Dynamic Sox5 protein expression during cranial ganglia development

Abstract

Sox5 is a member of the SoxD group of HMG-box transcription factors that, during the early stages of development, promotes neural crest generation. However, little is known about Sox5 function in neural crest derivatives such as the peripheral sensory nervous system. We have analysed the embryonic expression of Sox5 during chick cranial ganglia development, from the stages of ganglia condensation to those of differentiation. During this period, Sox5 expression is maintained in the crest-derived satellite glial cells in all the cranial ganglia. In contrast, Sox5 is only transiently expressed in a subpopulation of differentiating neurons of both neural crest and placode origin. This detailed analysis provides a good base to dissect the possible role of Sox5 in neural cell fate determination by future functional approaches. Developmental Dynamics 236:2702–2707, 2007. © 2007 Wiley-Liss, Inc.

INTRODUCTION

Cranial ganglia are derived from both neural crest cells and cranial neurogenic placodes (reviewed in Baker et al.,2001). To form the ganglia, both these cell types delaminate and migrate into the head mesenchyme where they aggregate. In the head of the chicken embryo, all the peripheral glial cells derive from the neural crest (Le Dourain and Kalcheim,1999), and they include the satellite glia of the cranial ganglia and the Schwann cells associated with the cranial nerves. However, there are differences between the cranial ganglia in terms of the origin of their neuronal population (Fig. 1). Thus, most neurons in the ciliary ganglion are derived from the neural crest, although a small population of neurons is of placodal origin (Lee et al.,2003). In contrast, the facial ganglion neurons are mostly derived from the otic placode, except for a minor population of crest-derived neurons from the hyoid stream. The trigeminal sensory ganglion constitutes the case of a clearly mixed origin ganglion, containing neurons of placodal origin that begin to differentiate in the most distal portions of the ophthalmic and maxillo-mandibular lobules, and neural crest–derived neurons that start to differentiate in the proximal portion of the ganglion (Noden,1978). In amniotes, the VIIth, IXth, and Xth cranial nerves each have a proximal and distal sensory ganglion and while the neurons of the distal ganglia are derived from epibranchial placodes (geniculate, petrosal, and nodose), the proximal domain is of neural crest origin (a minor part in the facial, superior, and jugular: Narayanan and Narayanan,1980; Ayer-Le Lievre and Le Douarin,1982).

Figure 1. Cranial ganglia organization in a HH28 chick embryo. The schematic diagram illustrates the cranial ganglia, their associated nerves (roman numbers) as well as their embryonic origins (from neural crest in green, placodal in red, and from the otic vesicle in blue). Anterior is on the right, posterior on the left; proximal is at the top and distal is at the bottom.

The Sox (Sry-related HMG box) family of transcription factors is involved in many aspects of central and peripheral nervous system development (CNS and PNS; Wegner and Stolt,2005). The Sox family is composed of at least 20 members in mammals that are classified into 11 groups (Bowles et al.,2000). The SoxB proteins (including Sox1, Sox2, and Sox3) are involved in establishing neuroectoderm competence and maintaining stem-cells in the central nervous system (Kishi et al.,2000; Bylund et al.,2003). In contrast, SoxE proteins (Sox8, Sox9, and Sox10) are required for the specification and terminal differentiation of macroglia in the CNS (Stolt et al.,2002,2003,2005). In the PNS, Sox10 is expressed in the neural crest cells as they delaminate from the neural tube, and during their migration, Sox10 ensures their survival, maintains their pluripotency, and suppresses neuronal differentiation (Kim et al.,2003). When the neural crest cells reach their final destination, Sox10 is switched off in the committed neurons but it is maintained in cells fated to become glia, contributing to their specification (Britsch et al.,2001).

Sox5 belongs to the SoxD group together with Sox6 and Sox13. In the embryonic chick nervous system, Sox5 is detected in the premigratory and migratory neural crest cells (Perez-Alcala et al.,2005) where it promotes the epithelial mesenchymal transition of neural crest cells prior to their delamination from the neural tube. More recently, it was shown that together with Sox6, Sox5 exerts an opposite effect on macroglia development to that of SoxE proteins, repressing oligodendrocyte specification and differentiation (Stolt et al.,2006). In the PNS, Sox5 expression is maintained in neural crest–derived Schwann cells that ensheath the cranial nerves, where it is co-expressed with P₀, an early marker of both myelinating and non-myelinating cells (Perez-Alcala et al.,2005). Whether Sox5 is more broadly required for cell fate specification in the PNS is still an open question. To begin to address this problem, we have analysed Sox5 expression during cranial ganglia development and compared it with that of Sox10. We show that most of the satellite glial cells express both factors while a subpopulation of differentiating neurons transiently expressed one or both Sox proteins. These data provide the basic frame to explore in the future the cooperation between Sox5 and Sox10 in cell fate determination in the PNS.

RESULTS AND DISCUSSION

In the Trigeminal Ganglion, Sox5 Is Expressed in the Cells Derived From the Neural Crest

The trigeminal ganglion is formed by neurons of neural crest and placode origin that are segregated and that differentiate progressively along the proximal-distal axis of the ganglion. In the chicken, trigeminal ganglion development begins at embryonic stage HH12 (Hamburger and Hamilton,1951) with the initial condensation of the neural crest cells followed by that of the cells of placodal origin (Covell and Noden,1989), which will be, however, the first neurons to differentiate. The initial stages of differentiation are marked by the expression of the Lim domain transcription factor, Islet1/2, followed by that of Brn3a, which appears later during neuronal maturation (Fedtsova et al.,2003). In HH17 embryos, neural crest cells that migrate to the region adjacent to the trigeminal placode and that are labelled with the neural crest marker HNK-1, also expressed Sox5 (Fig. 2A). In contrast, developing sensory neurons expressing Islet1/2 in the trigeminal placode and in the adjacent condensing trigeminal ganglion did not express Sox5 (Fig. 2B). Thus, in early stages of development of the trigeminal ganglion Sox5 expression seems to be restricted to neural crest–derived cells.

Figure 2. Sox5 is expressed in the neural crest–derived cells of the trigeminal ganglion. A,B: Transverse section of the head of a HH17 chick embryo at the level of the trigeminal placode (Tp). Neural crest–derived cells expressing HNK1 (red) co-express Sox5 (green) in A. Committed neurons delaminating from the placode constitute a different population expressing Islet1/2 (red) but not Sox5 (green) in B. C–J: Sagittal sections of HH28 chick heads showing the trigeminal ganglion. Anterior is on the right, posterior on the left; proximal is at the top and distal is at the bottom. C,D: p75 (red) coincides with Islet1/2 (green) in the committed neurons of the distal part of the maxillo-mandibular (Mx-md) and ophthalmic lobules (Opht). The insets show that p75 protein mostly accumulates in the cytoplasm while Islet1/2 is expressed in the nuclei. E–J: Sox5 (green) colocalizes with the neural differentiation markers Islet1/2 (red in E–G), Brn3a (red in H,I), and HuC/HuD (red in J) in the committed neurons in the proximal part of the trigeminal ganglion. Sox5 is also expressed in the Schwann cells of the Vth nerve (E and H) and in satellite glial cells (in G). F, G, and I are higher magnification pictures of the proximal (F,I) and distal (G) regions of the maxillo-mandibular lobe of the trigeminal ganglion. J shows a field in the proximal region of the trigeminal ganglion similar to that in F and I. White arrows point to neurons that coexpress Sox5 and HuC/D.

