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Keywords:

  • amacrine cells;
  • chick;
  • Neuropeptide Y;
  • retina;
  • Sox2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The retina has been extensively used as a model to study the mechanisms responsible for the production of different neural cell phenotypes. The importance of both extrinsic and intrinsic cues in these processes is now appreciated and numerous transcription factors have been identified which are required for both neuronal determination and cell differentiation. In this study we have analysed the expression of the transcription factor Sox2 during development of the chick retina. Expression was found in the proliferating cells of the retina during development and was down regulated by nearly all cell types as they started to differentiate and migrate to the different layers of the retina. In one cell type, however, Sox2 expression was retained after the cells have ceased division and migrated to their adult location. These cells formed two rows located on either side of the inner plexiform layer and were also positive for Neuropeptide Y, characteristics which indicate that they were a subpopulation of amacrine cells. The expression of Sox2 by only this population of post-mitotic neurones makes it possible to follow these cells as they migrate to their adult location and shows that they initially form a single row of cells which subsequently divides to form the double row seen in the adult tissue. We suggest that retained expression of Sox2 is involved in directing the differentiation of these cells and is an early marker of this cell type.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

The retina is a relatively simple laminated tissue composed of only six types of neurones and one type of glia, features which have made it an attractive model to study neuronal cell specification. As in other parts of the developing central nervous system, cell proliferation is restricted to a germinal layer from which differentiating post-mitotic cells originate (Prada et al. 1991). The resulting cell types generated segregate into three layers separated by two synaptic layers. These three nuclear layers are known as: the outer nuclear layer, which contains two classes of photoreceptors, rods and cones; the inner nuclear layer, which consists of horizontal, bipolar, amacrine neurons and displaced ganglion cells; and the ganglion cell layer, which contains predominantly ganglion cells and displaced amacrine cells. All cell types within the retina originate from one population of progenitor cells and lineage analysis has demonstrated that individual progenitor cells are multipotent and can generate different combinations of neurones and glia (Turner & Cepko, 1987).

Very little is known, however, about what determines the formation of different cell types. Transcription factors have been shown to play a key role in providing intrinsic information influencing cell fate decision (reviewed in Perron & Harris, 2000; Livesey & Cepko, 2001). In particular, individual transcription factors belonging to the bHLH gene family have been shown to be expressed in proliferating cell progenitors as well as being maintained by discrete cell types after they have ceased to divide. This suggests that these transcription factors may have a role in directing progenitors towards a particular cell fate. Examples of this type of expression pattern include amacrine cells, a major class of interneurons in the retina, which retain expression of Mash1 (Guillemot & Joyner, 1993) and NeuroD (Morrow et al. 1999), two different classes of bHLH genes that are broadly found in undifferentiated progenitor cells.

Sox2 belongs to the Sox transcription factor family first characterised 10 years ago with the identification of the Sex determining gene, Sry (Gubbay et al. 1990; Sinclair et al. 1990). Over 30 members of this highly conserved family of genes have been identified with expression in all animals from C. elegans to humans. Sox genes are characterised by a 79 amino acid high-mobility group (HMG) DNA binding domain (Gubbay et al. 1990) which recognises specific DNA sequences (reviewed in Wegner, 1999; Kamachi et al. 2000). Many Sox genes are expressed during embryonic development where they are involved in the formation of diverse tissues including the gonads, central nervous system, neural crest cells, skeletal muscle, haemopoetic and chondrogenic cells. From the pattern of their expression, which is often restricted to particular stages of cell differentiation, they are thought to participate in controlling progress along differentiation pathways (reviewed in Wegner, 1999).

Sox2 is expressed in the gut and lens as well as the developing nervous system where it initially appears shortly after neural induction (Kamachi et al. 1995, 1998; Rex et al. 1997; Streit et al. 1997; Ishii et al. 1998). After formation of the neural tube, expression was thought to be limited to the undifferentiated proliferating cells of the ventricular zone. Loss of cSox2 expression in the central nervous system occurred at the transition from an epithelial state to a migratory state, a change which coincides with the cessation of proliferation and initiation of differentiation (Uwanogho et al. 1995).

During the early development of the eye cSox2 is expressed in the neuroepithelium as the optic vesicles evaginate from the forebrain and is subsequently restricted to the neural retina (Uwanogho et al. 1995; Uchikawa et al. 1999). In this study we have followed the expression of cSox2 during the later stages of neural retina development. In agreement with the findings from other parts of the CNS, cSox2 is expressed in the proliferating, germinal layer throughout retina development. However, in contrast to previous studies, we show that cSox2 is maintained in a regularly arrayed population of cells which are no longer proliferating and have migrated away from the germinal layer. These cells contain Neuropeptide Y (NPY) and appear to be a population of displaced amacrine cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Animals

Fertilised white Leghorn chicken eggs were incubated at 39 °C in a humidified chamber. Embryos were staged in days according to the first day of incubation.

Labelling of dividing cells

Windows were made in the shells of eggs between day 7 and 18 of development, the vitelline membrane was removed and approximately 1 µL of 50 mm BrdU was injected into a vein. The eggs were sealed with sticky tape and incubated for a further 20 min before embryos were decapitated and fixed prior to analysis.

In situ hybridisation

The method was as described in Uwanogho et al. (1995) using an identical probe. Briefly, tissues were dissected and fixed in 4% paraformaldehyde in PBS at 4 °C overnight, dehydrated in ethanol and embedded in paraffin wax. Sections 10–15 µm thick were cut and mounted on TESPA (Sigma)-coated slides. Tissues were then dewaxed in histoclene and rehydrated through graded alcohol and rinsed twice in PBS for 5 min. The sections were digested with 10 mg mL−1 protein kinase (Sigma) for 10 min, rinsed three times in PBST, treated with 4% paraformaldehyde in PBS for 20 min, and washed twice in PBST. Hybridisation was done overnight at 70 °C in a humidified chamber, with a solution containing 1/500 digoxigenin-labelled single stranded RNA probe (sense or anti-sense). Sections were then rinsed under stringent conditions (twice for 15 min and once for 30 min with 50% formamide, 5× SSC, 1% SDS, at 65 °C, followed by three times for 30 min in 50% formamide, 2× SSC at the same temperature). Slides were then submerged in TBST (TBS + 0.1% Tween-20, TBS: 8 g NaCl + 25 mL/1 m Tris-HCl pH 7.5) three times for 5 min before DIG detection. The sections were incubated in 1% blocking reagent in PBST for 1 h and then transferred in 1/5000 dilution of pre-absorbed anti-DIG alkaline phosphatase conjugated fab fragment at 4 °C ON. Slides were washed three times for 20 min in PBST followed by two 5-min changes in freshly made NTMT buffer (0.1 m Tris–HCl {pH 9.5}, 50 mm MgCl2, 0.1 m NaCl, 0.1% Tween 20, 2 mm levamisole). The reaction was stopped by incubation in 1× TE (10 mm Tris–HCL pH 7.4, 1 mm EDTA pH 8) and slides processed for BrdU immunocytochemistry. Reactions were performed at room temperature unless stated otherwise.

Immunocytochemistry

All reactions were at room temperature, and the protocol was similar to that described by Uwanogho et al. (1995). For BrdU detection, sections were pretreated by digesting them in 0.4% pepsin in 0.01 m HCl for 40 min at 37 °C. The slides were then washed in water and placed into 2 m HCl at 37 °C for 15 min, and washed again in water and transferred into Scott’s water (0.04 m NaCO3, 0.08 m MgSO4) for 2 min. They were washed once more in water before BrdU immunostaining proceeded. All slides were then blocked in 20% serum in PBS, prior to incubation with primary antibody for 30 min (1/100 mouse anti-BrdU monoclonal antibody {Dako}, 1/50 rabbit NPY polyclonal antibody {Sigma} in PBS). For controls, slides were incubated for 30 min in PBS with no primary antibody. Slides were then rinsed in PBS and overlaid with biotinylated secondary antibody (1/100 sheep anti-mouse antibody for BrdU staining or 1/100 goat anti-rabbit antibody {Vector laboratories} for NPY) for 30 min. Following a brief rinse in PBS, slides were incubated in alkaline phosphatase-conjugated streptavidin diluted 1/250 in PBS for 30 min. Slides were then briefly rinsed in PBS before developing the colour in Fast Red (Sigma, prepared according to the manufacturer’s instructions) for BrdU detection or alkaline phosphatase streptavidin diluted 1/250 in PBS for NPY detection. The colour reaction was stopped in TE buffer after the suitable intensity appeared. No staining occurred in the absence of primary antibodies (controls).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Expression of cSox2 in the developing chick retina

In this study, in situ hybridisation for cSox2 was carried out from day 7 to 18 of development, a period during which neuronal differentiation and retinal layer formation is taking place. At day 7, cSox2-positive cells were detected in a central region of the retina (Fig. 1A), a pattern of expression which continued throughout development. Proliferating cells are only found in the centre of the retina and double staining for cSox2 and BrdU demonstrated that dividing cells were indeed located in the same region as cSox2-expressing cells. However, on the 9th day of incubation, in addition to the proliferating zone, a separate cSox2-positive cell population was observed in the centre of the inner plexiform layer, a region that separates the ganglion cell layer from the inner nuclear layer. These cells were organised as a single row of cells which were clearly positive for cSox2 expression after in situ hybridisation. This cell population did not incorporate any BrdU and was therefore post-mitotic. At 10 days, cells in the germinal region in the centre of the retina still expressed cSox2. However, the post-mitotic cSox2 population observed at 9 days outside the germinal region now formed two rows of cells located on either side of the inner plexiform layer (Fig. 1D). These cells were regularly arrayed, separated from each other by 1–5 cells, and exhibited in some regions a remarkably symmetrical arrangement on either side of the inner plexiform layer. This pattern was maintained at 11 days of development (Fig. 1B). By day 18 the retina has an adult structure but still retains a central region of proliferating cells. Cell division continues in this region in to the post hatching period when Müller glia and horizontal cells are generated (Fischer & Reh, 2000). cSox2 continues to be expressed by proliferating cells at day 18 (Fig. 1C) as well as the double row of post-mitotic cells on either side of the inner plexiform layer.

