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First published online 4 May 2005
doi: 10.1242/dev.01852

Development 132, 2623-2632 (2005)
Published by The Company of Biologists 2005
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The boundary cap: a source of neural crest stem cells that generate multiple sensory neuron subtypes

Jens Hjerling-Leffler1, Frédéric Marmigère1, Mikael Heglind2, Anna Cederberg2, Martin Koltzenburg3, Sven Enerbäck2 and Patrik Ernfors1,*

1 Unit of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden
2 Unit of Medical Genetics Department of Medical Biochemistry Göteborg University, 405 30 Göteborg, Sweden
3 Institute of Child Health, University College London, London WC1N 1EH, UK



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  		Fig. 1. The stem cells express neural crest and sensory lineage-specific markers. (A-D,F) Undifferentiated dissociated cells were plated 5 hours on PDL and immunocytochemically stained as indicated below. Cells were counterstained with nuclear Hoechst staining. All cells with an intact nucleus expressed the stem cell/progenitor marker nestin (red) and the low-affinity neurotrophin receptor p75NTR (green), as well as the GDNF family receptor Ret (red in F). (E) Expression of indicated transcription factors and receptors in the E11.5 dorsal root ganglion (DRG) and stem cells (SCs) determined by RT-PCR. Stem cells expressed mRNAs for the neuronal marker Ret as well as the sensory lineage markers Brn3a, Ngn1 and Ngn2. Notably the stem cells expressed the transcription factor Krox20, a transcription factor exclusively expressed by boundary cap cells lining the DREZ. The stem cells did not express mRNAs encoding CNS and kidney specific transcription factors Otx1, Pax2 and Pax5. Abbreviations: DRG, dorsal root ganglia; SCs, stem cells. RT-, reverse transcriptase negative. Scale bars: 25 µm.

 


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  		Fig. 2. The boundary cap contains NCSCs. (A) In situ hybridisation for Krox20 on a coronal section of E10.75 mouse embryo showing the dorsal part of the DRG and the boundary cap. (B) Immunocytochemistry for Maob (red) on adjacent section. (C) In situ for Krox20 on a coronal section of E11.75 mouse embryo. (D) Immunocytochemistry for Maob (red) on adjacent section. (E,F) Maob expression in dorsal root ganglion cells 2 hours (E) and 12 hours (F) after plating. (G) Enrichment of stem cells at 12 hours correlated with a several-fold increase of Maob+ cells. The enrichment is not due to transcriptional upregulation or proliferation of the Maob-positive population as it persists in the presence of actinomycin D and cytosine arabinoside (G). Notably, no change was seen in the absolute numbers of Maob-positive cells (data not shown). (H) A coronal section through an E11.5 thoracic dorsal root ganglion stained with Cresyl Violet with BC and dorsal root ganglion indicated. The dorsal root ganglion is outlined. The black lines indicate the parts isolated by micro-dissection of the dorsal root ganglia into a BC part (b) and a central part (c). (I) RT-PCR for Krox20 on dissected tissue with HPRT as internal reference for starting material. Krox20 mRNA is present only in the BC but not in the central part. (J) When cells from the BC and central parts were cultured in propagation medium separately, there was a significant difference in the number of stem cell clones produced from the different samples (***P<0.001, Mann-Whitney), showing that the stem cells are located in the BC. Abbreviations: Actino D, Actinomycin D; Ara-C, cytosine arabinoside; BC, boundary cap; c, central part; b, boundary cap part; DRG, dorsal root ganglia; SC, spinal cord. Scale bars: 50 µm.

