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doi: 10.1242/10.1242/dev.00416

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Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants

Sonia Garel, Kelly J. Huffman and John L. R. Rubenstein*

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, CA 94143-0984, USA



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  		Fig. 1. Levels of Fgf8 expression are reduced in Fgf8neo/neo embryos. (A-B') Whole-mount Fgf8 in situ hybridization on E9.5 wild-type (A,A') and Fgf8neo/neo (B,B') embryos. Lateral views (A,B) show that Fgf8 expression domains in the midbrain-hindbrain boundary (white arrows) and in the rostral midline (black arrowheads) are present but reduced in size and intensity in Fgf8neo/neo embryos. Frontal views of the same embryos (A',B') show changes in both the commissural plate (black arrowheads) and the olfactory placodes (black arrow), the two sites of Fgf8 expression in or in the vicinity of the telencephalic anlage. (C,D) Fgf8 in situ hybridization on horizontal sections of wild-type (C) and Fgf8neo/neo (D) E12 embryos. In Fgf8neo/neo embryos, the neocortex primordium and the bulks of the ventral telencephalic ganglionic eminences are present. The rostral midline is hypoplastic and the width of Fgf8 expression domain (black arrowheads) is reduced. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; Ncx, neocortex; Tel, telencephalon.

 


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  		Fig. 2. The subdivisions of the dorsal telencephalon are present in Fgf8neo/neo embryos. In situ hybridization on coronal sections of wild-type (A,C,E,G,I) and Fgf8neo/neo (B,D,F,H,J) E12.5 embryos performed with the indicated riboprobes. Otx2 expression in the choroids plexus (black arrows in A,B), Wnt3a expression in the cortical hem (black arrows in C,D), Lef1 (between arrowheads in E,F) and Emx2 (G,H) expression in the hippocampus primordium, and high levels of Pax6 expression in the ventral-most region of the neocortex (black arrows in I,J) do not appear modified in Fgf8neo/neo embryos. Emx2 in situ hybridization signal is slightly increased in the dorsal part of the neocortex (between arrowheads in G,H) in Fgf8neo/neo compared with wild-type embryos. The level of Pax6 expression in this same region is slightly less intense (between arrowheads in I,J). DT, dorsal thalamus; GE, ganglionic eminences; H, hippocampus; Ncx, neocortex.

 


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  		Fig. 3. Caudal-to-rostral gradients of expression are shifted rostrally in the dorsal telencephalon of E12/E13 Fgf8neo/neo embryos. Whole-mount in situ hybridization on dissected neural tubes of E12/E13 wild-type (A,A',C,C',E,G) and Fgf8neo/neo (B,B',D,D',F,H) embryos performed with the indicated probes. (A-B') The high caudodorsal to low rostroventral gradient of Emx2 expression in the dorsal telencephalon is detected in wild type on lateral (A) and dorsal (A') viewing of the telencephalic vessicules. Lateral (B) and dorsal views (B') of an Fgf8neo/neo embryo show the rostral shift in the anterior limit of this gradient (compare arrowheads in A and B). (C-D') The high caudoventral-to-low rostrodorsal gradient of COUP-TFI expression is visible on lateral (C) and dorsal (C') views of wild-type embryo. The anterior limit of high expression (white arrowheads) is shifted rostrally in Fgf8neo/neo embryos. (E,F) Dbx1 expression is normally restricted to a stripe in the caudal part of the ventral pallium (black arrowhead in E). The anterior limit of Dbx1 expression moves rostrally in Fgf8neo/neo embryos (black arrowhead in F). (G,H) FgfR3 high-expression is excluded from the rostral-most part of the telencephalon in control embryos (black arrowhead in G). This expression domain expands into the rostral telencephalon of Fgf8neo/neo embryos (black arrowhead in H). Tel, telencephalon.

 


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  		Fig. 4. Caudal gradients of gene expression are shifted rostrally in the dorsal telencephalon of E13.5 and E14.5 Fgf8neo/neo embryos. In situ hybridization on sagittal sections of wild-type (A,C,E) and Fgf8neo/neo embryos (B,D,F) with the indicated probes. The graded expression of COUP-TFI and Fgfr3 (black arrowheads) expands into the rostral neocortex. Pax6 expression appears normal in Fgf8neo/neo embryos (compare C with D). Note that the expression of these three genes outside of the neocortex is not affected in Fgf8neo/neo embryos except in the midbrain, which is dramatically reduced in size. M, midbrain; Ncx, neocortex; OB, olfactory bulb.

 


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  		Fig. 5. Molecular regionalization of the neocortex is perturbed in `mild' Fgf8neo/neo neonates. In situ hybridization performed on sagittal sections of wild-type or Fgf8neo/+ (A,C,E,G,I,K) and Fgf8neo/neo (B,D,F,H,J,L) newborns (A-F) and E18.5 embryos (K-J) with the indicated probes. Note that medial sections (A-F,G,H) of Fgf8neo/neo neonates show the presence of olfactory bulbs. (A-F) Medial sagittal sections of a wild-type brain show Id2 rostral superficial expression (Id2rs), Id2 parietal expression in layer 5 (Id25) and Id2 occipital superficial expression (Id2cs). The caudal limit of high Id2rs (black arrowhead in A) and rostral limit of Id25 (open arrowhead in A) coincide in wild type brains. In Fgf8neo/neo newborns, these two limits still coincide but both move rostrally towards the olfactory bulb (black and open arrowheads in B). The caudal limit of the superficial Cdh8 expression domain (Cdh8rs) in the frontal neocortex moves rostrally in Fgf8neo/neo newborns (compare black arrowheads in C and D). This modification correlates with a corresponding rostral shift in the high Cdh6 expression domain (black arrowheads in E and F). (G,H) In more lateral sections, Id2 strong caudal expression (Id2cs) shows a rostral shift in Fgf8neo/neo embryos (black arrowheads). The shift in the rostral Id2 domain is still observed (open arrowheads). (I-L) In medial (I,J) or lateral sections (K,L) there was no consistent changes in Epha7 or RZRß, although minor changes such as an increased spacing between the caudal and rostral Epha7-positive domains (black arrowheads in I,J) were observed in some Fgf8neo/neo embryos. 5, layer 5 expression; cs, caudal superficial expression; GE, ganglionic eminences; Ncx, neocortex; rs, rostral superficial expression; OB, olfactory bulb.