At HH26–HH28, the developing neurons of placodal origin are located at the distal ends of the ophthalmic and maxillo-mandibular lobules, where they mature in a distal-proximal gradient. We observed that this maturation correlated with the progressive accumulation of the neurotrophin receptor p75 in the developing neurons (Fig. 2C,D). In accordance with the lack of Sox5 expression in the trigeminal placode at early developmental stages, developing neurons expressing Islet1/2 and Brn3a at the distal ends of the trigeminal lobes did not express Sox5 (Fig. 2E,G,H, respectively). In the distal region of the ganglion, the only Sox5-expressing cells had morphologically distinct, small, and elongated nuclei that differed from those of the developing neurons (Fig. 2E,G,H). These small Sox5-expressing cells that populate the whole ganglion were satellite cells of neural-crest origin as confirmed by the expression of Sox10 (see Fig. 4). Similarly, Sox5 expression was also observed in Schwann cells of the Vth nerve (Fig. 2E), as previously described in the oculomotor nerve (Perez-Alcala et al.,2004).

Figure 4. Sox5 is co-expressed with Sox10 in the satellite glial cells of the cranial ganglia. A–D: Sagittal sections of HH20 chick heads showing the trigeminal ganglion. E–H: Higher magnification of the insets in A–D. Sox5 expression is shown in green (A, D, E, H), Sox10 in red (B, D, F, H), and Islet1/2 in blue (C, D, G, H) in the same slide. The merged image of the three proteins is shown in D, H. Anterior is on the right, posterior on the left; proximal is at the top and distal is at the bottom. Blue arrows point to cells co-expressing Sox5 and Islet1/2, while the pink arrows point to cells co-expressing Sox10 and Islet1/2, and the white arrows point to cells co-expressing Sox5, Sox10, and Islet1/2.

Between HH24 and HH31, the neural crest–derived neurons start to differentiate in the proximal region of the trigeminal ganglion (DÁmico-Martel and Noden,1980). Interestingly, in this proximal region at HH26–28, there was a narrow band of differentiating neurons that transiently co-expressed Sox5, Islet1/2, and Brn3a (Fig. 2E,F,H,I; data not shown). As Islet1/2 is also expressed in mitotically active neuronal cells (Begbie et al.,2002), we confirmed the expression of Sox5 in committed neurons by using the neuronal marker HuC/D (Cui and Goldstein,2000; Fig. 2J) Thus, Sox5 appears to be dynamically expressed in neural crest–derived neuroblasts although it is silenced when they differentiate into neurons. In conclusion, Sox5 is expressed in the crest-derived cells of the trigeminal ganglion, first in progenitors and then it is maintained transiently in committed neurons and more persistently in the peripheral glial cells.

Sox5 Is Transiently Expressed in Differentiating Neurons of Cranial Ganglia

We next analysed whether this pattern of Sox5 expression was conserved in other cranial ganglia, particularly in those of preponderant neural crest origin such as the ciliary ganglion. Neuroblasts in the ciliary ganglion start to differentiate at around HH19 when the first cells expressing Islet1/2 could be detected (Fig. 3A). However, Brn3a expression was not detected in this ganglion at any stage analysed from HH19 to HH29 (Fig. 3O and data not shown). More interestingly, none of the differentiating neural crest–derived neurons expressed Sox5 (Fig. 3A,E,J) but rather Sox5 expression was restricted to small satellite glial cells from HH19 onwards (Fig. 3E,J). Therefore, migratory neural crest cells from the trigeminal stream determined to be neurons silence Sox5 at some point before arriving at the condensing region of the ciliary ganglion. Accordingly, Sox5 seems to be excluded from the committed neurons of the ciliary ganglion, the only cranial ganglia formed by parasympathetic neurons and not by bipolar sensory neurons.

Figure 3. Sox5 is dynamically expressed in the committed neurons of the cranial ganglia. A–S: Sagittal sections of the chick head showing the ciliary, facial, geniculate, and petrosal ganglion at stages HH19, HH20, and HH24, and the superior ganglion at HH20 and HH24. Sox5 expression is shown in green, Islet1/2 in red (A–N) and Brn3a in red (O–S). Sox5 is expressed in neural crest–derived satellite glial cells in all the cranial ganglia, and it is transiently co-expressed with Islet1/2 in committed neurons in the geniculate, petrosal, and superior ganglia (white arrows in G, H, L–N) and with Brn3a in the superior ganglia (white arrows in S).

