Background
The neural crest (NC) is a transient embryonic tissue. NC cells delaminate from the dorsal neural tube as it closes [
1] and migrate to distinct locations, where they differentiate into various cell types, including neurons, glia, melanocytes, endocrine cells, and mesenchymal cells [
2‐
5].
The Sox proteins belong to the HMG (high mobility group) domain of transcription factors [
6,
7]. Sox-E is the earliest marker of a subset of cells at the border of the neural plate that will give rise to NC-lineage cells [
8]. Sox10, which is a member of the Sox-E family and shares high sequence homology with other Sox-E member transcription factors, regulates and coordinates diverse developmental processes such as organ development and cell survival and specification. Sox10 is highly expressed in the emerging NC and later in the developing glial cells of the peripheral nervous system (PNS) and central nervous system (CNS) [
9,
10]. Whether in mice or humans, Sox family protein deletions or mutations often result in developmental defects and congenital disease, and mutations of the human
SOX10 gene are associated with NC cell abnormalities [
9,
11‐
13].
Several transgenic mouse strains dedicated to tracing the NC lineage have already been developed, such as
Wnt1-Cre [
14],
Protein zero (P0)-Cre [
15], and
Ht-PA- Cre [
16] mice crossed with Cre-dependent reporter mice. The Cre recombinase expression was previously visualized by
LacZ, a β-galactosidase reporter gene inserted in the ROSA26 locus, that is expressed only after the loxP-flanked intervening sequence is excised by Cre [
17]. Once a specific promoter is activated, the cell is indelibly tagged with β-galactosidase. This kind of transgenic mouse is useful for monitoring the transient activation of various promoters, including the NC-specific promoter. Recently, mouse strains expressing a fluorescence-based reporter upon Cre-mediated conditional gene deletion have been developed for prospective cell sorting or direct observation without fixation [
18]; the CAG-CAT-EGFP reporter transgenic mouse strain expresses enhanced green fluorescent protein (EGFP) when the loxP-flanked CAT gene located between the modified chicken β-actin promoter (CAG promoter) and the EGFP gene [
18] is excised with Cre. In previous studies, we have used mice that enable Cre/loxP-mediated cell labeling with
LacZ or EGFP to analyze the NC lineage and to trace NC cells after their migration and differentiation [
5,
19‐
21].
However, for specific gene regulation analysis, transgenic or knock-in mouse lines that express a specific gene profile
in vivo are more useful, because the reporter gene is expressed only while the specific promoter or enhancer is active, and ceases when the promoter becomes inactive. Reporter mice have recently been developed to evaluate cell-type specification and maturation in the oligodendroglial lineage; these are the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (
CNP)-EGFP and myelin proteolipid protein (
PLP)-EGFP transgenic mouse lines [
22,
23]. The
CNP-EGFP transgenic mouse, in which the CNP promoter controls EGFP expression, has been used for the prospective identification of live oligodendroglial cells both
in vivo and
in vitro[
23]. The
PLP-EGFP transgenic mouse, in which EGFP expression is driven by the mouse
PLP gene promoter, has also been developed for investigating oligodendrocyte lineage cells without fixation and immunostaining [
22].
Sox10 expression is closely related to NC-lineage cells. The
Sox10LacZ/+[
24],
Sox10- rtTA [
25], and
Sox10- Cre [
26] mouse lines have all been reported to label NC cells and oligodendrocytes.
Sox10LacZ/+[
24], a mutant mouse targeting
Sox10, was generated by replacing the open reading frame of
Sox10 with
lacZ sequences. The
Sox10LacZ/+mutation causes haploinsufficiency, in which even heterozygous pups have the phenotype found in mice with a spontaneous mutation in the
Sox10 allele. Although
LacZ expression in this knock-in strain faithfully reflects the endogenous Sox10 expression, it is difficult to observe normal developmental behavior in the labeled cells because of the abnormal and pathological condition of the
Sox10
LacZ/LacZ
homozygous mice [
24]. Another unique reporter strain is the
Sox10- rtTA knock-in mouse [
25], in which a variant of the reverse tetracycline-controlled transactivator (rtTA) is inserted into the genomic
Sox10 locus, and the mice are crossed with the doxycycline-dependent
LacZ reporter line. This strain correctly recapitulates endogenous Sox10 expression in the NC and its derivatives, and also in oligodendrocytes. This inducible transgenic system is limited in its range of analysis, because the reporter gene expression is temporary and requires X-gal staining [
25]. The
Sox10- Cre transgenic mouse strain, designated as the S4F:Cre mouse, when crossed with the reporter line strain Rosa-
LacZ, identifies cells expressing Sox10, including NC-derived cells, oligodendrocytes, and cells in the ventral neural tube [
26]. These strains are powerful tools for tracing the progeny of Sox10-expressing cells in analyses of NC cell migration and oligodendroglial differentiation. However, permanent reporter gene expression does not permit the real-time analysis of Sox10 expression. To overcome these limitations, we generated a new
Sox10- Venus transgenic mouse, and confirmed that it enables the normal behavior of Sox10-expressing cells to be observed
in vivo.
Discussion
In this study, we developed and characterized the
Sox10- Venus BAC transgenic mouse. Analysis of the early embryonic stages showed that not only NC lineage cells, but also oligodendroglial cells were clearly labeled with high-intensity Venus fluorescence (Figure
1 and
2). Compared to other published transgenic and knock-in reporter mouse strains, the
Sox10- Venus mouse has advantages that make it invaluable for future studies.
The mice with NC-lineage tracing
Wnt1-Cre,
P0-Cre, and
Ht-PA-Cre, crossed with those with Cre/loxP-mediated cell labeling, Rosa-
LacZ and CAG-CAT-EGFP, can be used only for analyzing cell lineage or migration, because the target cells are irreversibly labeled [
5,
14‐
20]. Although
Sox10- Venus also labels NC-lineage cells, the fast on/off switching of Venus fluorescence is tightly correlated with endogenous Sox10 expression. Similarly, the reporter gene activity in
CNP- EGFP and
PLP- EGFP mice, which are transgenic mice that label oligodendroglial-lineage cells, truly reflects the
in vivo expression of specific genes driven by specific promoters or enhancers. The transgenic system of the
Sox10- Venus mouse is quite similar to these; however, the Venus fluorescence is brighter and more intense than EGFP fluorescence.
Although reporter gene activity occurs in NC cells and oligodendrocytes in all
Sox10 reporter mouse lines, i.e.,
Sox10
LacZ/+
,
Sox10- rtTA,
Sox10- Cre strains [
24‐
26], and the new
Sox10- Venus strain, each line has advantages and disadvantages.
LacZ knock-in
Sox10LacZ/+heterozygous pups are prone to spontaneous mutation phenotypes due to haploinsufficiency. Also, to observing the
LacZ expression in
Sox10LacZ/+mice requires additional visualization procedures, making live cell imaging difficult. The
Sox10- rtTA knock-in crossed with the inducible TRE-
LacZ transgenic is unique, but the reporter expression is transient and does not fluoresce, making it difficult to observe directly. The
Sox10- Cre/CAG-CAT-EGFP double transgenic mouse traces both NC and oligodendrocyte progeny, since it reports past as well as ongoing Sox10 expression. With this double transgenic mouse, it is possible to carry out cell sorting or live imaging.
The
Sox10- Venus strain overcomes most of the disadvantages of the above-mentioned
Sox10 reporter mouse lines. In this mouse, the intense Venus fluorescence can be directly observed from outside the embryo, without staining or enhancement procedures (Figure
1,
2, and movies in Additional Files
2 and
3). Venus expression faithfully reflects real-time endogenous Sox10 expression, with prompt on/off switching (Figure
3). Although the choice of strain obviously depends on the purpose of the study, we believe that the
Sox10- Venus mouse is the most appropriate reporter line for numerous fields of research.
Although
in vivo time-lapse imaging of oligodendroglial cell migration (including OPCs) has recently been reported, it has only been conducted in zebrafish [
33,
34]. Accurate time-lapse imaging has been difficult to conduct in mice until now. There are several issues that need to be considered in such studies. For instance, (1) Is the
normal and
natural behavior of the cells or tissues of interest being observed? In some cases, the invasive procedures required to complete the imaging, including surgical incision, tissue-slice culture, electroporation, dye injection, and virus infection, may affect the tissues observed. (2) To what degree does the transgenic manipulation affect the phenotype? In
Sox10LacZ/+mice, the resulting phenotype, coupled with haploinsufficiency, complicates analysis of the area of interest. Our analysis of the
Sox10- Venus transgenic line demonstrates that it has neither developmental defects nor ectopic Venus expression. Thus, the
Sox10- Venus strain appears to be a useful tool for investigating normal developmental processes via live cell monitoring, using directly observed fluorescence without invasive intervention procedures (Figure
1G, movies in Additional Files
2 and
3).
Sox10 is a well-known marker of neural crest stem cells (NCSCs), along with
slug, snail, and
p75[
35,
36]. There have been numerous reports recently of NCSCs surviving in a wide range of tissues through the entire lifespan of the animal, suggesting that NCSCs may have the potential to support the regeneration and recovery of damaged tissues. The Venus fluorescence in
Sox10- Venus mice will make it possible to prospectively sort Venus
+ cells by flow-cytometry and collect an enriched population of NCSCs. Since NCSCs are located in easily accessible peripheral tissues such as the skin and bone marrow, NCSCs have been receiving increasing attention for future clinical applications in cell transplantation therapy, because the feasibility of autologous transplantation is anticipated [
37‐
39]. Autologous cell transplantation therapy avoids the immunological and ethical concerns related to the use of embryonic stem cells. We hope that the
Sox10- Venus strain will prove to be a powerful tool for enhancing the progress of NCSC research.
The mouse is an excellent model system for studying human disease progression and pathogenesis. We demonstrated the usefulness of our new reporter mouse strain
Sox10- Venus for monitoring processes occurring after a traumatic disorder (Figure
5 and
6), and crossing it with mutant mouse lines may provide insight into the processes behind numerous developmental defects. As with the analysis conducted in CNP-EGFP mice to study the behavior of oligodendroglial cells in SCI [
30],
Sox10- Venus mice have potential applications not only for oligodendrocyte research, but also for all Sox10
+-tissue analyses, including disorders of the NC cells and peripheral nerves. The ability to visualize the processes of disease initiation and progression will help to shed light on the pathophysiology of many human diseases.
Acknowledgements
We are grateful to Dr. R.B. Darnell for the gift of the anti-Hu antibody. We are grateful to Drs. C. Hara, N. Kishi, N. Shimojima, M. Mori, A. Iwanami, and H. Kanki for their excellent technical instruction and for critical reading of the manuscript. We also thank all the members in the Okano Laboratory and Akazawa Laboratory for their encouragement and invaluable comments on this manuscript.
This work was supported by a Grant-in-Aid for Young Scientists and a Grant-in-Aid for Scientific Research (C) from The Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) to S.S. and F.R-M.; by a Keio University Grant-in-Aid for the Encouragement of Young Medical Scientists to S.S. and F.R-M.; by Keio Gijuku Academic Development Funds to S.S.; by a JST-SORST fellowship to F.R-M.; by grants from Research Foundation ITSUU Laboratory and Takeda Science Foundation to T. I.; and by a Grant-in-Aid from the Global COE Program of the MEXT to Keio University to H.O.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS, CA, and HO designed the project. SS, AY, FRM, SS, TI, YUI, NN, and MS performed experiments and analyzed the data. SS, AY, and FRM prepared the figures. SS, AY, FRM, HK, TI, MN, CA, and HO wrote the manuscript. MN, CA, and HO supervised the project. All authors read and approved the final manuscript.