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Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections

¹ Department of Psychiatry, Nina Ireland Laboratory of Developmental Neurobiology, Langley Porter Psychiatric Institute, University of California, San Francisco, USA
² Departament of Morphological Sciences, School of Medicine, University of Murcia, Spain

View larger version (105K): [in a new window]   Fig. 1. Expression of NKX2-1 in the developing forebrain. (A) Sagittal section through the brain of an E12.5 embryo showing the expression of NKX2-1 in the basal telencephalic and hypothalamic primordia. The broken lines indicate the approximate levels of the sections shown in (B,C). (B,C) Coronal sections that highlight the basal telencephalon and rostral diencephalon (B) and mid-diencephalon (C) of an E12.5 embryo showing expression of NKX2-1. (D-G) High-magnification images of coronal sections through the telencephalon (D,E) and diencephalon (F,G) of an E14.5 embryo showing expression of NKX2-1 at the level of the internal capsule (D) and close to cerebral peduncle at the level of the hypothalamus (F). (E,G) DAPI counterstained images of the sections showed in (D,F). The insets in E,G indicate the level from where the images were taken. (H) Schematic drawing showing the relationship between the paths followed by pyramidal tract (blue), corticothalamic (pink) and thalamocortical (purple) axons in relation to the territories patterned by Nkx2-1 (light pink). a/b, alar/basal boundary; AEP, anterior entopeduncular region; Chr, chiasmatic region; cp, cerebral peduncle; DT, dorsal thalamus; ET, eminentia thalami; GP, globus pallidus; H, hippocampus; Hb, hindbrain; Hyp, hypothalamus; ic, internal capsule; LGE, lateral ganglionic eminence; M, mesencephalon; MGE, medial ganglionic eminence; NCx, neocortex; ob, olfactory bulb; os, optic stalk; POa, preoptic area; PrH, peduncular region of the hypothalamus; p/sp, pallial/subpallial boundary; PT, pretectum; Str, striatum; V, layer 5 cortical neurons; VI, layer 6 cortical neurons; VT, ventral thalamus. Scale bar: 200 µm in A-C; 100 µm in D-G.

View larger version (97K): [in a new window]   Fig. 2. Patterning defects in the diencephalon of Nkx2-1 mutant mice. (A-H) Serial sagittal sections through the brain of E11.5 wild-type (A-C) and Nkx2-1 mutant embryos (E-G) showing mRNA expression of Nkx2-4 (A,E), Pax6 (B,F) and Sim1 (C,G). The white arrowhead indicates the pallial-subpallial boundary, whereas the black (with white border) arrowhead denotes the LGE-MGE boundary. Note ventral expansion of Pax6 in the telencephalon (black arrows) and of Pax6 and Sim1 in the diencephalon (white arrow). (D,H) Schematic drawings summarizing the expression of pattern of Nkx2-1, Nkx2-4, Pax6, SF1 and Sim1 in the forebrain of wild-type and Nkx2-1 mutant embryos. (I-L) Serial sagittal sections through the brain of E18.5 wild-type (I,J) and Nkx2-1 mutant fetuses (K,L) showing immunohistochemistry for GABA (I,K) and calbindin (J,L). a/b, alar-basal boundary; ac, anterior commissure; DT, dorsal thalamus; fr, fasciculus retroflexus; H, hippocampus; Hb, hindbrain; Hyp, hypothalamus; Hyp*, mutant hypothalamus; ic, internal capsule; LGE, lateral ganglionic eminence; LGE*, mutant lateral ganglionic eminence; MGE, medial ganglionic eminence; MGE*, mutant medial ganglionic eminence; MHb, medial habenula; MM, mammillary hypothalamus; mt, mammillothalamic tract; mtg, mammillotegmental tract; NCx, neocortex; ob, olfactory bulb; p/sp, pallial/subpallial boundary; P, pontine nuclei; PT, pretectum; PV, paraventricular hypothalamic region; R, reticular nucleus; S, septum; S*, mutant septum; SC, superior colliculus; Teg, mesencephalic tegmentum; VM, ventromedial hypothalamic nucleus; VT, ventral thalamus; zli, zona limitans intrathalamica. Scale bar: 400 µm in A-C,E-G; 500 µm in I-L.

View larger version (134K): [in a new window]   Fig. 3. Pathfinding defects of layer 5 cortical projections in the telencephalon of Nkx2-1 mutants. Coronal sections through the telencephalon of E14.5 (A,E) or E18.5 (B,C,F,G,H) wild-type (A-C) or Nkx2-1 mutant mice (E-H) showing anterogradely labeled fibers (A-C,E,F,H) and retrogradely labeled thalamic cells (B,C,H) after DiI crystal placements in the neocortex. Arrowheads point to corticofugal fibers following a normal course, whereas arrows indicate corticofugal fibers abnormally projecting towards the surface of the telencephalon. (G) Coronal section through the telencephalon of an E18.5 Nkx2-1 mutant mice showing retrogradely labeled cells in the cortex after a DiI crystal placement in the ventral surface of the telencephalon (at the approximate location of the arrow in F). The dotted line indicates the surface of the cortex. (D,I) Schematic drawings showing the paths followed by pyramidal tract (blue), corticothalamic (pink) and thalamocortical (purple) axons in wild-type (D) and Nkx2-1 mutant mice (I). The hatched areas (D,I) indicate the presumptive territory patterned by Nkx2-1 function. A, amygdala; c, cortical plate; cp, cerebral peduncle; DT, dorsal thalamus; GP, globus pallidus; H, hippocampus; ic, internal capsule; NCx, neocortex; Str, striatum; Str*, mutant striatum; V, layer 5 cortical neurons; VI, layer 6 cortical neurons; VT, ventral thalamus. Scale bar: 200 µm in A,E; 400 µm in B,C,F,H; 100 µm in G.

View larger version (85K): [in a new window]   Fig. 4. Normal pathfinding of layer 6 cortical projections in Nkx2-1 mutants. Coronal sections through the telencephalon of E18.5 wild-type (A,B) or Nkx2-1 mutant mice (C,D) showing the path followed by corticothalamic connections (A,C; white arrowheads) and retrogradely labeled cortical cells (B,D; open arrowheads) after DiI crystal placements in the dorsal thalamus. (C) The arrow points to the poor-cell region containing the abnormal tract formed by layer 5 cortical axons (not labeled in this experiment) present in Nkx2-1 mutants (see Fig. 3F). c, cortical plate; cp, cerebral peduncle; GP, globus pallidus; Str, striatum; Str*, mutant striatum; V, layer 5 cortical neurons; VI, layer 6 cortical neurons. Scale bar: 200 µm in A,C; 100 µm in B,D.

View larger version (79K): [in a new window]   Fig. 5. Pathfinding defects of layer 5 cortical projections in the diencephalon of Nkx2-1 mutants. Coronal (A,D) and horizontal (B,E) sections through the diencephalon (A,D) or pons (B,E) of E18.5 (A,B,D,E) wild-type (A,B) or Nkx2-1 mutant mice (D,E) showing anterogradely labeled fibers (A,B,D,E) and retrogradely labeled thalamic cells (A,D) after DiI crystal placements in the neocortex. The arrow indicates corticofugal fibers that follow an abnormal rostral path into the chiasmatic region of the hypothalamus. (C,F) The paths followed by pyramidal tract (blue), corticothalamic axons (pink) and thalamocortical axons (purple) in wild-type (C) and Nkx2-1 mutant mice (F). The hatched area indicates the presumptive territory patterned by Nkx2-1 function. Chr, chiasmatic region; Chr*, mutant chiasmatic region; cp, cerebral peduncle; DT, dorsal thalamus; fr, fasciculus retroflexus. Scale bar: 400 µm in A,B,D,E.

View larger version (72K): [in a new window]   Fig. 6. The basal telencephalon and hypothalamus contain a non-permissive activity for cortical axons. (A,C) Experimental paradigm for cortical-basal telencephalic (A) and cortical-hypothalamic (C) co-cultures in three-dimensional collagen matrices. Green slices and explants are derived from E13.5 GFP transgenic embryos, whereas gray slices and explants are derived from wild-type littermate embryos. (B,D) Explants after 72 hours in culture showing immunohistochemistry for GFP. The broken lines indicate the location of the non-GFP expressing explants. BT, basal telencephalon; Hyp, hypothalamus; NCx, neocortex.

View larger version (109K): [in a new window]   Fig. 7. Expression of Slits is altered in the forebrain of Nkx2-1 mutants. Serial coronal sections through the telencephalon (A,B,E,F) and diencephalon, and caudal telencephalon (C,D,G,H) of E13.5 wild-type (A-D) and Nkx2-1 (E-H) mutant embryos showing the expression of Slit1 (A,C,E,G) and Slit2 (B,D,F,H). Dorsal is upwards and the midline is to the right in all panels. Arrowheads point to regions of increased Slit1 expression, whereas arrows denote regions where Slit2 expression is either lost or decreased. AEP, anterior entopeduncular region; DT, dorsal thalamus; GP, globus pallidus; H, hippocampus; Hyp, hypothalamus; Hyp*, mutant hypothalamus; LGE, lateral ganglionic eminence; LGE*, mutant lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; POa, preoptic area; POa*, mutant preoptic area; Str, striatum; Str*, mutant striatum. Scale bar: 500 µm.

View larger version (91K): [in a new window]   Fig. 8. Distribution of mesotelencephalic dopaminergic fibers is altered in the diencephalon of Nkx2-1 mutants. (A,C) Sections horizontal to the diencephalon of E18.5 wild-type (A) and Nkx2-1 mutant (C) fetuses showing immunohistochemistry against tyrosine hydroxylase (TH). Caudal is upwards and rostral is downwards in both panels. The arrow points to the region where TH-positive fibers abnormally converge towards the rostral midline. (B,D) The path followed by dopaminergic mesotelencephalic axons (green) in wild-type (B) and Nkx2-1 mutant mice (D). The pink domains indicate the territories patterned by Nkx2-1 function. a/b, alar/basal boundary; AEP, anterior entopeduncular region; Chr, chiasmatic region; Chr*, mutant chiasmatic region; cp, cerebral peduncle; DT, dorsal thalamus; ET, eminentia thalami; GP, globus pallidus; H, hippocampus; Hb, hindbrain; Hyp, hypothalamus; Hyp*, mutant hypothalamus; ic, internal capsule; M, mesencephalon; mfb, medial forebrain bundle; NCx, neocortex; POa, preoptic area; POa*, mutant preoptic area; PrH, peduncular region of the hypothalamus; p/sp, pallial/subpallial boundary; PT, pretectum; ob, olfactory bulb; os, optic stalk; SC, superior colliculus; Str, striatum; Str*, mutant striatum; V, layer 5 cortical neurons; VI, layer 6 cortical neurons; VT, ventral thalamus. Scale bar: 400 µm.

View larger version (54K): [in a new window]   Fig. 9. A model for the guidance of cortical projections in Nkx2-1 mutant mice. (A,B) The paths followed by pyramidal tract (blue), corticothalamic (pink), thalamocortical (purple) and dopaminergic mesotelencephalic (green) axons in wild-type (A) and Nkx2-1 mutant mice (B). The broken lines indicate the territories patterned by Nkx2-1 function. (C-F) The expression of Slit genes in the ventricular zone of the subpallium in wild-type (C) and Nkx2-1 mutant mice (E), and the hypothesized consequences of their function on the guidance of layer 5 cortical axons in wild-type (D) and Nkx2-1 mutant mice (F). Blue positive signs indicate a permissive territory, whereas negative signs and red hatching denote a non-permissive territory. a/b, alar/basal boundary; AEP, anterior entopeduncular region; Chr, chiasmatic region; Chr*, mutant chiasmatic region; cp, cerebral peduncle; DT, dorsal thalamus; ET, eminentia thalami; GP, globus pallidus; H, hippocampus; Hb, hindbrain; Hyp, hypothalamus; Hyp*, mutant hypothalamus; ic, internal capsule; M, mesencephalon; NCx, neocortex; POa, preoptic area; PrH, peduncular region of the hypothalamus; p/sp, pallial/subpallial boundary; PT, pretectum; ob, olfactory bulb; os, optic stalk; Str, striatum; Str*, mutant striatum; V, layer 5 cortical neurons; VI, layer 6 cortical neurons; VT, ventral thalamus.