 


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  		Fig. 3. bNCSCs generate sensory neurons. (A) Example of a differentiated stem cell clone showing two populations of neurons, one double labelled with peripherin and ßIII-tubulin (Tuj1, yellow) and one only expressing ßIII-tubulin (red). (B) Example of a differentiated stem cell clone produced from the Fkh3lac-z /+ mice stained with X-gal, showing that the bNCSCs give rise to Fkh3+ cells of the sensory lineage. (C) Percentage clones positive for peripherin and Fkh3 (n=58 and 25, respectively). (D) Differentiated bNCSCs stained for CGRP (red) and peripherin (green) showing a mature sensory phenotype. Abbreviations: CGRP, calcitonin gene-related peptide Scale bars: in 100 µm in A,B; 25 µm in D.

 


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  		Fig. 4. Fkh3 specifically marks cells in the sensory lineage. (A,B) Whole-mount staining of a Fkh3 lac-z/+ mouse for ß-gal at E11.5 shows expression of ß-gal highly restricted to cranial and spinal sensory ganglia (schematic in B). (C) Coronal section through the lumbar region of the embryo showing that ß-gal is highly restricted to the sensory dorsal root ganglia at a cellular resolution. Abbreviations: Cerv, cervical; DRG, dorsal root ganglia; FGn, facial ganglion; JGn, jugular ganglion; Lumb, lumbar; Sacr, sacral; SGn, superior ganglion; TGn, trigeminal ganglion; Thor, thoracic. Scale bars: 1 mm in A; 100 µm in C.

 


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  		Fig. 5. bNCSCs show an intrinsic ability to differentiate into the sensory lineage. Comparison between bNCSCs and NCSCs isolated from the sciatic nerve from E12.5 mouse embryos (sNCSCs) established with the same protocol as the bNCSCs. (A) A 50 ng/ml Bmp2 treatment during a 5-day differentiation leads to increased neuronal differentiation in both types of NCSCs. Black bars are Bmp treated; white bars are untreated cultures. (B,C) ßIII-tubulin immunocytochemistry of NCSCs treated with 50 ng/ml of Bmp2. sNCSCs are larger in size. (D,E) Same cells as in B and C showing lack of immunoreactivity for tyrosine hydroxylase. (F,G) X-gal staining of cells differentiated for 5 days showing Fkh3-positive cells in the bNCSCs, but not in the sNCSCs. (H) Quantification of experiments in F,G (n=25 and n=45, respectively). Abbreviations: BC, boundary cap; SN, sciatic nerve; Bmp, bone morphogenic protein. Scale bars: in B, 25 µm for B,D; in C, 25 µm for C,E; 25 µm in F,G.

 


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  		Fig. 6. bNCSC-derived sensory neurons are physiologically functional and of different subclasses. Kinetic profiles of Ca2+ transients of single cells in bNCSC cultures, as well as in dissociated adult dorsal root ganglion cells. Each track indicates the change of the ratiometric fluorescence signal ({Delta}F) from a single neuron during its response to stimuli. The duration of the stimuli is depicted underneath the curve by black horizontal bars. (A) Three different cell types with a differential response to capsaicin and hypo-osmolarity showing the presence of both mechanoinsensitive and mechanosensitive nociceptors. The bottom traces could represent either a non-nociceptive mechanoreceptor or a mechanosensitive capsaicin-insensitive nociceptor. The vertical bars represent a {Delta}F of 0.1. (B) Three different cell types with a differential response to cold, menthol and capsaicin, showing the presence of both innocuous cool and noxious cold receptors, as well as a cold responsive cell not expressing TRP channels. The vertical bars represent a {Delta}F of 0.05. Abbreviations: bNCSC, boundary cap neural crest stem cell; DRG, dorsal root ganglion; Hypoosm, 30% hypoosmolarity; KCl, 100 mM potassium chloride.

 


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  		Fig. 7. A single bNCSC clone gives rise to multiple functional subclasses of sensory neurons. Cumulative graph of the percentage of clones producing multiple sensory subtypes. Twenty-five clones were analysed with ratiometric Ca2+ imaging and different subtypes (cells with different response profiles) within each clone was counted. The results show that a majority of the clones (72%) produce more than one sensory neuron subclass.

 

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© The Company of Biologists Ltd 2005