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

First published online 6 October 2004
doi: 10.1242/dev.01420

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 Plachta, N. Articles by Barde, Y.-A. Search for Related Content PubMed PubMed Citation Articles by Plachta, N. Articles by Barde, Y.-A.

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells

¹ Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
² Novartis Institutes for Biomedical Research, Neuroscience, CH-4002 Basel, Switzerland
³ Interdisciplinary Center for Neurosciences, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany

View larger version (60K): [in a new window]   Fig. 1. Characterization of CNS progenitors in RA-treated EBs. Cryosections (12 µm thick) of EBs after 4 days of RA treatment (see Materials and methods), double labelled with a nuclear stain (upper rows) and with the indicated markers (lower rows). The majority of the cells express nestin, Sox2, Rc2, Glast and Pax6, a profile characteristic of neurogenic RG cells. Note that the expression of these markers is evenly distributed throughout the EB. RA-treated EBs contain very few cells (<1%) expressing the markers Lim1/2, Mnr2 and Isl1, and no cells expressing Pax7, Nkx2.2 or Olig2. A 3-hour pulse with BrdU shows the presence of proliferative cells in RA-treated EBs. At least five EBs from different experiments were analyzed for each marker. Scale bar: 100 µm.

View larger version (94K): [in a new window]   Fig. 2. Progenitors from RA-treated EBs integrate in the chick neural tube. (A) A segment of one neural fold (400 µm long) was removed and discarded at the level of the forelimb bud of E2 chick embryos. EBs were dissociated with trypsin and implanted (see Materials and methods), and embryos were allowed to develop until E8. (B) Identification of donor cells by nuclear morphology. Nuclear staining with Hoechst of 16 µm-thick cryosections reveals differences in morphology between chick and mouse nuclei. (C,D) Transverse sections (16 µm thick) through chick embryos operated on as described in A. (C) Donor cells survive in large numbers and differentiate into GFP+ neurons throughout the operated side of the spinal cord. Left image shows nuclear staining revealing spinal cord morphology. Note that the operated side resembles the non-operated side in size and morphology. (D) GFP+ neurons populate the ventral spinal cord and extend axons towards the periphery. Axons can be observed projecting dorsally (arrowhead) and ventrally (arrow). GFP signal co-localizes with neurofilament (NF-M) staining (yellow). Scale bar: 10 µm in B; 100 µm in C,D.

View larger version (107K): [in a new window]   Fig. 3. Progenitors from RA-treated EBs differentiate into defined neuronal subtypes. (A-K) Transverse sections through E8 chick embryos operated on at E2, as described in Fig. 2, showing endogenous GFP signal and labelled for the indicated markers. (A) GFP+ neurons located at the middle spinal cord express Lim1/2 (merged yellow). Ventrally located GFP+ neurons express the motoneuron markers Mnr2 (B) and Isl1 (C). Note in B, the GFP+ axons (arrow) projecting from the MNR+/GFP+ cells towards the periphery. (D-I) Expression of neurotrophin receptors by donor cells. (D) p75 expression is detected in ventrally located GFP+ neurons and their axons (arrow). (E,F) Higher magnification of the GFP+ somata and the axons indicated by the arrow in D. (G) Expression of Trk receptors is detected in ventrally located GFP+ neurons using pan-Trk antibodies. (H,I) Higher magnification of the GFP+ somata and the axons indicated by the arrow in G. (J-K) Consecutive sections of the same embryo, double labelled by Hoechst (J,K), and by Lim1/2 (J') or Mnr2 (K'). GFP+ cells located at the middle spinal cord express Lim1/2 (J' shows a higher magnification of the same section), but do not express Mnr2 (K'). Scale bar: 50 µm in A-C,E,F,H,I; 100 µm in J-K'; 200 µm in D,G.

View larger version (91K): [in a new window]   Fig. 4. Progenitors from RA-treated EBs fail to acquire sensory neuron characteristics. (A) Host DRG (asterisk, Nomarsky image, left) is shown to be colonized by GFP+ cells (right). Donor cells colonized much of the host DRG, and the majority expressed GFP, indicating their neural differentiation. Note the lack of GFP+ processes. In this section (see also Fig. 6), mouse donor cells have migrated longitudinally, away from the site of implantation, and the spinal cord segment at this level is occupied mostly by chick cells. Expression of Brn3a (B) and Isl1 (C) in DRGs colonized by GFP+ cells. Note the absence of Brn3a and Isl1 expression by GFP+ cells (no merged yellow). (D) GFP+ cells express high levels of the p75 neurotrophin receptor in the DRG (merged yellow). (E) Expression of Trk receptors is detected in chick cells in the DRG (arrow), and in their axons (arrowhead) projecting to the spinal cord, but not in GFP+ donor cells. Cells expressing Brn3a or Isl1 always exhibited chick nuclear morphology (not shown). drez, dorsal root entry zone; sc, spinal cord. Scale bars: 100 µm in A; 50 µm in B-E.

View larger version (99K): [in a new window]   Fig. 5. Developmental potential of cells from RA-untreated EBs. ES cells were allowed to form EBs for 36 hours and then implanted into the chick neural tube as described in Fig. 2. (A) Cryosections (12 µm thick) through EBs double-labelled by Oct3/4 antibodies (upper row) and the nuclear staining Hoechst (lower row). After 36 hours, the majority of the cells within the EB express Oct3/4 (left column). After 8 days (4 without and 4 with RA), very few cells within the EB maintain Oct3/4 expression (right column). (B-F) Transverse sections through E8 chick embryos operated on as described previously, showing endogenous GFP signal and labelled for the indicated markers. (B) ES cell-derived GFP+ cells located at the middle spinal cord express the interneuron marker Lim1/2 (merged yellow). Ventrally located GFP+ cells express the motoneuron markers Mnr2 (C) and Isl1 (D). GFP+ cells in the ventral spinal cord elongate axons towards the periphery; both their somata (arrowheads) and axons (arrows) are labelled by p75 (E) and pan-Trk (F) antibodies. Scale bar: 100 µm in A; 25 µm in B-D; 100 µm in E,F.

View larger version (78K): [in a new window]   Fig. 6. Cells from RA-untreated EBs differentiate into DRG neurons and extend axons. (A) Host DRG (asterisk, Nomarsky image, A) is shown to be colonized by ES cell-derived GFP+ neurons (A'). Note GFP+ axons (arrow) projecting towards the spinal cord. (B,B') Donor axons are detected by endogenous expression of GFP (B') both in the DREZ (arrow) and in the spinal cord (arrowhead). (C-E) Expression of Brn3a (C), Isl1 (D) and p75 (E) by many GFP+ neurons (merged yellow) in host DRG. (F) Expression of pan-Trk in host spinal cord, ventral root, and in the DRG (asterisk), which is colonized by GFP+ cells. Higher magnification images of the same section show pan-Trk expression by GFP+ cells in the DRG (G), and also in their axons joining the ventral root (H). (I,J) GFP+ cells colonize both the spinal cord and the DRG (asterisk). I is stained for Mnr2; J for Lim1/2. GFP+ cells located only in the ventral spinal cord express Mnr2 (also shown in I' as a higher magnification of the same section), while GFP+ cells located throughout the spinal cord express Lim1/2. Note that no GFP+ cells express these markers in the DRG (asterisk). White lines delineate the spinal cord and DRG limits. drez, dorsal root entry zone; sc, spinal cord; vr, ventral root. Scale bar: 100 µm in A,F,I; 50 µm in B; 25 µm in C,E,G,H,J; 12.5 µm in D.