Developmental guidance of the retroflex tract at its bending point involves Robo1-Slit2-mediated floor plate repulsion

Abstract

The retroflex tract contains medial habenula efferents that target the hindbrain interpeduncular complex and surrounding areas. This tract displays a singular course. Initially, habenular axons extend ventralwards in front of the pretectum until they reach the basal plate. Next, they avoid crossing the local floor plate, sharply changing course caudalwards (the retroflexion alluded by the tract name) and navigate strictly antero-posteriorly across basal pretectum, midbrain and isthmus. Once they reach rhombomere 1, the habenular axons criss-cross the floor plate several times within the interpeduncular nuclear complex as they innervate it. Here we described the timing and details of growth phenomena as these axons navigate to their target. The first dorsoventral course apparently obeys Ntn1 attraction. We checked the role of local floor plate signaling in the decision to avoid the thalamic floor plate and bend caudalwards. Analyzing the altered floor and basal plates of Gli2 knockout mice, we found a contralateral projection of most habenular axons, plus ulterior bizarre navigation rostralwards. This crossing phenotype was due to a reduced expression of Slit repulsive cues, suggesting involvement of the floor-derived Robo-Slit system in the normal guidance of this tract. Using Slit and Robo mutant mice, open neural tube and co-culture assays, we determined that Robo1-Slit2 interaction is specifically required for impeding that medial habenular axons cross the thalamic floor plate. This pathfinding mechanism is essential to establish the functionally important habenulo-interpeduncular connection.

Keywords: Axon guidance; Floor plate; Habenula; Retroflex tract; Robo-Slit.

Figure 1. Symmetric trajectory of the rft

(A) Sagittal section of a X-gal-stained Pou4f1-tauLacZ adult brain. The black dotted lines define the boundaries between different neuromeres (p2, p1, mb, ist, r1). The red dotted line indicates the alar-basal boundary. (B–E) Coronal sections of an E18.5 embryo, ordered from rostral to caudal levels, showing nuclear staining (DAPI) and differential fluorescent tracing of the two habenular efferent tracts (rft). We inserted DiI (right Hb) and DiA (left Hb) crystals. Abbreviations: IPc, caudal interpeduncular nucleus; IPr, rostral interpeduncular nucleus; Ist, isthmus; Mb, midbrain; p1, prosomere 1 (pretectum); p2, prosomere 2 (thalamus), rft, retroflex tract; RMTg, rostromedial tegmental nucleus r1, rhombomere 1. Scale bar: 200μm.

Figure 2. Embryonic development of the rft

(A, C, E) Sagittal sections of E11.5 (A, paraffin), E12.5 (C, vibratome) and E13.5 (E, vibratome) immunoreacted against DCC. (B, D, F) Top view of E11.5 (B), E12.5 (D) and E13.5 (F) ONTs showing DiI and DiA labeling of rft. (B′, D′, F′) Higher magnification images obtained by confocal microscopy of B, D and F (dotted boxed regions). The dotted lines define the border of the ONTs, the boundaries between the prosomeres and the floor plate. The arrows in F′ point to scattered floor plate-crossing axons. Abbreviations: fp, floor plate; Hb, habenula; Ist, Isthmus; Mb, Midbrain; pc, posterior commissure; p1, prosomere1; p2, prosomere2; PT, pretectum; rft, retroflex tract; Th, Thalamus. Scale bar: 200μm and 100μm in F′.

Figure 3. Altered rft trajectory in the Gli2 mutant

(A) Vibratome horizontal section through midbrain, diencephalon and hypothalamus of an E18.5 Gli2^−/− embryo stained with α–DCC. This image is the result of the superposition of two consecutive sections, in order to show the overall rostralward trajectory of the aberrant rft tracts. (B) This insert shows a high magnification image of a vibratome coronal section of an E18.5 Gli2^−/− brain in which the right and left Hb were labeled with DiI (red) and DiA (green), respectively. (C) Vibratome coronal section of an E18.5 wt embryo stained with α–DCC. (D) Top view of an E13.5 Gli2^−/− ONT labeled with DiI (right Hb, red) and DiA (left Hb, green). (D′) Higher magnification of dotted boxed region in (D) obtained by confocal microscopy. E: Schematic diagram showing the altered trajectory of the rft in Gli2^−/− mice (compare Fig. 1F). The dotted line in (A) and (D) defines the boundaries between the different neuromeres. The dotted line in (B) and (D′) represents the midline. Ca, caudal; Hb, habenula; Hyp, hypothalamus; Ist, Isthmus; Mb, Midbrain; pc, posterior commissure; p1, prosomere1; p2, prosomere2; p3, prosomere 3; ro, rostral; rft, retroflex tract. Scale bar: 200μm and 50 μm in B and D′.

Figure 4. Abnormal expression of signaling cues in Gli2^−/− embryos

(A–H) Adjacent horizontal paraffin sections of one E12.5 wild type specimen (A, C, E, G) and one Gli2 mutant embryo (B, D, F, H) at comparable section levels. (A–B) Immunohistochemistry against DCC. (C–H) In situ hybridization for Ntn1 (C, D), Slit1 (E, F) and Slit2 (G, H). The arrows in A and B show the localization of the rft in a wild type embryo (A) and in a Gli2 mutant (B). The arrow in D, F and H shows the alteration of the expression of these floor plate guidance cues where the rft adopts an abnormal trajectory. Hyp, hypothalamus; Mb, Midbrain. Scale bar: 200μm.

Figure 5. Slit2 as the main repulsive cue for the rft

(A–C) E12.5 habenular explants were cultured with COS cells expressing GFP (A), Slit2 (B) or Slit1 (C). (D) COS cells transfected with GFP (control, 0,954±0,077; n=15) and Slit1 (1,027±0,1242; n=9) had no repulsive effect on habenular axons. In contrast, the presence of Slit2 produced a strong repulsion (0,384± 0,173; n=12). For statistical analysis Student’s t-test, or Mann-Whitney’s test were used, depending on the distribution of the data. Values are given as ± s.d. *p was < 0.001 for Slit2 compared with control. (E) Vibratome coronal section from an E18.5 Slit1 mutant embryo labeled against DCC, showing normal rft tracts. (F) Transversal paraffin section from an E18.5 Slit1-Slit2 double mutant embryo immunolabeled against DCC. The arrow in F indicates the marked crossing of fibers of the left rft to the contralateral side of the brain. Abbreviations: rft, retroflex tract. Scale bar: 200μm.

Figure 6. Robo1 mediates the Slit2 repulsion of the mHb axons

(A–B) In situ hybridization for Robo1 (A, A′) and Robo2 (B, B′) in a paraffin coronal section of an E12.5 (A, B) and E18.5 (A′, B′) embryo, showing expression in the Hb. (C, D) E12.5 Hb explants from Robo1^+/− (C) and Robo1^−/− (D) embryos. They were cultured with COS cells expressing Slit2. (E) The repulsion of Slit2 (0,573±0,289, n= 11) disappeared in absence of Robo1 (1,063±0,204 n= 11). For statistical analysis Student t- test was used. Values are given ± s.d. *p was < 0.001 for Robo1^−/− compared with Robo1^+/−. (F–G) Vibratome coronal section from an E18.5 Robo1 (F) and Robo2 (G) mutant embryo labeled against DCC. The arrow in (F) indicates the presence of DCC labeled axons entering the midline. (H) Top view of an E13.5 Robo1 mutant ONT labeled with DiI (right Hb, red) and DiA (left Hb, green). (H′) Higher magnification of the boxed region in (H), obtained by confocal microscopy. Abbreviations: Hb, habenula; mHb, medial habenula; rft, retroflex tract; Th, thalamus. Scale bar: 200μm and 100μm in H′.