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 Mansouri, A. Articles by Gruss, P. Search for Related Content PubMed PubMed Citation Articles by Mansouri, A. Articles by Gruss, P. Social Bookmarking                         What's this?

Pax3 acts cell autonomously in the neural tube and somites by controlling cell surface properties

¹ Max-Planck Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg 11, D-37077 Göttingen, Germany
² Génétique du Développement des Mélanocytes, Institut CURIE – Section Recherche, UMR146 CNRS, Bat 110, Centre Universitaire, F-91405 Orsay, France

View larger version (75K): [in a new window]   Fig. 1. The expression pattern of the lacZ gene from the mutated Pax3 allele recapitulates the Pax3 expression pattern. Whole mount X-gal staining at different developmental stages. Embryos in A and B are heterozygous, embryos shown in E and F are homozygous for the mutation (A,E: E10; B,F: E10.75). Exencephaly and spina bifida are indicated in black arrows in E and F. The lateral dermomyotome in E and F is disorganized and limb muscle is detected only in A and B, as indicated by black arrows. The lack of limb muscle is also documented by transverse sections at the limb level in G and H (higher magnification of G) and corresponding sections of heterozygous embryos in C and D (higher magnification of C) (E10.5). In D, myoblasts are indicated by black arrows. DM, dermomyotome; NT, neural tube; sp, spina bifida; +/-, heterozygous; -/-, homozygous. Scale bars: 1 mm in A,B,E,F; 500 µm in C,G; 200 µm in D,H.

View larger version (21K): [in a new window]   Fig. 2. The aggregation procedure using Sp2H and Sp2G embryos. The scheme for one of the aggregation procedures using Sp2H and the allele generated by knocking-in the lacZ gene into the Pax3 locus. Briefly, heterozygous animals (Sp2H/+ and Sp2G/+) were intercrossed and morulae were prepared at E2.5 of gestation. In parallel, embryos from NMRI (albino) wild-type intercrosses were also prepared. Embryos from both matings were denuded from the zona pellucida using acidic tyrode solution (Hogan et al., 1994) and subjected to aggregation as indicated. The embryos from Sp2H and Sp2G crosses are on a mixed 129/sV and C57Bl/6 background and presented in gray. The embryos were left to develop overnight to blastocysts and subsequently transferred to foster mothers. Chimeric embryos were prepared at the appropriate time. DNA was prepared from embryonic membranes and both alleles are detected by PCR. Embryos that are positive for both alleles are supposed to contain homozygous Pax3-/- cells. The embryos that are positive only for lacZ are considered to consist of wild-type and Pax3+/- cells and are used as controls. In older embryos, the degree of chimerism is also monitored by eye pigmentation.

View larger version (48K): [in a new window]   Fig. 3. Highly chimeric (>80%) embryos exhibit with high incidence a Splotch phenotype. Most of the shown embryos (E10.5-E11.5) are highly chimeric as revealed by the strong X-gal staining. Also strong eye pigmentation can be recognized in some embryos. The chimeras were generated by aggregating Pax3-/- ES cells to NMRI morulae. Arrows point to spina bifida or exencephaly (neural tube defects), as described in Splotch embryos. Scale bar: 1mm.

View larger version (50K): [in a new window]   Fig. 4. Defects in the neural tube and dermomyotome of chimeric embryos are not rescued by wild-type cells. High chimeric embryos exhibit spina bifida and/or exencephaly. The remarkable contribution of mutant cells to the dermomyotome, documented by the X-gal staining, results in disorganized somites. In addition, the muscle precursor cells are not able to migrate into the limb (A,B) when compared with the embryo in C (chimera generated with Pax3+/- cells), where blue cells can be observed in the limb (white arrow). Black (A) and white (B) arrowheads show the disorganized dermomyotome. Black arrows in C indicate the regular and organized dermomyotome marked by Pax3+/- cells. Scale bars: 1 mm in A,C; 0.5 mm in B.

View larger version (87K): [in a new window]   Fig. 5. Segregation of mutant Pax3-/-from wild-type cells in the neural tube and dermomyotome. Whole-mount X-gal staining of several chimeric embryos of different stages and transverse sections of chimeric embryos at the limb and trunk level. The embryos shown in A-C are derived from ES+/- cells and exhibit no segregation of wild-type and mutant cells in the dermomyotome; corresponding embryos from ES-/- cells, however, exhibit segregation in the dermomyotome after E10 (G,H,K,L,O) and in the neural tube in J,M-O. The few white cells (+/+) in the neural tube are indicated by double white arrows on a transverse section of E10.25 embryo (O). The chimeras shown in D,E,I,J were generated according to the scheme in Fig. 2 using Sp2H and Sp2G mice. From this aggregation, segregation of wild-type and mutant -/- cells is shown at E12.5 (J). Black arrows in D,E,I,J indicate the DRG. In I, the DRG are affected and smaller in size. In the older embryo (J), wild-type cells rescue them, shown in transverse section of a highly chimeric embryo of E12.5 (chimerism in this embryo was determined by eye pigmentation, data not shown). DM, dermomyotome; DRG, dorsal root ganglia; +/-, heterozygous; -/-, homozygous. Scale bars: 400 µm in A-C,F-H,L,N; 200 µm in D,E,I,J; 300 µm in K; 600 µm in M; 100 µm in O.