image

Figure 1. Endogenous expression of cSox2 in the developing chick retina. A, B, C, D and E are cross-sections through the retinas at days 7, 11, 18 and 10 of development, respectively. Endogenous cSox2 mRNA was revealed by in situ hybridisation using anti-sense digoxigenin-labelled riboprobes. Sense probes showed no staining. The cells expressing cSox2 appeared in blue. Proliferating cell nuclei were labelled with BrdU and stained in red. D and E: Comparison between cSox2-positive cells and NPY immunoreactive cells, respectively, at day 10 of development (both revealed in blue). Lack of staining in the absence of primary antibodies demonstrates specificity of the staining over normal background (pictures not shown). GCL: ganglion cell layer, INL: inner nuclear cell layer, ONL: outer nuclear cell layer, PE: pigmented epithelium, GL: germinal layer. Arrows indicate cSox2-positive cells in the IPL. Scale bar = 18.8 µm.

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The cSox2 population on either side of the inner plexiform layer are NPY positive

From their location, the cSox2-positive cells on either side of the inner plexiform layer could be a subpopulation of amacrine cells which can be identified by the presence of NPY in their cytoplasm. Immunostaining to detect NPY was performed on days 10 and 18 of development. NPY immunoreative cells were found in the ganglion cell layer and as two rows of cells on either side of the inner plexiform layer. The location, size and numbers of NPY immunoreative cells in the later location corresponded closely with the cSox2-positive cells found in this region (Fig. 1D,E).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

We have analysed the expression pattern of the cSox2 gene in the chick retina from day 7 to day 18 of development. Expression of cSox2 was initially restricted to the germinal region of the retina where it was expressed in undifferentiated, proliferating cells. Previous studies on the developing CNS have demonstrated that loss of cSox2 expression correlates with the transition from proliferation to migration (Uwanogho et al. 1995; Rex et al. 1997). In agreement with this, our studies showed that cSox2 expression was lost by the majority of cells as they migrated away from the central proliferative region and differentiated. However, starting at day 7 of development, a cSox2-positive, non-proliferating cell population was observed. This population was first detected as a single row of cells in the inner plexiform layer but by day 10 consisted of two rows located on either side of the plexiform layer. The expression of cSox2 by these post-mitotic cells appears to define a unique population of cells in the developing CNS and suggests that continued expression of cSox2 as these cells migrate and differentiate may be involved in setting them aside from other cells.

A population of amacrine cells which forms two parallel rows on either side of the inner plexiform layer has been described in mouse, cat and chick (Brunn et al. 1986; Hustler & Chalupa, 1995; Sinclair & Niremberg, 2001). This population is immunoreative to NPY and sends processes to sublayers within the IPL (Brunn et al. 1986). These cells are thought to function as inhibitory interneurones during inner plexiform layer processing (Sinclair & Nirenberg, 2001). Immunonstaining developing retinas for NPY showed that this population of NPY immunoreative amacrine cells is present at day 10. The location and number of NPY immunoreative cells on either side of the IPL matches the location of cSox2-positive cells suggesting that cSox2 expression is an early marker for this population of amacrine cells.

As cSox2 expression is retained by these cells in contrast to all other cell types, it is possible to follow their changing distribution as they migrate to their adult location during retina development. As described above the single row of cells seen at day 9 has become a double row on either side of the IPL by day 10. This change in distribution could occur by the migration of additional cSox2-positive cells from the proliferative region, by migration of cells present in the centre of the IPL to the margins of the IPL or by expansion of the IPL separating cSox2-positive cells. As the numbers of cSox2 cells appears to increase between day 9 and 10 it is likely that the first suggestion is correct.

This study indicates how a subpopulation may be set aside by selective regulation of transcription factor expression. The retention of transcription factors normally only expressed in proliferating or undifferentiated cells may be a commonly used mechanism to specify a discrete neuronal cell population during development. For example, cSox11, another member of the Sox family, is an early marker of cell differentiation. This gene is expressed as cells leave the germinal layer and begin to differentiate. However, in the retina, cSox11 is maintained in the ganglion cells, as opposed to the photoreceptors or interneurons in which cSox11 is only expressed for a short transient period of time (our own data, not published). Sox genes are expressed by undifferentiated cells and appear to be retained in specific subpopulations of cells as they differentiate. The maintenance of specific intrinsic signals within cells undergoing differentiation seems therefore to be essential for cells to acquire specific phenotypes.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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