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doi: 10.1242/10.1242/dev.00487

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The isthmic organizer signal FGF8 is required for cell survival in the prospective midbrain and cerebellum

¹ Department of Anatomy and Program in Developmental Biology, School of Medicine, University of California at San Francisco, San Francisco, CA 94143-0452, USA
² Institute of Neurosciences, University Miguel Hernandez, San Juan, Alicante, Spain 03550
³ GSF-Research Center, Institute for Developmental Genetics, 85764 Munich/Neuherberg, Max Planck Institute of Psychiatry, 80804 Munich, Germany

View larger version (73K): [in a new window]   Fig. 1. Analysis of Cre-mediated inactivation of Fgf8 in MHB KO mutants. (A) Schematic representation of the mutant alleles carried by En1Cre/+, Fgf8flox/null embryos (MHB KO mutants) and a reporter gene used to detect Cre activity. Cre-mediated recombination of the R26R reporter gene deletes the neo sequences flanked by loxP sites, and allows expression of lacZ. Cre-mediated recombination of Fgf8flox deletes crucial exons, creating the Fgf8null allele. (B,C) Dorsal views of embryos at the somite stages (s) indicated, assayed for En1 expression by RNA whole-mount in situ hybridization (B), or for Cre-mediated recombination by staining for ß-galactosidase (ß-GAL) activity (C). (D) Control and MHB KO mutants hybridized with a probe that detects Fgf8 exon 3. Embryos at the 7 somite stage are shown in lateral view only; those at the 11 somite stage are shown in both lateral (left) and dorsal (right) view. Red asterisks indicate the regions in which Fgf8 expression is detected in control, but not in mutant embryos. frt, frt site present in the Fgf8flox and Fgf8null alleles, as described by Meyers et al. (Meyers et al., 1998); pro, promoter; s, somite stage.

View larger version (72K): [in a new window]   Fig. 2. Analysis of mes-met derivatives in Fgf8 loss of function mutants. (A-F) Analysis of midbrain, isthmus and cerebellum phenotypes at E17.5. (A,B) Lateral views of control embryos and MHB KO mutants at E17.5. The arrow in B points to the part of the head that is misshapen due to the absence of the midbrain and cerebellum. Note that craniofacial development appears normal in the mutant embryo. (A',B') Dorsal views of the brains from the embryos shown in A and B. (C-F) Paramedial sagittal sections of control, Fgf8neo/neo, Fgf8neo/null and MHB KO mutant brains at E17.5, stained with Cressyl Violet. The arrow in each panel points to the caudal limit of the posterior commissure, which marks the rostral limit of the midbrain. The bulk of the dorsal midbrain (tectum) is subdivided into the superior colliculus (rostral) and inferior colliculus (caudal). (C'-F') Lateral sagittal sections of the same brains, assayed by immunohistochemistry with an antibody against tyrosine hydroxylase, which specifically stains nuclei in the ventrolateral midbrain (tegmentum) and anterior hindbrain. The pontine nuclei are present in all samples, but are outside of the field in the control. (G-J) Embryos at E10.5, assayed by whole-mount immunohistochemistry with an anti-neurofilament antibody to label the cranial nerves and ganglia. III, oculomotor nerve; IV, trochlear nerve; V, trigeminal nerve and ganglia; VII, facial nerve and ganglia; VIII, acoustic nerve and ganglia; Cb, cerebellum; CP, choroid plexus; Di, diencephalon; IC, inferior colliculus; LoC, locus ceruleus; Mb, midbrain; OB, olfactory bulb; PC, posterior commissure; PN, pontine nuclei; PPT, posterior pretectal nucleus; Pt, pretectum; SC, superior colliculus; SNC, substantia nigra pars compacta; Tel, telencephalon; VTA, ventral tegmental area.

View larger version (76K): [in a new window]   Fig. 3. Analysis of cell death in Fgf8 MHB KO mutants. (A-D) Whole-mount control and MHB KO embryos assayed for cell death by staining with Nile Blue Sulfate (NBS) or by TUNEL at the stages indicated. All embryos are shown in dorsal view, except for the TUNEL-stained mutant (20 somite stage) and control (24 somite stage) embryos, which are shown in lateral view. Many dying cells are detected in control embryos, particularly at the lateral edges of the neural plate, which will fuse to form the dorsal midline of the neural tube. The schematic diagrams summarize the results of the assays. Areas of normal and ectopic cell death are indicated in blue and red, respectively. Pink and orange arrowheads indicate the approximate rostral border of the mesencephalon and caudal border of the anterior metencephalon (r1), respectively. These borders were determined by referring to morphological landmarks in the developing brain, including the preotic sulcus at the r2/r3 boundary at the 7-12 somite stage (arrow), as well as the locations of the descendants of En1- expressing cells, as marked by lacZ expression in En1Cre/+ embryos carrying the R26R reporter gene (Fig. 1C, and data not shown). The green arrowhead indicates the approximate location of the isthmic organizer at the midbrain/hindbrain boundary, which was identified by referring to gene expression patterns in control and mutant embryos (Fig. 5, and data not shown). At the 19-20 somite stage, the midbrain vesicle is obvious in the NBS stained control embryo (between the pink and green arrowheads), but appears to be entirely deleted in the Fgf8 MHB KO embryo (indicated by half pink, half green arrowhead). In the mutant embryos, the remaining mes/met tissue, in which ectopic cell death is readily detected, can be identified as rostral metencephalon by the presence of the rhombic lips. Note that at the 14-15 and 19-20 somite stages, the views of the mutant and control embryos are not strictly equivalent, because the curvature of the brain is decreased in the mutants as a result of tissue loss. Consequently, the rostral end of the mesencephalon is not visible in the photographs of the control embryos. Moreover, although the distance between the green and orange arrowheads is essentially the same in mutant and control embryos, it appears different because they are viewed from different angles. Ot, otocyst; RL, rhombic lip; V, trigeminal ganglia.

View larger version (58K): [in a new window]   Fig. 4. Analysis of cell death in Wnt1-/-, En1-/- and Fgf8 MHB KO mutants. (A-C) Dorsal views of control and mutant embryos of the genotypes indicated, stained in whole mount with NBS. Schematic diagrams and arrowheads are as described in Fig. 3. In C, note that the midbrain appears partially truncated in the En1-/- embryo, mostly deleted in the Wnt1-/- embryo, and completely absent in the Fgf8 MHB KO embryo (indicated by half pink, half green arrowhead). *, partial deletion; Fb, forebrain; Hb, hindbrain; Mb, midbrain.

View larger version (96K): [in a new window]   Fig. 5. Analysis of gene expression in Fgf8 MHB KO mutants. Control embryos and MHB KO mutants at the somite stages indicated (s) were assayed by whole-mount RNA in situ hybridization using the probes indicated. Red arrows in A,C,E,G indicate the regions of gene expression in control embryos that are affected in MHB KO mutants. In M and N, the control and mutant embryos are both heterozygous for En1cre. In O,P,W,X, note the distance between the caudal limit of the Otx2 expression domain (open arrowhead) and the r2/3 border (black arrowhead), adjacent to the Hoxa2 expression domain in r3. Black arrows in R,T,V indicate regions in MHB KO mutants in which a small amount of gene expression is detected.