View larger version (121K): [in a new window]   Fig. 6. Contribution of Pax3-deficient cells to the dermomyotome do not affect epithelial formation at E9.5. The high contribution of mutant cells to the dermomyotome at E9.5 for Pax3+/- and Pax3-/- ES cells is shown on frontal sections. The epithelial architecture looks normal indicating that Pax3 is not required for the initiation process. This is in good correlation with earlier findings showing that truncation of the lateral dermomytome occurs after E9.5 in Splotch embryos (Daston et al., 1996). (A) Section from a chimera generated with Pax3+/- cells. (B) Section from a chimera generated with Pax3-/- cells. DM, dermomyotome; SC, spinal cord. Scale bar: 100 µm.

View larger version (148K): [in a new window]   Fig. 7. The high contribution of mutant Pax3 cells to the dermomyotome results in a failure to maintain an organized epithelium. Transverse sections showing the dermomyotome of chimeric embryos at E11 at the limb level. (A-C) The dermomyotome appears highly organized. (A,C) the blue cells refer to chimeras generated from Pax3+/- ES cells. (B) Section from a chimera derived from Pax3-/- ES cells: there is no contribution of blue cells to the dermomyotome; however, the neural tube contains mutant cells (data not shown). In D-I, several transverse sections at the limb level are shown from chimeric E11 embryos with high contribution to the somite. They document the disorganized architecture of the epithelium made from blue Pax3-/- cells. Four embryos were analyzed. DM, dermomyotome. Scale bar: 100 µm.

View larger version (101K): [in a new window]   Fig. 8. Fgf8 is not detected in the dermomyotome of Splotch embryos. Whole-mount in situ hybridization showing the expression pattern of Fgf8 in control and Splotch embryos. Fgf8, which is normally expressed in the dermomyotome, is not detectable in the somite of Splotch and high chimeric (>80% of Pax3-/- cells) embryos at all stages analyzed (E9.0-E10.5). The expression in the hindbrain and limb bud is not affected in Splotch embryos. (A) Control embryo (E9.5); (B) Splotch2H embryo (E9.0); (C) Sagittal section of embryo in A showing the expression of Fgf8 in the caudal and rostral lips of the dermomyotome as indicated by black arrowheads, rostral is to the left; (D) chimeric embryo (generated with Pax3-/- ES cells) stained for ß-galactosidase and hybridized with Fgf8 probe; hybridization in the limb bud is indicated by arrows; in the somites only the X-gal staining is detectable. DM, dermomyotome. Scale bars: 500 µm in A,B,D; 100 µm in C.

View larger version (130K): [in a new window]   Fig. 9. Apoptosis is detected in Pax3-deficient cells of the lateral dermomyotome but not in the neural tube. Cell survival is analyzed in the somites and in the neural tube of chimeric embryos using the TUNEL assay. (A,D,G) Apoptosis staining using TUNEL assay; (B,E,H) corresponding adjacent sections showing X-gal staining; and (C,F,I) corresponding adjacent sections showing DAPI staining. (A-C) Chimera with blue Pax3-/- cells in the dermomyotome showing apoptosis only in the lateral dermomyotome (white arrow). In wild-type embryos (D-F) no apoptosis is detected. (G-I) TUNEL assay in the spinal cord of a chimeric embryo as indicated by the X-gal staining (H). No apoptosis is detectable in Pax3-/- cells (blue cells) of the neural tube. Three embryos analyzed for somite and four for spinal cord. Scale bar: 100 µm.

View larger version (48K): [in a new window]   Fig. 10. Pax3-deficient neural crest cells are able to migrate in chimeric environment of mouse and chick. (A-D) Several embryos show the migration of Pax3-deficient neural crest cells in an environment mixed with wild-type cells. All the presented chimeras are made using aggregation of Pax3-deficient ES cells. (A,B) The migration of the neural crest cell is indicated by white arrows in two different chimeric embryos at E9.0 and E9.75, and stained in whole mount for ß-galactosidase. At E11.5, DRG are detected in embryo C, as revealed by whole-mount X-gal staining; white arrowheads point to some of the ganglia between forelimb and hindlimb. In D, a frontal section from an E9.0 embryo documents the migration of neural crest cells at the hindbrain and at the more caudal neural tube level (arrows). In E,F, grafted Pax3-deficient ES cells migrate on the DL and on the DV neural crest cell pathway of the chicken embryo. (E) Transverse section of a chicken embryo (in red) fixed 18 hours after the graft of Pax3/9 homozygous ES cells (in green). These embryos were analyzed by optical scanning using a confocal scanning fluorescence microscope. ES cells were previously labeled with a vital green fluorescent marker (CFSE). After fixation, embryos were immunostained with an anti-laminin antibody that recognised the basal lamina of the embryo (red). (F) DL view of the trunk of an X-gal-stained embryo fixed 18 hours after grafting of Pax3/9 homozygous ES cells. The cells located on the DV pathway of neural crest cells are not visible. The black bar on the side of the neural tube indicates the grafting site. (G) Statistical analysis of embryos grafted with Pax3/6 heterozygous ES cells (n=24) or Pax3/9 homozygous ES cells (n=26). The diameter of the pie charts represents the percentage of positive grafted embryos. The embryos were classified as follows: embryos with no migrating cells (pink areas), embryos containing cells migrating solely on the DV pathway (blue areas), embryos containing cells migrating on the DL and DV pathways (green areas). (H) Analysis of embryos containing cells migrating on the DL and DV pathways. For each embryo, the cells were migrating preferentially either on the DV pathway (maj DL) or on the DV pathway (maj DV). D, dermomyotome; NT, neural tube; S, somite. Scale bars: 300 µm in A-C; 100 µm in D; 15 µm in E; 200 µm in F.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter