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First published online 23 January 2008
doi: 10.1242/dev.011569

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Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

M. Albert Basson^1,2,3,*, Diego Echevarria^4,*, Christina Petersen Ahn^1,*, Anamaria Sudarov⁵, Alexandra L. Joyner⁵, Ivor J. Mason², Salvador Martinez⁴ and Gail R. Martin^1,

¹ Department of Anatomy and Program in Developmental Biology, University of California, San Francisco, CA 94158, USA.
² MRC Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK.
³ Department of Craniofacial Development, King's College London, London SE1 9RT, UK.
⁴ Instituto de Neurociencias de Alicante, UMH-CSIC, 03550-San Juan de Alicante, Spain.
⁵ Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

View larger version (82K): [in this window] [in a new window]   Fig. 1. Spry2-GOF, a conditional Spry2 gain-of-function allele, and its expression following recombination by En1Cre. (A) Schematic diagram illustrating the Spry2-GOF transgene. The CAGG promoter drives expression of the β-Geo gene, which is followed by a triple polyadenylation sequence (3x pA). Cre-mediated recombination deletes β-Geo, and mouse Spry2 and human placental alkaline phosphatase (PLAP) cDNAs are expressed as a bicistronic mRNA containing an internal ribosome entry site (IRES) that directs translation of PLAP. (B) Assays in whole mount for β-Geo expression (β-GAL activity) in embryos hemizygous for Spry2-GOF. (C-K) Analysis of expression of the recombined Spry2-GOF transgene in En1Cre/+;Spry2-GOF (mes/r1-S2GOF) mutants. (C-E) Assays for PLAP activity in embryos and postnatal brain at the stages indicated. Blue staining identifies cells in which recombination of the transgene occurred. The arrow in D indicates two ventrolateral stripes in the spinal cord. (F-K) RNA in situ hybridization assays in whole mount at the stages indicated, using a mouse Spry2 probe. The broken white lines outline the left side of mes/r1 in the control embryos, where the probe detects endogenous Spry2 expression. In mes/r1-S2GOF embryos, the Spry2 RNA detected represents the sum of expression from the endogenous Spry2 gene and the recombined transgene. Regions in which there is ectopic Spry2 expression in mes and in r1 are indicated by broken red lines (I,K) and by a red arrow (K), respectively. AER, apical ectodermal ridge of the limb bud; BA1, first branchial arch; CbV, cerebellar vermis; CbH, cerebellar hemispheres; Mb, midbrain; mes; mesencephalon; r1, rhombomere 1; so, somites.

View larger version (111K): [in this window] [in a new window]   Fig. 2. Expression of a single copy of the recombined Spry2-GOF allele in mes/r1 causes absence of the inferior colliculus and anterior vermis. (A,B,E-I) Midsagittal sections of brains of the genotypes indicated, collected at the stages denoted and stained with Cresyl Violet or Hematoxylin and Eosin (I). Anterior is towards the left, posterior is towards the right. In control embryos, the posterior midbrain and anterior hindbrain regions that are absent in the mutant embryos are demarcated by red and yellow broken lines, respectively; in the mutant embryos, the regions where the missing midbrain and anterior hindbrain tissue would normally be found are indicated by red and yellow asterisks, respectively. (C,D) Higher power view of the tissue shown in A and B, respectively, stained with an antibody against calretinin (CR). The CR-negative inferior colliculus in the control is indicated by a broken red line. In the mutant, this CR-negative region, as well as the isthmus and velum medullare are absent (arrowhead). (G-I) Comparison of vermis development in control, mes/r1-S2GOF, and Fgf17-/-; Fgf8+/- animals at 3 weeks of age. CP, choroid plexus; IC, inferior colliculus; SC, superior colliculus; VM, velum medullare. The lobules of the vermis are indicated by Roman numerals, as described by Inouye and Oda (Inouye and Oda, 1980).

View larger version (103K): [in this window] [in a new window]   Fig. 3. Decreasing Fgf8 gene dose in mes/r1-S2GOF embryos results in absence of the vermis. (A,B) Dorsal views of brains collected from E17.5 embryos of the genotypes indicated. In the mutant brain, the region where the vermis would normally be found is indicated by a yellow asterisk. (C-E) Low-magnification views of midsagittal sections of E17.5 control and mes/r1-S2GOF;F8 mutants, stained with Cresyl Violet. (D,E) Examples of the group I (milder) and group II (more severe) brain phenotypes observed in the mutants. The broken red and yellow lines and red and yellow asterisks are explained in the legend to Fig. 2A,B,E,F. The areas outlined in C-E are shown at higher magnification in F-H, respectively. (F-H) High magnification views of basal structures. When present, nIII and nIV are circled with broken lines. (I-K) High magnification views of more lateral sagittal sections of the same brains, assayed by immunohistochemistry with an antibody against tyrosine hydroxylase, which specifically stains nuclei in the lateral posterior diencephalon, midbrain and anterior hindbrain. The broken red line indicates the boundary between diencephalon and midbrain. (L,M) Dorsal views of intact brains collected from P21 mice of the genotypes indicated, stained with ink. The broken line in L outlines the vermis, which is absent in the mutant brain. CbV, cerebellar vermis; CbH, cerebellar hemispheres; CP, choroid plexus; Di, diencephalon; IC, inferior colliculus; Is, isthmus; LoC, locus ceruleus; mlf, medial longitudinal fascicle; nIII, oculomotor nucleus; nIV, trochlear nucleus; PPT, posterior pretectal nucleus; SC, superior colliculus; SN-VTA, substantia nigra-ventral tegmental area.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Reducing FGF signaling from the isthmic organizer results in cell death in the anterior mesencephalon. (A-E) Lateral views of embryos of the genotypes indicated, collected at the somite stages denoted and labeled with LysotrackerT (LysoT), which marks regions in which dead cells are abundant. The approximate position of the isthmic constriction (Is) is indicated. (C') The embryo shown in C was fixed after labeling with LysoT and assayed by RNA in situ hybridization for Pax6 expression. The broken yellow line indicates the posterior limit of Pax6 expression, which lies at the border between the prospective forebrain (diencephalon, Di) and midbrain (mesencephalon, Mes). In control embryos (A,D), LysoT labeling is detected where neural crest (NC) is migrating into the first branchial arch (BA1), but there is relatively little labeling in the neuroepithelium. By contrast, in the mutant embryos (B,C,E) there is extensive LysoT labeling in the mes (white arrows). (F-K) Embryos of the genotypes indicated, collected at the somite stages indicated, were assayed in whole mount by RNA in situ hybridization using a probe for Spry1 or Efna2. The extent of the expression domain from the mes/r1 boundary (red circle) towards the forebrain is indicated by a broken red arrow.

View larger version (111K): [in this window] [in a new window]   Fig. 5. The roof plate is expanded and Grem1 expression is reduced in mes/r1-S2GOF;F8 embryos. (A-D') Transverse sections through r1 of a control and a mes/r1-S2GOF;F8 embryo at 48 somites (E11.75), stained with Hematoxylin and Eosin. Every third or fourth section in the series was assayed by RNA in situ hybridization with a probe for Otx2, to locate the posterior limit of the mesencephalon (not shown). The broken lines in the inset in A illustrate the approximate levels at which the anterior (A) and posterior (P) sections were cut, i.e. within 40-100 and 130-250 µm of the posterior limit of the Otx2 expression domain (indicated in blue), respectively. The sections in all panels are shown at the same magnification. The regions demarcated by broken boxes in A-D are shown at fourfold higher magnification in A'-D', respectively. The mediolateral width of the dorsal midline domain in which the neuroepithelium is only two or three cell layers thick is indicated by red brackets (A'-D'). (E-L,O,P) RNA in situ hybridization assays in whole mount using the probes indicated. Dorsal views of embryos of the genotypes indicated, collected at the stages denoted. Anterior is towards the top, posterior towards the bottom. (E-I) The roof plate that bisects the Fgf8 expression domain is indicated by arrowheads. In control and mes/r1-S2GOF embryos, the Fgf8-negative roof plate is difficult to discern; it is considerably expanded mediolaterally in the mes/r1-S2GOF;F8 embryo (open arrowhead in G). The lateral and ventral aspect of the Fgf8 expression domain in r1 is visible in E and G on the left side, through the roof plate. (J-L) A broken red line is drawn at the same distance posterior to the isthmic constriction at the mes/r1 boundary (black arrow). Bmp7 expression marks the roof plate, and the roof plate widens closer to the isthmic constriction in the mes/r1-S2GOF;F8 embryo than in the control and mes/r1-S2GOF embryos. (M,N) Transverse sections through anterior r1 at approximately the same AP level as in A and B, hybridized with a probe for Msx1. The mediolateral extent of the Msx1 expression domain from the center of the roof plate (open circle) is indicated by a broken red arrow. (O,P) Grem1 expression is readily detected at the dorsal midline of r1 in the control embryo, but is barely detected in r1 of the mes/r1-S2GOF;F8 embryo (red asterisk).

View larger version (29K): [in this window] [in a new window]   Fig. 6. A model to explain the phenotypes obtained when FGF signaling is progressively reduced in mes/r1. Schematic diagrams illustrate dorsal views of mes/r1 in embryos of the genotypes indicated at the somite stages denoted. The intensity of the blue color in the triangle to the left of a diagram illustrates the level of FGF signaling. (A) The regions in which cells are dying or have already died are indicated by red stippling. (B) The regions that contain the progenitors (p) of the superior colliculus (SC), inferior colliculus (IC), cerebellar vermis (CbV) and cerebellar hemispheres (CbH) are indicated. The phenotypes observed in mutants that attain progressively lower levels of FGF signaling are illustrated. In mes/r1-S2GOF embryos, the anterior mes has been lost, and the surviving posterior cells are specified to an anterior (SC) fate. Consequently the IC does not develop. In mes/r1-S2GOF;F8 embryos, the effects on the mes are similar and, in addition, the vermis fails to develop because roof plate (RP) expansion in r1 results in a loss of CbV progenitors. (C) In the wild-type embryo, FGF signaling induces/maintains the expression of Grem1, which functions to inhibit BMP signaling and expression of the BMP effector MSX1. This pathway maintains the normal balance of vermis and roof plate development. In the mes/r1-S2GOF;F8 embryo, the reduction in the level of FGF signaling results in a severe reduction in Grem1 expression. In the absence of this antagonist, the level of BMP signaling and therefore MSX1 expression increases and an expanded roof plate develops where the vermis progenitors normally reside.