Neurons in the superior and jugular ganglia are also derived from the neural crest, and among these, only a few neurons that express Islet1/2, Brn3a, or HuC/D also expressed Sox5 (Fig. 3N,S, and data not shown). Again, Sox5 was predominantly expressed in the narrow nuclei of the satellite glial cells (Fig. 3N,S). In conclusion, Sox5 is not always expressed in committed neurons of neural crest origin in the developing cranial ganglia.

We then explored the expression of Sox5 in the cranial ganglia of placodal origin from HH19 to HH28. Most of the neuroblasts in the facial ganglion are derived from the auditory placode, except for those neurons in the proximal part of the ganglion. The facial neural precursors delaminate from the most ventral region of the otic vesicle and rapidly differentiate into neurons that express Islet1/2 and Brn3a (Huang et al.,2001; and Fig. 3B,F,K,P). The majority of these placode-derived neurons did not express Sox5 at any stage analysed from HH19 to HH28 (Fig. 3B,F,K,P and data not shown). Thus, in the facial ganglion Sox5 expression seems to be mostly restricted to satellite glial cells.

We next examined whether Sox5 protein was present in committed neurons in ganglia entirely derived from the epibranchial placode, the distal ganglia of the VIIth-IXth-Xth nerves (Fig. 3). By stage HH24, Islet1/2 was expressed by committed neurons in the geniculate (Fig. 3L), petrose (Fig. 3M), and nodose ganglia (data not shown), although just a few of them expressed Brn3a at this developmental stages (Fig. 3Q,R, and data not shown). Interestingly, Sox5 was expressed by a small proportion of the committed neurons that contain Islet1/2 in the geniculate ganglion (Fig. 3L), and in a smaller proportion of similar cells in the petrose and in the nodose ganglia (Fig. 3M, and data not shown). Furthermore, Sox5 was not observed in late differentiating Brn3a-expressing cells in the geniculate ganglion or the petrose ganglion (Fig. 3Q,R), again indicating that there was an inverse correlation between Sox5 expression and the differentiation of sensory neurons as occurred in the trigeminal ganglion. Nevertheless, we also assessed whether Sox5 expression started in the epibranchial placode or in the committed neurons once they had arrived at the condensing epibranchial ganglia. Surprisingly, at HH20 we found that a significant proportion of the cells that expressed Islet1/2 co-expressed Sox5, not only in the geniculate ganglion (Fig. 3G) but also in the petrose and nodose ganglia as well (Fig. 3H, and data not shown). However, a few hours before, when the epibranchial ganglia had just started to condense at stage HH19, Sox5 was not detected in the committed neurons expressing Islet1/2.

In conclusion, Sox5 is expressed by the neural crest–derived microglia in all the cranial ganglia. Furthermore, Sox5 is transiently expressed in committed sensory neurons in all the ganglia independently of their neural crest cells or placodal origin. This transient expression may reflect Sox5 participation in the differentiation program of sensory neurons. In contrast, Sox5 was absent from committed parasympathetic neurons of the ciliary ganglion.

Sox5 Is Co-Expressed With Sox10 in the Satellite Glial Cells of the Cranial Ganglia

The HMG-box factor Sox10 is another Sox family member that is expressed as the neural crest cells delaminate from the neural tube. Sox10 expression is maintained in the glial and melanocyte lineages, but it is turned off in many other neural crest cell derivatives (Kuhlbrodt et al.,1998). Moreover, Sox10 has been described as a key regulator of peripheral glial development and, indeed, Schwann cells or satellite cells are not generated in mice with a targeted null mutation of Sox10 (Britsch et al.,2001). To demonstrate that the small microglia-like cells expressing Sox5 were bona fide satellite cells, the expression of Sox10 was analysed in conjunction with that of Sox5 and Islet1/2.

