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1 Supporting Information Creuzet /pnas Fig. S1. Migrating CNC cells express Bmp inhibitors. (A) HNK1 immunolabeling of migrating CNC cells at 7 ss. (B) At this stage, Fgf8 is strongly expressed in the presumptive territory of the ANR and in the isthmus. Additionally, Fgf8 transcripts are present in the superficial ectoderm and the anterior intestinal portal. (C) Merged picture shows that, shortly after the onset of their migration at 6 ss, i.e., 90 min later, CNC cells surround the domain of Fgf8 expression in the ANR. (D-G) Gremlin and Noggin expression at neurula (5 ss) and pharyngula (25 ss) stages. (D) Gremlin transcripts are accumulated in the cephalic neural folds at 5 ss before the onset of CNC cell migration. (E) At 25 ss, CNC cells, which cover the prosencephalic vesicles, strongly express Gremlin.(F) Similarly, Noggin is activated in CNC at 5 ss. (G) However, at 25 ss, Noggin activation concerns the CNC cells that have populated the nasofrontal and maxillomandibular buds; its expression is particularly important in the retro-ocular area as well as in regions flanking the mesencephalic and diencephalic vesicles. 1of6
2 Fig. S2. Modulation of morphogen activity by CNC cells at neurula stage. (A and B) Bmp2 expression of 7-ss control (A) and CNC-ablated (B) embryos is not altered by the operation. (C and D). Shh (sonic hedgehog) transcripts are equally present in notocord and Pcp of control (C) and CNC-ablated (D) 7-ss embryos. (E-J) Bmp7 expression in the Pcp of control (E and E ), dsnoggin-treated (F,G, and G ), and dsnoggingremlin-treated embryos (F,H and H ). Examination of the ventral side of dissected embryos revealed that knocking down Bmp inhibitors entails a rostral expansion of Bmp7 expression in the Pcp (G and H ; dotted lines; compare with E ). However, such an induction remains limited compared with the induction of Bmp7 resulting from CNC ablation (I, J, and J ). 2of6
3 Fig. S3. Transfection of Noggin-RCAS in ANR in CNC-ablated embryos. Comparison of Fgf8 expression and CNC cell migration (HNK1 Mab labeling) in control (A, D, and G), CNC-ablated (B, E, and H), and CNC-ablated and Noggin-treated (C, F, and I) embryos. Fgf8 expression (D) is abolished by CNC ablation (E) but restored when Noggin (C, in green) is transfected in ANR. This coincides with the massive progression of CNC cells coming from the edge of the excised territory (I; compare to H). Fgf8 activation in ANR resulting from the transfection of Noggin-RCAS concurs in restoring, to a large extent, the development of the experimental brain (K; compare with control in J). However, rescued brains are hypoplastic and harbor midline defects: the optic tectum remains as a unique lobe and the interhemispheric structures are underdeveloped. 3of6
4 Fig. S4. Respective effects of Gremlin and Noggin uptake on brain morphogenesis. (A) Schematic representation of the operation: retroviral constructs (in red) are injected in the neural groove and headed for bilateral CNC transfection by a triangular electrical field at 5 to 6 ss in quail embryo. Transfected neural folds are then engrafted into un-transfected stage-matched recipient embryo. (B-G) Gross anatomy of isolated brains at E8 taken from control (B and C), Gremlin-treated (D and E), and Noggin-treated embryo; lateral (B, D, and F) and ventral (C, E, and G) aspects of brain preparations are shown. Despite subtle differences, each of the Bmp inhibitors, when separately overexpressed in CNC cells, slightly stimulates longitudinal and transverse growth of mesencephalic and prosencephalic vesicles. 4of6
5 Fig. S5. Changes borne on face and brain by GremlinNoggin-RCAS transfected CNC cells. (A and B) Frontal view of E4 control (A) and experimental (B) embryos. The latter has been engrafted with GremlinNoggin-RCAS-transfected CNC and exhibits a considerable expansion of the telencephalic vesicles. (C) Measurements of cerebral growth on profile view: lens-dorsal telencephalon distance (n 1), lens-epiphysis (n 2), lens-optic tectum (n 3), and optic vesicle diameter (n 4). (D) Measurements of facial growth on frontal view: outer inter-canthal distance (n 6), inter-alar distance (n 7), inter-commissural distance (n 8), and mandibular arch length (n 9). (E) Graph representing the percentage of changes on brain (in red) and face (in cyan) development, respectively, following Gremlin and Noggin overexpression in CNC (n 10). Whereas the facial morphogenesis is slightly increased ( 3%), the growth of cephalic and optic vesicles is strongly stimulated under the influence of transfected CNC (up to 30%). (F and G) Gross anatomy of E8 control (F) and experimental (G) embryos show that the morphogenesis of the upper and lower beaks is barely modified by the uptake of Bmp antagonists in CNC cells (compared with eye morphogenesis). 5of6
6 Fig. S6. Schematic representation of the multiple role of CNC cells in brain development. The CNC cells interact with the adjacent neuroepithelium to inhibit Bmp4 effect and maintain neural tube closure and counteract on Bmp7 expression in the Pcp to promote brain vesicle expansion and Fgf8 induction in ANR. In addition, CNC cells thwart Bmp4 activity around the ANR to favor the maintenance of Fgf8 expression in the prosencephalic organizer, thus ensuring the proper patterning of the telencephalic hemispheres. 6of6
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