![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 19, Issue 4, 1575-1586, April 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany; and Department of Molecular Biology of the Cell I, German Cancer Research Center, D-69120 Heidelberg, Germany
Submitted August 13, 2007;
Revised January 22, 2008;
Accepted February 5, 2008
Monitoring Editor: Marianne Bronner-Fraser
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). At the dorsal aorta, neural crest cells are instructed by bone morphogenetic protein (BMP) signals to develop into catecholaminergic precursors, which are marked by expression of a set of transcription factors, including paired homeobox 2b (Phox2b), mammalian achaete scute homolog 1 (Mash1), Gata3, and Hand2 (for a recent review, see Goridis and Rohrer, 2002; Huber, 2006). It has been proposed that sympathoadrenal precursors represent a single homogeneous population of fate-restricted cells that give rise to both neurons in sympathetic ganglia and chromaffin cells in the adrenal gland. Recent results, however, suggest that there may be distinct precursors of sympathetic neurons and chromaffin cells already at very early times and that the precursor cell pool at the dorsal aorta as well as the neural crest cells entering the adrenal anlagen are heterogeneous (Ernsberger et al., 2005; Huber, 2006). After entry into the adrenal anlage, precursor cells differentiate further into chromaffin cells under the influence of local signals. What these signals are, is not fully understood after recent results have shown that glucocorticoids from the developing adrenal cortex are not required for most phases of chromaffin development as postulated for a long time from in vitro experiments (Finotto et al., 1999; Gut et al., 2005).
Transcription factors of group E of the Sry-box (Sox) protein family (i.e., Sox8, Sox9, and Sox10, jointly referred to as SoxE) are strongly expressed in the early neural crest, and they have been shown to influence neural crest development at multiple stages (Wegner, 1999; Wegner and Stolt, 2005; McCauley and Bronner-Fraser, 2006). In the mouse and in chicken, Sox9 is already expressed in the premigratory neural crest. Loss-of-function studies argue for a role of Sox9 in the survival of neural crest stem cells, whereas gain-of-function studies show that Sox9 is part of the transcriptional network that confers neural crest stem cell identity (Cheung and Briscoe, 2003; Cheung et al., 2005). Later roles in the epithelial-mesenchymal transition and the ectomesenchymal specification of the cranial crest have also been reported (Mori-Akiyama et al., 2003; Cheung et al., 2005).
Sox9 also induces expression of Sox10 in neural crest cells (Cheung et al., 2005). Whereas most neural crest cells loose Sox9 expression upon delamination, Sox10 continues to be expressed in migrating neural crest cells, and it is required for their survival, the maintenance of their pluripotency, and their specification to nonectomesenchymal derivatives such as melanocytes, peripheral glia, and cells of the enteric nervous system (for review, see Wegner and Stolt, 2005; Kelsh, 2006). This important role of Sox10 is also evident from the fact that heterozygous mutations in humans lead to neurocristopathies, most frequently a combination of Waardenburg syndrome and Hirschsprung disease (Pingault et al., 1998).
Although Sox8 is also broadly expressed in the early neural crest and several neural crest derivatives (Sock et al., 2001), the function of this third SoxE protein is less clear in the mouse. In Xenopus, however, Sox8 has many of the neural crest functions attributed to Sox9 in mouse or chicken (O'Donnell et al., 2006).
Taking the wide expression of SoxE proteins throughout the neural crest and their importance into account, it is reasonable to assume that they may also influence development of the adrenal gland. However, no systematic analysis has been undertaken so far to clarify their role in adrenal development. It is only known that Sox8 and Sox10 are expressed in the embryonic adrenal medulla, that their expression is strongly down-regulated in the adult (Kuhlbrodt et al., 1998; Sock et al., 2001; Deal et al., 2006), and that an adrenal medulla is missing at the time of birth in homozygous dominant megacolon mice (Sox10dom/dom) that express a truncated version of the Sox10 protein with possible dominant-negative function (Kapur, 1999). Therefore, we embarked on a study of adrenal development in mice carrying various SoxE gene mutations and clarified which SoxE protein contributed at what stage to adrenal development. This study has also implications for the genealogy of the chromaffin cell and its relation to a common sympathoadrenal precursor.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), Sox10Sox8ki (Kellerer et al., 2006), Sox10aa1 (Schreiner et al., 2007), Sox10
K2 (Schreiner et al., 2007), Sox10loxP (Schreiner and Wegner, unpublished data), and Sox8lacZ (Sock et al., 2001). For the conditional deletion of the Sox10loxP allele, a DBH::Cre BAC transgene was used (Parlato et al., 2007). Genotyping was performed by polymerase chain reaction (PCR) as described previously (Britsch et al., 2001; Sock et al., 2001; Kellerer et al., 2006; Parlato et al., 2007; Schreiner et al., 2007).
Tissue Preparation, Immunohistochemistry, and Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL)
Embryos were obtained at 10.5 days postcoitum (dpc), 11.5 dpc, 12.5 dpc, and 18.5 dpc from staged pregnancies. After fixation in paraformaldehyde, cryoprotection, and freezing at –80°C in Lung Tissue Freezing Medium (Leica, Nussloch, Germany), genotyped, age-matched embryos were transversally sectioned on a cryotome. Sections (10 µm) from the region in which the adrenal gland forms or had formed were used for immunohistochemistry according to standard protocols as described previously (Stolt et al., 2002, 2003). The following primary antibodies were used in various combinations: anti-Sox8 guinea pig antiserum (1:1000 dilution; Stolt et al., 2005), anti-Sox10 guinea pig antiserum (1:1000 dilution; Maka et al., 2005), affinity-purified anti-Sox10 rabbit antiserum (1:7500 dilution; Stolt et al., 2003), affinity-purified anti-Sox9 rabbit antiserum (1:2000 dilution; Stolt et al., 2003), anti-p75 rabbit antiserum (1:500 dilution; Promega. Madison, WI), anti-tyrosine hydroxylase (TH) rabbit antiserum (1:1000 dilution; BIOMOL Research Laboratories, Plymouth Meeting, PA), anti-phenylethanolamine N-methyltransferase (PNMT) rabbit antiserum (1:500 dilution; Immunostar, Hudson, WI), anti-VMAT-1 rabbit antiserum (1:1000 dilution; Weihe et al., 1994), anti-3β-hydroxysteroid dehydrogenase (HSD) rabbit antiserum (1:500 dilution; gift of A. H. Payne, Stanford School of Medicine), anti-steroidogenic factor 1 (SF1) rabbit antiserum (1:1000 dilution; gift of K. Morohashi, National Institute for Basic Biology, Japan), anti-Phox2b rabbit antiserum (1:500 dilution; gift of C. Goridis, Ecole Normale Superieure, Paris), anti-β-galactosidase rabbit antiserum (1:500 dilution; MP Biomedicals, Irvine, CA), or anti-β-galactosidase goat antiserum (1:500 dilution; BioTrend, Köln, Germany). Secondary antibodies conjugated to Alexa 488, cyanine (Cy)2, and Cy3 immunofluorescent dyes (Dianova, Hamburg, Germany) were used for detection. TUNEL assays were performed according to the manufacturer's protocol (Chemicon International, Temecula, CA). Samples were analyzed and documented using a Leica inverted microscope (DMIRB) equipped with a cooled MicroMax charge-coupled device camera (Princeton Instruments, Trenton, NJ).
Quantifications
After immunohistochemistry and documentation, the number of immunoreactive cells in the adrenal gland was counted when nuclear markers were stained. For all other markers, the immunoreactive area of the adrenal gland was determined morphometrically using NIH ImageJ software (http://rsb.info.nih.gov/ij/). The size of the labeled region was measured by summarizing suprathreshold zones regardless of variations in brightness. At 12.5 dpc, the adrenal gland was identified as the region containing SF1-positive cells, and results from quantifications are shown as the actual mean number of immunoreactive cells or the actual mean size of the immunoreactive area per section. At 18.5 dpc, the complete area of the adrenal gland was calculated in parallel to the immunoreactive areas, with the outer margin of the β-HSD immunoreactivity serving to demarcate the adrenal gland. Quantifications for nuclear markers at this embryo stage are shown as mean number of positive cells per unit area of the complete adrenal gland. Immunoreactive areas for all other markers were expressed as percentage of the whole adrenal gland at 18.5 dpc. The combined immunoreactivity for β-HSD as a cortical marker, and TH as a medullary marker accounted for 80 ± 2% of the total adrenal gland area, with the remaining 
20% corresponding to intercellular space and connective tissue. To determine the proportion of cortex versus medulla, the sum of the mean values for the β-HSD labeled and the TH-labeled areas was set to 100% in each genotype. Data were obtained from at least five adrenal glands for each genotype.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
; Huber et al., 2005), and its expression precedes that of enzymes required for noradrenaline biosynthesis, such as dopamine-β-hydroxylase (DBH) or TH. As a consequence, only a fraction of the Phox2b-positive cells contained TH (data not shown). TH-expressing cells, however, lacked Sox8 or Sox10 immunoreactivity (Figure 1, E and J). We therefore conclude that Sox8 and Sox10 are down-regulated in the neural crest-derived Phox2b-positive precursors before or concomitant with the onset of TH expression. Our results furthermore point to a considerable heterogeneity of neural crest-derived cells within the early adrenal gland. The similar labeling pattern obtained for Sox8 and Sox10 argued that both Sox proteins are coexpressed during adrenal gland development at 12.5 dpc. This was confirmed by coimmunohistochemistry (Figure 1G). Sox9, in contrast, was not detected in the adrenal anlage or in the neural crest cells migrating toward it (Figure 1B). Because we also failed to detect Sox9 at later embryonic stages in the adrenal gland (data not shown), Sox9 seems not to be expressed in the adrenal gland, and it is thus unlikely to exert direct effects on its development.
