Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(3):e33105.
doi: 10.1371/journal.pone.0033105. Epub 2012 Mar 8.

Evidence that descending cortical axons are essential for thalamocortical axons to cross the pallial-subpallial boundary in the embryonic forebrain

Yijing Chen  1 , Dario Magnani, Thomas Theil, Thomas Pratt, David J Price
Affiliations

Evidence that descending cortical axons are essential for thalamocortical axons to cross the pallial-subpallial boundary in the embryonic forebrain

Yijing Chen et al. PLoS One. 2012.

Abstract

Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cortex-specific deletion of Apc disrupts the generation of differentiating neurons.
(A–D) Map2 immunohistochemistry on coronal sections of E13.5 control and conditional null mutant forebrain. Areas boxed in A and B are shown at higher magnification in C and D, respectively. (A,C) in the pallium of controls, Map2 was detected through the cortical plate (CP) and the intermediate zone (IZ), both of which are thicker laterally; (B,D) in contrast, there were relatively few Map2-positive cells in the pallium of conditional null mutants (PZ, proliferative zone; T, thalamus). (E,F) Neurofilament (NF) immunohistochemistry on coronal sections of E13.5 control and conditional mutant forebrain: (E) a superficial layer of immunostaining was detected in the pallium of control brains but (F) not of conditional mutant brains. Scale bars: A&B, 100 µm; C–F, 100 µm.
Figure 2
Figure 2. Cortex-specific deletion of Apc prevents the production of descending cortical efferents.
Injections of axonal tracers into the cortex of control and conditional null mutant embryos: areas boxed in A are shown at higher magnification of single optical sections in C,E,G and areas boxed in B are shown at higher magnification in D,F. (A,C,E,G) In E13.5 controls, cortical efferents descended across the PSPB: arrows indicate axons and growth cones descending from labelled cortical cell bodies (arrowhead in G). (B,D,F) In E13.5 conditional mutants, there were no cortical efferents crossing the PSPB (arrow in B indicates a group of pallial cells that were labelled retrogradely from the injection site). (H,I) Tag1 immunohistochemistry on coronal sections of E15.5 control and conditional mutant forebrain: (H) large numbers of descending Tag1 positive cortical efferents were detected in the control brains but (I) not in the conditional mutant brains. Scale bars: A&B, 100 µm; C–F, 100 µm; H&I, 100 µm.
Figure 3
Figure 3. Corticothalamic and thalamocortical axons were not detected in E16.5–E18.5 conditional null mutants.
(A,B) Placements of axonal tracers into the E16.5 cortex of control and conditional null mutant embryos: areas boxed in A and B are shown at higher magnification in C and D, respectively. (A,C) Cortical placements into control brains at E16.5 labelled large bundles of axons that were travelling through the ventral telencephalon. (B, D) Cortical placements into conditional null mutants at E16.5 failed to label more than the occasional axon navigating in the direction of the PSPB. (E) At E18.5, cortical placements labelled thalamic (T) nuclei in controls. (F) Cortical placements into conditional null mutants at E18.5 failed to produce any labelling of the thalamus. Nuclei were counterstained with TO-PRO-3 iodide. Scale bars: A&B, 500 µm; C&D, 100 µm; E&F, 500 µm.
Figure 4
Figure 4. Thalamic axons failed to reach the cortex in Apc conditional null mutants.
(A,D) At E15.5 and E18.5, injections of tracer in control thalamus labelled large bundles of axons projecting from the thalamus (T) through the subpallium and the intermediate zone of the cortex. (B,C) In E15.5 conditional null mutants, thick bundles of axons were observed traversing the subpallium but they stopped before crossing the PSPB and entering the cortex; instead, they changed direction and turned ventrally. (E,F) In E18.5 conditional null mutants, labelled thalamic axons deflected away from the PSPB and cortex. (G–I) In these E14.5 embryos, DiI (red) was injected in rostral thalamus whereas DiA (green) was injected in caudal thalamus (the DiA injection sites are not seen in these planes of section). Nuclei were counterstained with TO-PRO-3 iodide. (G) In controls, the thalamic axons from the rostral thalamus maintain their position medial to axons from the caudal thalamus as they traverse the subpallium. (H,I) Results from two conditional null mutants show that the same order is maintained up to the point at which the thalamic axons halt their progress toward the cortex. Scale bar: A–I, 100 µm.
Figure 5
Figure 5. The expression of maker genes of PSPB is similar between control and conditional null mutant.
(A) A LacZ reporter allele shows that Emx1Cre causes cortical recombination as far ventrally as the angle region at E13.5. (B,C) Expression of APC around the angle region (blue arrowhead) in (B) control and (C) conditional null mutants at E13.5, confirming loss of APC dorsal to the angle region, in the region labelled for β-galactosidase in A. (D) The E13.5 PSPB (white arrowhead), situated ventral to the angle region of the cortex (blue arrowhead), is marked by the transition from high (dorsal) to lower (ventral) expression of the transcription factor Pax6 and by cells of the lateral cortical stream (LCS). (E) In the conditional null mutant, Pax6 expression at the PSPB is similar to the control in D. (F,G) At E13.5, the expression pattern of the transcription factor Mash1 is similar between the control in F and the conditional null mutant in G. Expresses is in the subpallium and displays a sharp boundary respecting the medial edge of the PSPB (white arrowhead). (H,I) Similar to the control in H, the E13.5 PSPB in a conditional null mutant in I is marked by the lateral edge of the expression domain of ventrally-expressed transcription factor Gsh2 and a prominent RC2-expressing radial glial palisade from this region flanks the PSPB (bracket). Scale bars: A, 200 µm; B&C, 200 µm; D–I, 200 µm.
Figure 6
Figure 6. Thalamic axons stop before the radial glial palisade that flanks the PSPB.
(A,C,E) In controls, L1 labelled thalamocortical axons pass the radial glial palisade and the PSPB to enter the cortex. (B,D,F) In conditional null mutants, L1 labelled thalamic axons stop at the lateral edge of the radial glial palisade. Boxed areas in A and B are shown at higher magnification in C and D. Scale bars: A&B, 200 µm; C&D, 200 µm; E&F, 200 µm.
Figure 7
Figure 7. Control and Apc−/− angle region express molecules that stimulate thalamic growth.
(A, B) In situ hybridizations show expression of Ig-Nrg1 with highest intensity around the angle region (arrowheads). Scale bar: 200 µm. (C) This diagram shows the regions from which explants were taken for co-culture experiments: Cx, cortex; An, angle region; T, thalamus; Hy, hypothalamus. (D–I) Examples show six cultures, each containing thalamus and a different co-cultured tissue or, in H, thalamus alone.
Figure 8
Figure 8. The cortex of conditional null mutants maintains the ability to promote thalamic axonal growth.
Quantitative analyses show that neither the angle region nor more dorsal cortex of conditional null mutants have a reduced ability to promote thalamic axonal growth. (A) Concentric lines 100, 170, 240, 310 and 380 µm from the edge of the thalamic explant, divided into four quadrants, one facing towards the co-cultured explant, the other facing away. Within selected quadrants or along the entire length of each line, the percentages of the lengths that were crossed by axons were calculated. (B) Average (± sem) values for growth towards or away from co-cultured tissue, measured along the inner-most lines, as shown in A by black and white arrows. The only tissue that caused a significant difference in outgrowth on the two sides was hypothalamus (p<0.05; Student's t-test; n = 13), which is known to be repulsive to thalamic axons. (C,D) Average (± sem) values for growth across the entire length of each concentric line at each distance from each explant's edge (excluding parts covered by the co-cultured explant). (C) Results from thalamic explants cultured either alone or with null mutant or control angle regions (numbers of cultures in brackets). (D) Results from thalamic explants cultured either alone or with null mutant or control lateral cortical regions (numbers of cultures in brackets).
Figure 9
Figure 9. Replacing null mutant cortex with control cortex allows thalamic axons to cross the PSPB.
(A,B) Thalamus from either control or conditional null embryos transplanted to the diencephalic-telencephalic border of control slices generated axons (DiI labelled, red) that crossed the PSPB in close association with descending cortical axons (DiA labelled, green). (C) When thalamus was transplanted to the diencephalic-telencephalic border of slices from conditional null embryos, thalamic axons traversed the subpallium but did not cross the PSPB. Injections of tracer into the cortex of these co-cultures revealed that, as expected from in vivo work, the conditional null cortex produced no descending axons (inset C′). (D) When thalamus was transplanted to the diencephalic-telencephalic border of slices from conditional null embryos whose cortex was replaced by control cortex, thalamic axons crossed the PSPB in close association with descending cortical axons provided from the control cortex. (E–H) Thalamic axons only are shown from the boxed areas in A–D. (I–L) Higher magnification images from the boxed areas in A–D. Nuclei were counterstained with TO-PRO-3 iodide. The diagrams underneath each column of photographs summarize the experimental paradigms and their outcomes. Scale bars: A–D, 50 µm; E–L, 25 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bentley D, Caudy M. Pioneer axons lose directed growth after selective killing of guidepost cells. Nature. 1983;304:62–65. - PubMed
    1. McConnell SK, Ghosh A, Shatz CJ. Subplate neurons pioneer the first axon pathway from the cerebral cortex. Science. 1989;245:978–982. - PubMed
    1. Molnar Z, Blakemore C. Lack of regional specificity for connections formed between thalamus and cortex in coculture. Nature. 1991;351:475–477. - PubMed
    1. Metin C, Godement P. The ganglionic eminence may be an intermediate target for corticofugal and thalamocortical axons. J Neurosci. 1996;16:3219–3235. - PMC - PubMed
    1. Tuttle R, Nakagawa Y, Johnson JE, O'Leary DD. Defects in thalamocortical axon pathfinding correlate with altered cell domains in Mash-1-deficient mice. Development. 1999;126:1903–1916. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources