991). However, ChAT immunoreactive cells have been observed in the ventricular zone of the forebrain in mouse (Schambra et al. 1989) as early as E13.5 and at E15 in the VZ and subventricular zone (SVZ) of the striatum, whilst transient populations of acetylcholinesterase-positive cells have been observed in diencephalic ventricular and migratory zones (Schlaggar et al. 1993). Thus, it is likely that acetylcholine is present in the neuroepithelium before the development of those neurons that give rise to cholinergic projections to the forebrain and therefore has the potential to activate M1 muscarinic receptors on neuronal progenitor cells. The consequence of such activation is unknown at present.

Having established that the M1 gene is expressed on neuroepithelial cells, we were interested to see if transcription of the M1 gene in primary neural cells was governed by the same regulatory cis elements previously reported to regulate M1 transcription in cell lines (Wood et al. 1999). Characterisation of genomic regulatory elements are often carried out using reporter gene assays in cell lines, but it is not always clear what relationship exists between transcriptional mechanisms operative in immortalised cell lines and those operative in native cells. In previous studies, we have identified regulatory regions within the first exon of the rat M1 gene that are required for cell-specific expression in the IMR32 neural cell line. These studies characterised two adjacent regions at the 3′ end of the first exon that were required and that could enhance expression from a heterologous promoter (Wood et al. 1999). The results gained from our studies on cultured neuroepithelial cells show a similar profile, indicating that common regulatory elements are required to drive cell-specific expression in both cell lines and in primary neuroepithelial cells, although the enhancer construct containing the polypyrimidine tract and conserved region (pGL3 +396/+569 InR) acted as a much stronger enhancer in neuroepithelial cells than in cell lines. Although, it is clear that these regulatory sites are necessary for cell-specific expression, we anticipate that it is unlikely that they are sufficient, but these data do serve to underline the usefulness of cultured progenitor cells to identify bona fide regulatory elements.

In summary, we conclude that M1 receptors are expressed on both neuroepthelial cells and on neurons in the embryonic rat forebrain. We show that this embryonic expression is governed, at least in part, by elements in the first exon that are also responsible for regulating expression in neural cell lines. These observations give further substance to the idea that muscarinic receptors play a role in early neurogenesis in addition to their classical role in mediating synaptic neurotransmission.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Frozen sections
  5. In situ hybridisation
  6. Cell culture
  7. Immunohistochemistry
  8. Reporter gene assays
  9. Results
  10. The M1 gene is expressed on both the ventricular and marginal zones of the embryonic rat brain
  11. The M1 receptor is expressed on both neurons and neuroepithelial cells in culture
  12. Regulatory elements in the first exon are responsible for driving M1 expression in neuroepithelial cells
  13. Discussion
  14. Acknowledgements
  15. References