We analysed the cranial ganglia at HH20 when, as described above, Sox5 expression is more complex and it is clearly present in different types of cells that do and do not express Islet1/2 (Fig. 4). In the trigeminal ganglia at HH20, the majority of cells labelled for Sox5 with elongated nuclei co-expressed Sox10 at high levels, confirming these cells to be microglia (Fig. 4A,B,D,E,F,H). Again, a few differentiating neurons in the proximal region co-expressed Islet1/2 and Sox5 (Fig. 4E,G,H; cyan arrows) and surprisingly, another group of neurons with Islet1/2 also expressed Sox10, or both Sox5 and Sox10, albeit at low levels (Fig. 4F–H; magenta arrows and Fig. 4E–H). A similar situation was observed in the jugular or superior ganglion, where most satellite glial cells expressing Sox10 also expressed Sox5, and where a few of the cells labelled for Sox5, Sox10, or both also expressed Islet1/2 (data not shown).

In conclusion, members of both the SoxD and SoxE gene families are expressed in neural crest–derived satellite and Schwann glial cells of the cranial ganglia (data presented here; Perez-Alcala et al.,2004; Britsch et al.,2001; and data not shown). Furthermore, Sox5 and Sox10 are transiently expressed in a small population of committed neurons. Given the phenotypic variety among the different mature cranial ganglia, during development ganglion specific differences are evident in the pattern of Sox5 expression in committed neurons. Thus, Sox5 is absent in ciliary ganglion neurons, it is very rare in facial ganglion neurons and it is expressed in a small population of maturing neurons of neural crest or placodal origin.

Sox10 has been proposed to preserve not only glial but also neuronal potential in neural crest stem cells by counteracting or inducing lineage commitment signals. This is reflected by the fact that Sox10 is required to induce the neurogenic transcription factors MASH1 and PHOX2B (Kim et al.,2003). Our data open the avenue to explore by functional assays if, similarly to Sox10, Sox5 could play a dual role in cell fate determination during the development of the cranial ganglia.

EXPERIMENTAL PROCEDURES

Embryos

Fertilised chicken eggs were purchased from the Granja Santa Isabel (Córdoba, Spain). The eggs were incubated, opened, and staged according to Hamburger and Hamilton (1951). Subsequently, they were dissected and fixed for a few hours in 4% paraformaldehyde prepared in phosphate buffered saline (PBS) at 4°C.

Immunohistochemistry

Immunohistochemistry was performed on 15-μm frozen sections according to the protocol described previously (Perez-Alcala et al.,2005). The primary antibodies were used at the following concentrations: rabbit anti-LSox5, 1:4,000 (Perez-Alcala et al.,2005); mouse anti-Islet-1/2, 1:1,000 (40.2D6, DSHB); mouse anti-Brn3a, 1:1,000 (Chemicon); rabbit anti-p75 ^ICD 1:2,000 (9992, intracellular domain; gift from M. Chao); mouse anti-HNK-1, 1:4,000 (prepared from a cell line obtained from ATCC); mouse anti-HuC/HuD (16A11; Invitrogen) and goat anti-Sox10 1:1500 (gift from M. Wegner). The Cy2-, Cy3-, or Cy5-conjugated secondary antibodies were used at a dilution of 1:1,000 (Jackson). After immunohistochemistry, the sections were mounted in Citifluor (Citifluor Ltd., UK) and photographed using a Nikon fluorescence microscope (eclipse 80i) with a Nikon digital camera, or using a Leica spectral confocal microscope.

Acknowledgements

We thank B. Lázaro for technical assistance, and P. Bovolenta and R. Díez del Corral for comments on the manuscript. We also thank M. Chao for the gift of the anti-p75^ICD antiserum and M. Wegner for the gift of the Sox10 antibody. The other monoclonal antibodies used in this work were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa.