QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Paratore, C. Articles by Sommer, L. Search for Related Content PubMed PubMed Citation Articles by Paratore, C. Articles by Sommer, L. Social Bookmarking                         What's this?

Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling

¹ Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zurich, Switzerland
² Institute of Biochemistry, University of Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany

View larger version (96K): [in a new window]   Fig. 1. Expression of Sox10 protein in neural crest stem cells and peripheral glia. Neural crest stem cells (NCSCs) were replated from neural crest explants, fixed after 4 hours, and immunolabeled using anti-p75 antibody (visualized by Cy3 fluorescence) (A) and anti-Sox10 antibody (visualized by FITC fluorescence) (B). Note that virtually all freshly isolated NCSCs are double-positive for p75 and Sox10. Sister cultures of replated NCSCs were allowed to develop in conditions (Materials and Methods) that predominantly generate S100-positive glia (D), neurofilament160 (NF)-positive neurons (G) or smooth muscle actin (SMA)-positive non-neural cells (J). Double-labeling of the cultures for Sox10 revealed that Sox10 expression is maintained in the glial lineage (E) while it is downregulated in neuronal (H) and non-neural (K) cells. (C,F,I,L) Corresponding phase-contrast pictures.

View larger version (51K): [in a new window]   Fig. 2. Sox10-dependent survival of undifferentiated postmigratory neural crest cells. (A-C) Apoptotic cell death of undifferentiated postmigratory Sox10–/– neural crest cells. Transverse cryosections through DRG from E11 embryos were triple labeled for p75 (FITC fluorescence), NF (Cy5 fluorescence) and TUNEL (Cy3 fluorescence), and analyzed by confocal microscopy. A single confocal plane is shown. Apoptosis as assayed by TUNEL labeling was frequent in undifferentiated p75-positive DRG cells of Sox10 homozygous (–/–) mouse mutants (arrow in B) but not of wild-type (+/+) littermates. The association of nuclear TUNEL staining with p75 labeling (on the cell surface) was particularly apparent when various confocal planes were analyzed (not shown). Note that NF-positive differentiated neurons were usually not associated with apoptotic figures at this stage (arrowhead in B). Scale bar: 10 µm. (C) Quantification of apoptotic figures in DRG of wild-type (+/+) and Sox10–/– (–/–) embryos. Each bar represents the number (mean±s.d.) of apoptotic nuclei in DRG per section. Two independent experiments using non-sibling embryos were performed, scoring 13 consecutive thoracic sections per experiment. (D) NRG1 is a Sox10-dependent survival factor of undifferentiated postmigratory neural crest cells. Postmigratory neural crest cells prepared from wild-type (+/+) or Sox10–/– (–/–) DRG were plated at clonal density, the position of single neural crest cells was mapped, and clones were allowed to develop in differentiation medium (‘no NRG1’) or in differentiation medium supplemented with NRG1 (‘NRG1’). The numbers indicate the percentage of clones lost owing to cell death of the founder neural crest cell. The data were obtained from four independent experiments counting 60 clones each. Note that cell death of wild-type cells but not of Sox10–/– mutant cells was significantly reduced in the presence of NRG1.

View larger version (67K): [in a new window]   Fig. 3. Loss of glial potential in Sox10–/– undifferentiated neural crest cells. (A-J) Cells isolated from DRG of wild-type or Sox10–/– mutant E13 embryos were either incubated in differentiation medium (‘no add’) or in differentiation medium supplemented with NRG1 (‘+NRG1’). In both conditions, surviving undifferentiated Sox10–/– mutant p75-positive cells (C,D) were not able to produce any O4-positive glia (F,G) but adopted the morphology of smooth muscle-like non-neural cells (I,J). Wild-type cells generated mainly O4-positive glia cells (E). (K) Quantitative clonal analysis of undifferentiated cells incubated in the presence of NRG1. G indicates clones containing exclusively glial cells; G/NN, clones containing glia and non-neural smooth muscle-like cells; NN, clones containing exclusively non-neural smooth muscle-like cells. For the quantitative analysis, expression of p75, NF160 and absence of these markers were used to distinguish between different fates. Numbers are given as percentage of all founder cells originally plated. Note that the indicated numbers of clone phenotypes do not sum to 100% because of cell death of a proportion of the founder cells (see Fig. 2). The data are expressed as mean±s.d. of four independent experiments. 60 clones were scored per experiment.

View larger version (42K): [in a new window]   Fig. 4. Haploinsufficiency of Sox10 affects glial fate acquisition but not survival of neural crest cells. Undifferentiated p75-positive cells isolated from DRG of wild-type and Sox10+/– mutant E13 embryos (A-D) were incubated at clonal density either in differentiation medium (‘no add’) or in differentiation medium supplemented with NRG1 (+NRG1). Wild-type cells produced mainly O4-positive glia-containing clones (E). Neurofilament-positive cells were not observed in either wild-type or in Sox10+/– clones (H-J). Sox10+/– clones contained no O4-positive cells (F,G). (N,O) Quantitative analysis (see legend to Fig. 3K). ‘Death’ indicates loss of clones. Note that glial fate specification is impaired in both conditions while cell death in Sox10+/– mutant cells is similar to wild-type cells. NRG1 is acting as a survival factor for Sox10+/– mutant cells and wild-type cells, independently of fate decisions. In both conditions a glial to non-neural cell fate switch was observed in the Sox10+/– experiments. (N) represents the data of four independent experiments and (O) the data of three independent experiments. Fifty clones were scored per experiment.

View larger version (77K): [in a new window]   Fig. 5. Neurogenesis of Sox10+/– mutant cells in high density cultures. Undifferentiated p75-positive cells isolated from DRG of wild-type and Sox10+/– mutant E13 embryos were incubated at high density in differentiation medium. Wild-type cells produced O4-positive glia (A) while de novo neurogenesis did not occur. Arrowhead in C indicates a NF-positive sensory neuron present at time of isolation. Only few O4-positive glia were generated in Sox10+/– mutant cultures (arrow in E). Many mutant cells formed aggregates of NF-positive neuronal cells (arrow in G). Insets in G,H show a neuronal cluster at higher magnification. After prolonged incubation, neuronal cells acquired features of fully differentiated neurons expressing peripherin (Per) (arrow in I).

View larger version (115K): [in a new window]   Fig. 6. Glial fate decision by Sox10+/– mutant cells is dependent on community effects and the presence of the instructive growth factor NRG1. (A,B) Normal expression of ErbB3 mRNA in wild type and Sox10+/– mutant embryos at E13. In situ hybridization analysis on transverse sections showed normal expression in DRG and peripheral nerves (arrow) of wild type and Sox10+/– animals. nt, neural tube. Scale bar: 20 µm. (C-F) Undifferentiated neural crest-derived cells isolated from DRG of wild type and Sox10+/– mutant E13 embryos were incubated at high density in differentiation medium supplemented with NRG1. Under these conditions, Sox10+/– mutant cells were able to differentiate into GFAP-positive glia (C,D) that after prolonged incubation expressed the glial differentiation marker O4 (E,F).

View larger version (22K): [in a new window]   Fig. 7. Summary of context-dependent fates: fate decisions are dependent on levels of Sox10, presence of soluble extracellular factors such as NRG1 and short-range cell-cell interactions. Summary of the observed phenotypes. Postmigratory neural crest cells isolated from wild-type DRG produce predominantly glia-containing clones in differentiation medium and in differentiation medium supplemented with NRG1, irrespective of their cellular context. By contrast, single Sox10 heterozygous cells undergo a fate switch and produce only non-neural cells at the expense of the glial fate. Moreover, in high density cultures that allow short range cell-cell interactions to occur, Sox10 heterozygous cells undergo limited gliogenesis while neurogenesis is a prominent fate. The promotion of gliogenesis in Sox10 heterozygous cells is dependent on the synergistic activities of short range cell-cell interactions and the presence of NRG1. In summary, extracellular signals influencing cell fate decisions are differentially interpreted by postmigratory neural crest cells, depending on the cellular context and the level of Sox10.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter