Heads of wild-type (A) and PGK:Cre;Dbx1^DTA (B) E12.5 mouse embryos. Mutants are exencephalic and display craniofacial malformations. (C–H) Alternate sagittal sections of wild-type (C, E, G) and PGK:Cre;Dbx1^DTA (D, F, H) E12.5 embryos subjected to Nissl staining (C, D), emx2 (E, F) and foxg1 (G, H) in situ hybridisation. (I–J) Coronal sections of wild-type (I) and PGK:Cre;Dbx1^DTA (J) E12.5 embryos stained by Nissl. The antero-posterior level at which coronal sections were collected is indicated by yellow dashed lines in C and D. The arrow in J points to the remaining telencephalon in ablated embryos. (K–M) High magnification of these telencephalic tissues in PGK:Cre;Dbx1^DTA E12.5 embryo following emx2 (K), ngn2 (L) and dlx1 (M) in situ hybridisation. Scale bars A: 500 µm, C, I: 400 µm, K: 200 µm.

Side (A, B, E, F) and front (C, D) views of wild-type (A, C, E) and PGK:Cre;Dbx1^DTA (B, D, F) E9.5 embryos (18–19ss) subjected to foxg1 (A–D) and wnt1 (E, F) whole mount in situ hybridisation. Most of neural tissues anterior to the midbrain-hindbrain boundary (arrow in F) are missing in ablated mutants. Scale bar: 200 µm.

(A–J) Dbx1 expression analysed by whole-mount in situ hybridisation. B and D are dorsal views of the embryos presented in A and C respectively, rostral is up. F, H and J are frontal views of the embryos shown in E, G and I respectively. Antero-posterior subdivisions of the neural plate/tube are indicated. Mb: midbrain, Fb: Forebrain, Mes: mesencephalon, Di: diencephalon, Tel: telencephalon. Arrows in E, G and I point to the vDi, dDM and FE subsets respectively; the arrowhead in I shows the ANR subset. (K, L) Coronal sections through the forebrain of a 12ss embryo showing dbx1 expression in the ANR (K) and facial ectoderm (L). (M) Coronal section collected at the midbrain level of a 3ss embryo illustrating dbx1 mRNA expression; the neural plate is delineated by dashed lines. (N) Coronal section collected at the level of the midbrain of a 6ss Dbx1^Cre;R26^YFP embryo and stained for YFP (green) and DAPI (blue). (O) Quantification of dbx1 expression by qPCR. Asterisks indicate stages at which dbx1 could not be amplified. The relative expression values (normalised to 1 at 2ss and expressed in arbitrary units) are indicated on top of each bar as mean ± s.e.m. (P, Q) Coronal sections of a 20ss (E9.5) Dbx1^Cre;R26^YFP embryo at the level of the telencephalon (P) and di/mesencephalon (Q), stained for YFP (green) and DAPI (blue). YFP is detected in regions that express dbx1 at earlier stages: ANR (red arrowhead in P), FE (white arrowheads in P), vDi and dDM (red and white arrowheads in P respectively) but also in cells located in the head mesenchyme (arrows in P). (R–W) Examples of YFP⁺ cells found in the head mesenchyme of Dbx1^Cre;R26^YFP embryos at 8ss (R–T) and 11ss (U–W) revealed by DAB immunostaining. Sections in R, S and U were collected at telencephalic levels, T, V and W at diencephalic levels. Neural tissues, surface ectoderm and endoderm are surrounded by black, blue and green dash lines respectively. (X) Coronal sections of a 8ss Dbx1^Cre;R26^YFP embryo immunostained for YFP (green) and the neural crest cells marker AP2α (red); cell nuclei are stained by DAPI (blue). Some of the YFP⁺ cells found in the neural plate are AP2α⁻ (arrowhead in X₁) whereas YFP⁺ cells located at the most lateral aspect of the neural plate or in the mesenchyme (arrows in X₁–X₃) are AP2α⁺. Images X₁–X₃ correspond to single confocal planes. (Y) Schematic drawing of dbx1 expression pattern. Scale bars: A: 200 µm; K, M, N, R, X: 50 µm; P: 100 µm.