 


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  		Fig. 6. Complementary Id2 and Cdh6 expression domains show a molecular shift in the orbitofrontal and frontal neocortex of Fgf8neo/neo newborns. Id2 (A-H) and Cdh6 (A'-H') in situ hybridization were performed on adjacent 100 µm sections (A-H and A'-H') from the rostral brain of a wild-type (A-D') and a Fgf8neo/neo (E-H') newborn. In wild type, Id2 and Cdh6 expression delimit two complementary zones (black arrowheads in A-D'): a rostral and medial domain of high Id2rs, low Id25 and low Cdh6 expression, and a lateral parietal domain of low Id2rs, high Id25 and Cdh6 expression. These two zones are still observed in Fgf8neo/neo newborns (black arrowheads in E-H'). However the rostral and medial domain is severely reduced and the lateral parietal domain expands both medially and rostrally (E-H'), even into the rostralmost neocortex (E,E'). 5, layer 5 expression; Ncx, neocortex; rs, rostral superficial expression; OB, olfactory bulb.

 


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  		Fig. 7. Topographic organization of thalamocortical projections in Fgf8neo/neo neonates. DiI and DiA crystals or single DiI crystals were placed in the occipital (Occ) and parietal (Par) neocortex (A-B'), the caudal parietal neocortex (C-D'), the parietal neocortex (E-F') and the rostral parietal and frontal (Fr) neocortex (G-H'). In A-H, crystal placements (red and green arrowheads) are shown in lateral (A,B) or dorsal views (C-H) of the brain hemispheres. These placements are shown before dye diffusion, except in A,B. (A'-H') Dorsal thalamus coronal sections of the corresponding brains after dye diffusion. The retroflexus tract (white arrowhead) and the external medullary lamina (broken line) are morphological landmarks used to position and identify presumptive thalamic nuclei. Sections are counterstained with Hoechst. (A-B') DiI injection in the occipital neocortex and parietal neocortex retrogradely stain the same presumptive thalamic nuclei in Fgf8neo/neo brains. The dorsal lateral geniculate nucleus (dLGN) is labeled in pink/red with DiI and the lateral part of the ventroposterior complex (VP) is labeled in green with DiA. (C-D') Large DiI injections in the caudal parietal neocortex normally label cells in the lateral VP domain and spread into the presumptive lateral posterior nucleus (LP) (open arrowheads) in both wild type and mutant neonates. In this particular Fgf8neo/neo neonate, the injection also labeled a few cells located more lateroventrally in the dLGN (arrows). (E-F') Small injections of DiI and DiA into the parietal neocortex label two overlapping domains within the VP complex in both control and Fgf8neo/neo newborns. (G-H') Small injections of DiI into the orbitofrontal/frontal neocortex and of DiA in the rostral parietal neocortex label a medial VP domain as well as a more medial domain in both wild-type and Fgf8neo/neo newborns. White arrowheads in C',D',E',F',G',H' indicate the position of the fasciculus retroflexus. dLGN, dorsal lateral geniculate nucleus; LP, lateroposterior nucleus; M, midbrain; n/n, Fgf8neo/neo; Ncx, neocortex; OB, olfactory bulb; VP, ventroposterior nucleus.

 


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  		Fig. 8. Normal thalamocortical projections with the rostral neocortex of `severe' Fgf8neo/neo newborns. In situ hybridization on sagittal sections (A-D) and DiI/DiA axonal tracing (E-L) were performed on the two hemispheres of one wild-type newborn (A,C,E,G,I,K) and one `severe' Fgf8neo/neo newborn lacking olfactory bulbs (B,D,F,H,J,L). (A-D) The superficial Cdh8 rostral expression (arrowhead in A) is absent in the rostral neocortex of the Fgf8neo/neo, as seen in a high-magnification view of the rostral neocortex (inset). In the same Fgf8neo/neo brain, the anterior limit of high Cdh6 expression moves into the most rostral and medial neocortex (arrowheads in C and D). (E,F) Lateral views show that DiI and DiA crystals (red and green arrowheads) were placed in the frontal (Fr) and parietal (Par) neocortices and into the parietal and frontal neocortex of the wild-type and Fgf8neo/neo brain, respectively (E,F). Approximate levels of the coronal sections presented in G,I,K and H,J,L are indicated by white lines. (G-J) Dark field pictures of coronal sections showing the position of the dye crystals (red and green arrowheads) in the frontal (G,H) and parietal (I,J) neocortices. Note that DiI and DiA crystals have inverted positions in wild-type and Fgf8neo/neo newborns. (K,L) Coronal sections through the dorsal thalamus, counterstained with Hoechst, show very similar retrograde labeling in the wild type and Fgf8neo/neo brain (arrowheads indicate the fasciculus retroflexus. Ncx, neocortex; OB, olfactory bulb.

 

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