At 18.5 dpc, Sox10 expression in the adrenal gland was less prominent than at 12.5 dpc (Figure 2A). The remaining Sox10 immunoreactivity was found in the central region of the adrenal gland that was not stained with β-HSD or SF1 as two markers of the adrenal cortex (Figure 2A; data not shown). Despite its occurrence in the adrenal medulla, Sox10 was not expressed in chromaffin cells, which at this time not only express TH but also the vesicular monoamine transporter (VMAT)-1 and PNMT (Figure 2, C–E). The identity of the Sox10-expressing cells could not fully be clarified, because they were not Phox2b-positive and because p75 immunoreactivity was no longer a reliable indicator of neural crest cells at 18.5 dpc in the adrenal gland (data not shown). It is unlikely, however, that the Sox10-positive cells simply represent Schwann cells of invading nerves because most of the Sox10-expressing cells were negative for Gfap, Oct6, or Krox20 as Schwann cell markers (data not shown). From their appearance and their failure to express chromaffin or Schwann cell markers, Sox10-positive cells thus likely correspond to a residual precursor pool in the adrenal medulla.
|
). Most likely, β-galactosidase persists longer in the adrenal gland than the Sox8 protein once expression from the Sox8 locus subsides.
Sox10 Is a Major Determinant of Early Adrenal Gland Development
To determine the role of Sox10, we analyzed adrenal gland development in constitutively Sox10-deficient (i.e., Sox10lacZ/lacZ) mice. As expected there was no Sox10 immunoreactivity at 12.5 dpc throughout the embryo, including the region in which the adrenal gland started to form as an aggregate of SF1-positive cells (compare Figure 3, A and Z, with B and A'). We also failed to detect any Sox8-, β-galactosidase, or Phox2b-positive cells in the adrenal anlage (compare Figure 3, F, K, and P, with G, L, and Q). As a consequence, TH-positive cells were also absent at 12.5 dpc (Figure 3, U and V). Furthermore, none of these markers was expressed where sympathetic ganglia normally form in the vicinity of the adrenal gland (compare Figure 4, A–C with D–F), indicating that the complete sympathoadrenal neural crest lineage was heavily affected at this trunk level. This corroborates previous reports in which a cranio-caudal gradient had been observed for defects in the sympathetic ganglion chain of Sox10-deficient embryos at 12.5 dpc with rostral ganglia hypoplastic and caudal ganglia absent (Kapur, 1999; Britsch et al., 2001).
|
|
|
), but it also points to a so far unknown Sox10 requirement for induction and/or maintenance of Sox8 expression in the sympathoadrenal neural crest lineage. Instead, many cells at the dorsal aorta were apoptotic in Sox10lacZ/lacZ embryos as evident from TUNEL assays, whereas apoptosis rates were low in the corresponding wild-type controls (Figure 6, D and H). As a consequence of this increased apoptosis in Sox10lacZ/lacZ embryos, very few neural crest cells were found migrating toward the adrenal anlage at 11.5 dpc compared with the wild type (Figure 6, I and M). These cells did not express Sox8 nor Phox2b (compare Figure 6, J and K, with N and O). Because apoptotic cells were still present at increased rates on the migratory route in Sox10lacZ/lacZ embryos (Figure 6, L and P), most if not all Sox10-deficient neural crest cells die before reaching the adrenal anlage. We conclude that the absence of an adrenal medulla in Sox10lacZ/lacZ embryos is due to dramatically increased apoptosis in those neural crest cells destined to populate the adrenal gland. Neural crest cells furthermore fail to turn on Phox2b as an essential regulator of their development.
|
|
The normal adrenal development in Sox8lacZ/lacZ embryos may be accounted for by the ability of Sox10 to compensate for the loss of Sox8. Because lower Sox10 levels should be less likely to compensate, we also studied adrenal development in mice with Sox8 loss and simultaneously reduced Sox10 dosage. At 12.5 dpc, we consistently detected in these Sox10+/lacZ, Sox8lacZ/lacZ compound mutants a decrease of neural crest-derived cells in the adrenal anlage with all available markers, including Sox10, β-galactosidase, Phox2b, and TH (compare Figure 3, A, F, K, P, and U with E, J, O, T, and Y). Slight reductions were also observed in the sympathetic ganglia at this trunk level (Figure 4, M–O). Because none of these differences was detected in age-matched Sox10+/lacZ embryos (Figure 3, D, I, N, S, and X and Figure 4, J–L), the neural crest cell reduction must be attributed to the additional loss of Sox8.
The phenotype in Sox10+/lacZ, Sox8lacZ/lacZ compound mutants was still apparent when adrenal morphology and marker gene expression were analyzed at 18.5 dpc. The number of Sox10-positive cells was slightly reduced (compare Figure 5A with E). The area occupied by TH-positive chromaffin cells in the adrenal medulla was decreased by 5–7% compared with age-matched Sox10 heterozygotes and wild-type littermates (compare Figure 5K with N and O; quantified in Figure 7F). Similar decreases were also observed for PNMT and VMAT-1 as additional chromaffin markers (Figures 5, P, S, T, U, X, and Y, and 7E). The β-HSD–positive area of the adrenal gland was reciprocally increased by 3–5% (Figures 5, F, I, and J, and 7G). The medulla thus suffered an overall reduction from 22.5% of the total adrenal gland to 14.8% in the compound mutant (Figure 7H). We thus conclude that there is a minor role for Sox8 in adrenal gland development which is only revealed on a sensitized background with reduced Sox10 levels.
Differences in Gene Regulation and in Protein Properties Both Contribute to the Different Roles of Sox8 and Sox10 in Adrenal Gland Development
Sox10 can compensate for the loss of Sox8 in adrenal gland development, whereas Sox8 cannot compensate for the loss of Sox10 in the corresponding null mutant because of the observed dependence of its expression on Sox10. Whether there are additional functional differences between these two highly related Sox proteins in adrenal development was studied in a mouse mutant in which the Sox10 gene was replaced by Sox8. These Sox10Sox8ki/Sox8ki mice express Sox8 in all cells in which Sox10 is normally expressed and in levels comparable to Sox10 (Kellerer et al., 2006).
Despite the absence of Sox10 (compare Figure 8, A and B), neural crest-derived precursor cells were detected in the adrenal anlage of Sox10Sox8ki/Sox8ki embryos at 12.5 dpc by expression of Sox8, Phox2b, and TH (Figure 8, G, L, and Q) as well as in sympathetic ganglia (Figure 4, V–X). This argues that Sox8 can indeed substitute for Sox10 when expressed in a Sox10-specific pattern. However, all available markers were present in fewer cells than in the wild type (Figures 7, A–C, and 8, F, G, K, L, P, and Q), indicating that the rescue in Sox10Sox8ki/Sox8ki embryos is not complete.
|
|
), whereas the Sox10K2 allele lacks a region coding for a cell-specific transactivation and putative protein–protein interaction domain in the central Sox10 portion (Schreiner et al., 2007).
When adrenal gland development was studied in the two hypomorphic mouse mutants, significant defects were detected. Already at 12.5 dpc, the number of Sox10-, Sox8-, or Phox2b-positive precursor cells within the adrenal anlage was significantly reduced (Figures 7, A and B, and 8, C, D, H, I, M, and N) as was the number of TH-positive cells (Figures 7C and 8, R and S). This early reduction again translated into proportionate reductions in the size of the adrenal medulla at 18.5 dpc (Figures 7H and 9, H and I), the number of remaining precursors (Figures 7D and 9, C and D) and the number and/or occupied area of TH-, PNMT- or VMAT-1–positive chromaffin cells (Figures 7, E–G, and 9, M, N, R, S, W, and X). Interestingly, adrenal gland defects were severer in the hypomorphic mutants than in Sox10Sox8ki/Sox8ki embryos at both 12.5 and 18.5 dpc, indicating that Sox10 replacement was less detrimental than Sox10 mutation. Between the mutants, Sox10K2/K2 embryos showed a slightly stronger phenotype than Sox10aa1/aa1 embryos (compare Figure 9, C, H, M, R, and W with D, I, N, S, and X), with the remaining area occupied by chromaffin cells in the Sox10K2/K2 embryos being 8–11% of the wild type (Figure 7, E and F). Taking into account that the two regions targeted in the hypomorphic Sox10 alleles are present and highly conserved in Sox8, it is likely that their presence in Sox8 contributes to its rescue activity in the replacement mutant. Interestingly, sympathetic ganglia were also severely reduced in size at this trunk level (Figure 4, P–U), whereas ganglia of the rostral sympathetic chain are only mildly affected (Schreiner et al., 2007).
Sox10 Is Not Involved in Later Stages of Chromaffin Development
The severe and early adrenal gland defect in Sox10lacZ/lacZ mice makes it impossible to analyze additional later roles of Sox10 in adrenal gland development. To rule out such later roles in chromaffin development, we used a conditional Sox10 allele in combination with a DBH::Cre transgene, which deletes Sox10 efficiently from cells of the adrenal medulla at a time when these cells reach the chromaffin precursor stage and turn on catecholamine-synthesizing enzymes. Because previous analyses had shown that Sox10 expression is normally down-regulated in neural crest-derived cells of the adrenal medulla upon their acquisition of these enzymes, alterations in adrenal gland development of Sox10loxP/loxP, DBH::Cre mice were not expected. This was indeed the case both at early (Figure 8, E, J, O, and T) and at late (Figure 9, E, J, O, T, and Y) embryonic ages thus also proving genetically that Sox10 function is restricted to the neural crest derived precursor that has not yet acquired expression of catecholamine-synthesizing enzymes.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In contrast to Sox9, both Sox8 and Sox10 occur in those neural crest cells that give rise to the adrenal medulla. Sox10 in particular is important for adrenal development because no adrenal medulla develops in the absence of Sox10. The lack of an adrenal medulla has been reported previously in Sox10dom/dom mice (Kapur, 1999). Because of the presence of a shortened Sox10 protein with fully functional DNA binding activity in this mouse mutant (Herbarth et al., 1998; Southard-Smith et al., 1998; Kim et al., 2003), it has, however, been unclear whether loss of the adrenal medulla is caused by the absence of Sox10 or by the presence of a dominant-negative Sox10 version that interferes with the action of other functionally similar proteins such as Sox8. Here, we show that the absence of Sox10 is fully sufficient for the loss of the adrenal medulla. Sympathetic ganglia were also absent in the vicinity of the adrenal gland, arguing for a general essential role of Sox10 in the sympathoadrenal neural crest lineage at this axial level. This requirement for Sox10 is less stringent in other regions, as ganglia in the rostral sympathetic chain are hypoplastic, but nevertheless form in the absence of Sox10 (Kapur, 1999; Britsch et al., 2001).
The stage at which Sox10 becomes essential for adrenal development has also not been analyzed so far. Our results prove that Sox10 functions at a very early stage even before the neural crest-derived precursors have entered the adrenal anlage. Sox10 furthermore mainly functions as a survival factor. Such a role for Sox10 in survival has also been documented in other parts of the neural crest (Southard-Smith et al., 1998; Kapur, 1999; Britsch et al., 2001; Maka et al., 2005). Kim et al. (2003), however, had previously failed to detect a survival function for Sox10 in the sympathoadrenal neural crest at 9.5 dpc. Taking into account that our analysis was performed at 10.5 dpc, Sox10 may become active in its survival function between these time points. Alternatively, the sympathoadrenal neural crest cells may have been studied in different trunk regions, and the different results may simply reflect a different dependence of neural crest cell survival on Sox10 along the rostrocaudal axis.
A role of Sox10 in the induction of Phox2b expression has previously been postulated (Kim et al., 2003). Sympathoadrenal neural crest cells including those on their way to the adrenal anlage normally start to express Mash1 and Phox2b (Lo et al., 1998; Pattyn et al., 1999) under the influence of BMPs from the dorsal aorta (Reissmann et al., 1996; Schneider et al., 1999). The fact that sympathoadrenal neural crest cells at those axial levels where the adrenal medulla forms mostly fail to express Phox2b in Sox10-deficient embryos is compatible with the postulated role of Sox10 in Phox2b induction. In view of the large increase in cell death, the low number of Phox2b-expressing cells might, however, as well be caused by apoptosis of neural crest cells that start to express Phox2b. Increased apoptosis in Sox10-deficient sympathoadrenal neural crest, in contrast, is unlikely to be secondary to the loss of Phox2b expression, because sympathoadrenal neural crest cells are still present in significant numbers in the adrenal glands of Phox2b-deficient embryos at 13.5 dpc and later (Huber et al., 2005).
Although severe adrenal gland phenotypes have also been observed in Phox2b-deficient mice (Huber et al., 2005) or in mice with mutations in other Phox2b-dependent components of the sympathoadrenal transcriptional network (Huber et al., 2002; Moriguchi et al., 2006), Sox10-deficient mice are the only mice in which the adrenal anlage is not colonized by neural crest cells. In all other reported cases, sympathoadrenal neural crest cells are initially present within the adrenal anlage, but they become arrested at various stages of chromaffin development and eventually die. This phenotypic difference is compatible with Sox10 being genetically the most upstream factor. Despite the complete absence of sympathoadrenal neural crest cells in the adrenal glands, the cortex forms in Sox10-deficient mice, thus proving genetically that cortical development is largely independent of signals from the neural crest.
Sox10 expression and function is furthermore restricted to early neural crest derived precursors for chromaffin cells. It looses its importance in the chromaffin cell lineage with the acquisition of catecholaminergic traits. This coincides with down-regulation of its expression. Further proof for a restricted Sox10 function in the early precursor comes from the fact that specific ablation of Sox10 in DBH-expressing cells has no consequence on adrenal development. Accordingly, Sox10 is only transiently coexpressed with the early regulators of sympathoadrenal development such as Mash1 and Phox2b (Guillemot et al., 1993; Pattyn et al., 1999) and not with later regulators such as Gata3 and Hand2 (Lim et al., 2000; Lucas et al., 2006). Sox10 should thus be limited to interactions with the early ones.
Despite its down-regulation in cells with catecholaminergic traits, Sox10-positive cells remain in the adrenal medulla at least until the time of birth. This argues for the presence of a precursor cell population in this tissue. Its characterization and developmental potential needs to be analyzed. It is also unclear whether these cells are still present in the adult adrenal medulla, because our previous failure to detect Sox10 by Northern blotting (Kuhlbrodt et al., 1998) may simply be due to the insensitivity of the method.
The essential requirement for Sox10 contrasts dramatically with the very minor role of Sox8 that could only be visualized as a slight decrease in neural crest and chromaffin precursor cells, when one of the two Sox10 alleles was additionally deleted. This is surprising because Sox8 and Sox10 are coexpressed in precursor cells of the adrenal medulla during the decisive early phases of embryogenesis. Although replacement of Sox10 by Sox8 in the mouse reveals slight functional differences between the related proteins, one major reason for the different impact of Sox8 and Sox10 on adrenal gland development seems to lie in the fact that Sox8 expression in the sympathoadrenal neural crest depends on Sox10, but not vice versa. As a consequence, Sox10 deletion leads to a simultaneous loss of Sox8 expression, whereas Sox8 deletion leaves Sox10 expression unaffected. This dependence of Sox8 on Sox10 is cell type specific because it has not been observed in oligodendroglial cells that also express Sox8 and Sox10 (Stolt et al., 2004). Furthermore, Sox10 alone is not sufficient to maintain Sox8 expression as Sox8 is down-regulated in neural crest-derived cells of the adrenal gland from 14.5 dpc onward despite the continued presence of Sox10.
In its strictest interpretation, the model of a common sympathoadrenal precursor posits that sympathoadrenal neural crest cells are uniformly specified at the dorsal aorta, and then continue their migration as Phox2b- and Mash1-positive cells to their destination to give rise to all sympathetic neurons and chromaffin cells. However, what we observe is that both during migration from dorsal aorta to adrenal anlage and within the adrenal anlage itself, the neural crest population is quite heterogenous with some cells expressing Sox10 and p75; others expressing Sox10, Phox2b, and p75; and yet others expressing Phox2b without Sox10. Although the latter may be derived from previously Phox2b- and Sox10-expressing cells that have turned off Sox10 expression in the course of their development to chromaffin precursors, the Sox10-positive, but Phox2b-negative cells are difficult to reconcile with the sympathoadrenal precursor model both at this early stage and later in the adrenal medulla at 18.5 dpc. Thus, it seems that the neural crest cells migrating into the adrenal anlage are a heterogenous population and that they may undergo specification at different times, not solely under the influence of signals from the dorsal aorta, but also driven by local signals at their final destination, the adrenal anlage, as postulated previously (Ernsberger et al., 2005; Huber, 2006).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Michael Wegner (m.wegner{at}biochem.uni-erlangen.de)
Abbreviations used: β-HSD, β-hydroxysteroid-dehydrogenase; DBH, dopamine-β-hydroxylase; dpc, days postcoitum; Mash1, mammalian achaete scute homologue 1; Phox2b, paired homeobox 2b; PNMT, phenylethanolamine N-methyltransferase; SF1, steroidogenic factor 1; Sox, Sry-box; TH, tyrosine hydroxylase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; VMAT, vesicular monoamine transporter.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cheung, M., and Briscoe, J. (2003). Neural crest development is regulated by the transcription factor Sox9. Development 130, 5681–5693.
Cheung, M., Chaboissier, M.-C., Mynett, A., Hirst, E., Schedl, A., and Briscoe, J. (2005). The transcriptional control of trunk neural crest induction, survival, and delamination. Dev. Cell 8, 179–192.[CrossRef][Medline]
Deal, K. K., Cantrell, V. A., Chandler, R. L., Saunders, T. L., Mortlock, D. P., and Southard-Smith, E. M. (2006). Distant regulatory elements in a Sox10-betaGEO BAC transgene are required for expression of Sox10 in the enteric nervous system and other neural crest-derived tissues. Dev. Dyn 235, 1413–1432.[CrossRef][Medline]
Ernsberger, U., Esposito, L., Partimo, S., Huber, K., Franke, A., Bixby, J. L., Kalcheim, C., and Unsicker, K. (2005). Expression of neuronal markers suggests heterogeneity of chick sympathoadrenal cells prior to invasion of the adrenal anlagen. Cell Tissue Res 319, 1–13.[CrossRef][Medline]
Finotto, S. et al. (1999). Analysis of mice carrying targeted mutations of the glucocorticoid receptor gene argues against an essential role of glucocorticoid signalling for generating adrenal chromaffin cells. Development 126, 2935–2944.[Abstract]
Goridis, C., and Rohrer, H. (2002). Specification of catecholaminergic and serotonergic neurons. Nat. Rev. Neurosci 3, 531–541.[CrossRef][Medline]
Guillemot, F., Lo, L.-C., Johnson, J. E., Auerbach, A., Anderson, D. J., and Joyner, A. L. (1993). Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75, 463–476.[CrossRef][Medline]
Gut, P., Huber, K., Lohr, J., Bruhl, B., Oberle, S., Treier, M., Ernsberger, U., Kalcheim, C., and Unsicker, K. (2005). Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation and differentiation of chromaffin cells. Development 132, 4611–4619.
Herbarth, B., Pingault, V., Bondurand, N., Kuhlbrodt, K., Hermans-Borgmeyer, I., Puliti, A., Lemort, N., Goossens, M., and Wegner, M. (1998). Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease. Proc. Natl. Acad. Sci. USA 95, 5161–5165.
Huber, K. (2006). The sympathoadrenal cell lineage: specification, diversification, and new perspectives. Dev. Biol 298, 335–343.[CrossRef][Medline]
Huber, K., Bruhl, B., Guillemot, F., Olson, E. N., Ernsberger, U., and Unsicker, K. (2002). Development of chromaffin cells depends on MASH1 function. Development 129, 4729–4738.[Medline]
Huber, K., Karch, N., Ernsberger, U., Goridis, C., and Unsicker, K. (2005). The role of Phox2B in chromaffin cell development. Dev. Biol 279, 501–508.[CrossRef][Medline]
Kapur, R. P. (1999). Early death of neural crest cells is responsible for total enteric aganglionosis in Sox10(Dom)/Sox10(Dom) mouse embryos. Pediatr. Dev. Pathol 2, 559–569.[CrossRef][Medline]
Kellerer, S., Schreiner, S., Stolt, C. C., Bösl, M. R., and Wegner, M. (2006). Functional equivalency of transcription factors Sox8 and Sox10 is tissue-specific. Development 133, 2875–2886.
Kelsh, R. N. (2006). Sorting out Sox10 functions in neural crest development. Bioessays 28, 788–798.[CrossRef][Medline]
Kim, J., Lo, L., Dormand, E., and Anderson, D. J. (2003). SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron 38, 17–31.[CrossRef][Medline]
Kuhlbrodt, K., Herbarth, B., Sock, E., Hermans-Borgmeyer, I., and Wegner, M. (1998). Sox10, a novel transcriptional modulator in glial cells. J. Neurosci 18, 237–250.
Lim, K.-C., Lakshmanan, G., Crawford, S. E., Gu, Y., Grosveld, F., and Engel, J. D. (2000). Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nature 25, 209–212.[CrossRef]
Lo, L., Tiveron, M. C., and Anderson, D. J. (1998). Mash1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity. Development 125, 609–620.[Abstract]
Loring, J. F., and Erickson, C. A. (1987). Neural crest cell migratory pathways in the trunk of the chick embryo. Dev. Biol 121, 220–236.[CrossRef][Medline]
Lucas, M. E., Muller, F., Rudiger, R., Henion, P. D., and Rohrer, H. (2006). The bHLH transcription factor hand2 is essential for noradrenergic differentiation of sympathetic neurons. Development 133, 4015–4024.
Maka, M., Stolt, C. C., and Wegner, M. (2005). Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect. Dev. Biol 277, 155–169.[CrossRef][Medline]
McCauley, D. W., and Bronner-Fraser, M. (2006). Importance of SoxE in neural crest development and the evolution of the pharynx. Nature 441, 750–752.[CrossRef][Medline]
Mori-Akiyama, Y., Akiyama, H., Rowitch, D. H., and de Crombrugghe, B. (2003). Sox9 is required for determination of the chondrogenic cell lineage in the cranial neural crest. Proc. Natl. Acad. Sci. USA 100, 9360–9365.
Moriguchi, T. et al. (2006). Gata3 participates in a complex transcriptional feedback network to regulate sympathoadrenal differentiation. Development 133, 3871–3881.
O'Donnell, M., Hong, C. S., Huang, X., Delnicki, R. J., and Saint-Jeannet, J. P. (2006). Functional analysis of Sox8 during neural crest development in Xenopus. Development 133, 3817–3826.
Parlato, R., Otto, C., Begus, Y., Stotz, S., and Schutz, G. (2007). Specific ablation of the transcription factor CREB in sympathetic neurons surprisingly protects against developmentally regulated apoptosis. Development 134, 1663–1670.
Pattyn, A., Morin, X., Cremer, H., Goridis, C., and Brunet, J. F. (1999). The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399, 366–370.[CrossRef][Medline]
Pingault, V. et al. (1998). Sox10 mutations in patients with Waardenburg-Hirschsprung disease. Nat. Genet 18, 171–173.[CrossRef][Medline]
Reissmann, E., Ernsberger, U., Francis-West, P. H., Rueger, D., Brickell, P. M., and Rohrer, H. (1996). Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons. Development 122, 2079–2088.[Abstract]
Schlierf, B., Ludwig, A., Klenovsek, K., and Wegner, M. (2002). Cooperative binding of Sox10 to DNA: requirements and consequences. Nucleic Acids Res 30, 5509–5516.
Schneider, C., Wicht, H., Enderich, J., Wegner, M., and Rohrer, H. (1999). Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons. Neuron 24, 861–870.[CrossRef][Medline]
Schreiner, S., Cossais, F., Fischer, K., Scholz, S., Bösl, M. R., Holtmann, B., Sendtner, M., and Wegner, M. (2007). Hypomorphic Sox10 alleles reveal novel protein functions and unravel developmental differences in glial lineages. Development 134, 3271–3281.
Sock, E., Schmidt, K., Hermanns-Borgmeyer, I., Bösl, M. R., and Wegner, M. (2001). Idiopathic weight reduction in mice deficient in the high-mobility-group transcription factor Sox8. Mol. Cell Biol 21, 6951–6959.
Southard-Smith, E. M., Kos, L., and Pavan, W. J. (1998). Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat. Genet 18, 60–64.[CrossRef][Medline]
Stolt, C. C., Lommes, P., Friedrich, R. P., and Wegner, M. (2004). Transcription factors Sox8 and Sox10 perform non-equivalent roles during oligodendrocyte development despite functional redundancy. Development 131, 2349–2358.
Stolt, C. C., Lommes, P., Sock, E., Chaboissier, M.-C., Schedl, A., and Wegner, M. (2003). The Sox9 transcription factor determines glial fate choice in the developing spinal cord. Genes Dev 17, 1677–1689.
Stolt, C. C., Rehberg, S., Ader, M., Lommes, P., Riethmacher, D., Schachner, M., Bartsch, U., and Wegner, M. (2002). Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10. Genes Dev 16, 165–170.
Stolt, C. C., Schmitt, S., Lommes, P., Sock, E., and Wegner, M. (2005). Impact of transcription factor Sox8 on oligodendrocyte specification in the mouse embryonic spinal cord. Dev. Biol 281, 323–331.
Wegner, M. (1999). From head to toes: the multiple facets of Sox proteins. Nucleic Acids Res 27, 1409–1420.
Wegner, M., and Stolt, C. C. (2005). From stem cells to neurons and glia: a soxist's view of neural development. Trends Neurosci 28, 583–588.[CrossRef][Medline]
Weihe, E., Schafer, M. K., Erickson, J. D., and Eiden, L. E. (1994). Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat. J. Mol. Neurosci 5, 149–164.[Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