(A–H) Whole-mount in situ hybridisation of 5 to 6ss control (A–D) and PGK:Cre;Dbx1^DTA (E–H) embryos using probes against six3 (A, E), emx2 (B, F), pax6 (C, G) and otx1 (D, H). (I–J) Coronal sections collected at the level of the midbrain of 5ss control (I) and PGK:Cre;Dbx1^DTA (J) embryos immunostained for phosphorylated histone-3 (PH3, green) and DAPI (blue). (K–L) Higher magnification of the DAPI staining, pyknotic nuclei are highly fluorescent. Quantifications (M, N) revealed a significant reduction in the number of PH3⁺ cells in the neural plate, but not in the mesenchyme, of mutant compared to controls (M) as well as a significant increase in the number of pyknotic cells per section (N). * p<0.05 in M and p<0.02 in N; n = 3 controls and 4 mutant embryos (4 to 8ss). Scale bars: A: 200 µm; I: 50 µm.

Whole-mount TUNEL staining of 2ss (A–D) and 11–13ss (I–L) control (A, C, I, K) and PGK:Cre;Dbx1^DTA (B, D, J, L) embryos. C and D are dorsal views of the embryos shown in A and B respectively. (E–H) are cryosections of the embryos shown in A and B at the level of the forebrain (E, F) and midbrain (G, H) as indicated by dashed lines in C and D. K and L are front views of the embryos shown in I and J respectively. M and N are cryosections collected at the level of the diencephalon of these embryos as indicated by dashed lines in I and J. Neural tissues are surrounded by a dashed line for better visualisation. Scale bars: A, I: 200 µm; E, M: 100 µm.

Whole-mount TUNEL staining of 3 to 4ss (A–D) and 8 to 9ss (I–L) control (A, I), Nkx2.1:Cre;Dbx1^DTA (B, J), Foxg1^Cre;Dbx1^DTA (C, K) and Wnt1:Cre;Dbx1^DTA (D, L) embryos. E–H and M–P are cryosections of the embryos shown in A–D and I–L collected at the level indicated by a dashed line. Neural tissues are surrounded by a dashed line for better visualisation. (Q–T) Immunostaining for activated Caspase-3 (red), YFP (green) and DAPI (blue) on coronal cryosections of Nkx2.1:Cre;R26^YFP;Dbx1^DTA (Q), Foxg1^Cre;R26^YFP;Dbx1^DTA (R), Wnt1:Cre;R26^YFP;Dbx1^DTA (S) and control (T) embryos. Images in Q–T correspond to a single confocal plane. Arrows point to Caspase-3⁺/YFP⁺ cells, arrowheads indicate Caspase-3⁺/YFP⁻ cells. Scale bars: A, I: 200 µm; E, M, Q, R: 100 µm; S, T: 50 µm.

Heads of wild-type (A), Nkx2.1:Cre;Dbx1^DTA (B), Foxg1^Cre;Dbx1^DTA (C) and Wnt1:Cre;Dbx1^DTA (D) E12.5 mouse embryos. (E–H) Nissl staining of coronal sections collected at the level of the eyes of the embryos shown in A–D. Scale bars: 500 µm.

(A) Head of an E12.5 Triple-Cre;Dbx1^DTA embryo. The red arrowhead indicates the forebrain. (B) Nissl-stained coronal section of an E12.5 Triple-Cre;Dbx1^DTA embryo at the level of the eyes. (C–E) Dorsal views of whole-mount in situ hybridisation for dbx1 on wild-type (C), PGK:Cre;Dbx1^DTA (D), Triple-Cre;Dbx1^DTA (E) 4ss embryos. A significant number of dbx1-expressing cells (arrows) appear to escape ablation in Triple-Cre ablated embryos. The strong staining in D corresponds to the (out of focus) rhombencephalic expression of dbx1, and appears obvious because revelation time had to be increased in PGK:Cre ablated embryos in order to visualise the very few positive cells that remain. (F–H) Whole-mount TUNEL staining on wild-type (F), PGK:Cre;Dbx1^DTA (G), Triple-Cre;Dbx1^DTA (H) 4ss embryos. I–K are sections of these embryos collected at the levels indicated by dashed lines. Less apoptotic cells are found in Triple-Cre than in PGK:Cre ablated embryos. (L–Q) Whole-mount in situ hybridisation for sox10 on wild-type (L, O), PGK:Cre;Dbx1^DTA (M, P) and Triple-Cre;Dbx1^DTA (N, Q) embryos at 4ss (L–N) and 8ss (O–Q). At 4ss, PGK:Cre;Dbx1^DTA embryos show a strong decrease in the number of sox10⁺ premigratory CNCCs whereas Triple-Cre;Dbx1^DTA are less severely affected. At 8ss, very few sox10⁺ CNCCs have invaded the cephalic mesenchyme in PGK:Cre;Dbx1^DTA whereas Triple-Cre;Dbx1^DTA are not as severely affected. Scale bars: A, B: 500 µm; C, O: 200 µm; I: 100 µm.