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First published online 16 November 2005
doi: 10.1242/dev.02142

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Sim1 and Sim2 are required for the correct targeting of mammillary body axons

Research Center, Hôpital Sainte-Justine, 3175 Cote Ste-Catherine, Montréal, Quebec H3T 1C5, Canada

View larger version (80K): [in a new window]   Fig. 1. Co-expression of Sim1 and Sim2 in the developing mammillary body. Adjacent coronal sections through the prospective MB of E10.5 (A,B), E11.5 (C,D), E12.5 (E,F) and E14.5 (G,H) wild-type embryos were hybridized either with Sim1 (A,C,E,G) or Sim2 (B,D,F,H). (A,B) At E10.5, Sim1 is expressed in the lateral aspect of the neuroepithelium, which presumably corresponds to the mantle layer, but is expressed less strongly in the medial aspect (bracket), which corresponds to the ventricular layer (A). Sim2 is mainly expressed in this medial domain (B). (C-F) At E11.5 and E12.5, Sim1 is expressed strongly in the mantle layer, which corresponds to the prospective MB, but also weakly in the ventricular layer (bracket, C). Sim2 is expressed in the ventricular layer and in the medial aspect of the mantle layer of the prospective MB. (G,H) At E14.5, Sim1 shows the same expression pattern. However, Sim2 expression has decreased in intensity and becomes restricted to the ventricular layer. The arrows indicate the domains of Sim1/Sim2 expression in the prospective MB.

View larger version (19K): [in a new window]   Fig. 2. Organization of the mammillary body projections. The left side of the brain is shown from a sagittal perspective. Rostral is to the right. The principal mammillary tract (PMT) gives rise to the mammillotegmental (MTEG) and mammillothalamic tract (MTT).

View larger version (105K): [in a new window]   Fig. 3. MTEG and MTT development affected by Sim1/Sim2 gene dosage. E18.5 brains of various genotypes were sectioned sagittally and stained with Haematoxylin. The upper panels (A-C,G-I) represent medial sections containing the PMT, whereas the lower panels correspond to lateral sections that include the MTT and the PMT. The MTEG is not readily detectable on sagittal sections because of its orientation. The PMT is indicated by arrows, the MTT by arrowheads. The PMT and MTT are well developed in Sim1+/-;Sim2+/- (A,D) and Sim1+/+;Sim2-/- (B,E) embryos, whereas they are thinner in Sim1-/-;Sim2+/+ (C,F), Sim1+/-;Sim2-/- (G,J) and Sim1-/-;Sim2+/- (H,K) embryos. The PMT and MTT were barely detectable in Sim1-/-;Sim2-/- embryos (I,L). In all cases, the MB was histologically present.

View larger version (58K): [in a new window]   Fig. 4. Creation of a Sim1 allele expressing Tau-lacZ. (A) Schematic representation of the Sim1 locus (wt), of the targeting vector (HR), and of the Sim1 mutant allele (m). Homologous recombination replaces the initiation codon and the basic HLH domain with a Tau-lacZ fusion gene. The 5' external probe is indicated. B, BamHI; H, HindIII. (B) Southern blot analysis of genomic DNA from Sim1+/+, Sim1tlz/+ and Sim1tlz/tlz mice. The 5' probe detects a wild-type 5.2-kb BamHI fragment and a mutant 4.5-kb BamHI fragment. (C,D) Sagittal sections through the MB of Sim1+/+;Sim2+/+ and Sim1tlz/tlz;Sim2-/- E18.5 embryos that have been stained with Haematoxylin. The MTT and the PMT are not detectable in the MB of embryos homozygous for the Sim1- (C) or the Sim1tlz (D) allele.

View larger version (96K): [in a new window]   Fig. 5. ß-galactosidase staining of mammillary body axonal projections in E14.5 Sim1/Sim2 mutant embryos. E14.5 brains of various genotypes were stained for ß-galactosidase activity and sectioned coronally. For each brain, four consecutive sections are shown, the most anterior being at the top of the figure. PMTs are indicated by arrows, whereas the abnormally targeted axons are indicated by arrowheads. The loss of Sim1 function is associated with a decrease of the PMT and the emergence of MB axons directed towards the midline. Sim2 also contributes to this phenotype, as the axonal abnormalities are more severe in Sim1tlz/tlz;Sim2-/- than in Sim1tlz/tlz;Sim2+/+ embryos.

View larger version (92K): [in a new window]   Fig. 6. Abnormal targeting of mammillary body axons as revealed by DiI labelling. Crystals of DiI were inserted into E14.5 brains of Sim1+/-;Sim2+/- (A,B) and Sim1-/-;Sim2-/- (C,D) embryos, and the brains sectioned after an incubation period of 2 weeks. (B,D) Higher magnification images of A (B) and C (D). The white line in B and D corresponds to the midline. The PMT (arrowhead) is recognizable in Sim1+/-;Sim2+/- but not in the double mutant. A few axons appear to progress towards the midline in Sim1+/-;Sim2+/- embryos (arrow), whereas the majority of them do so in Sim1-/-;Sim2-/- embryos.

View larger version (121K): [in a new window]   Fig. 7. ß-galactosidase staining of mammillary body axonal projections in E11.5 Sim1/Sim2 mutant embryos. E11.5 brains with various genotypes, as indicated, were stained for ß-galactosidase activity and sectioned coronally. Axonal bundles (arrows) are easily recognizable in Sim1tlz/+;Sim2+/- (A) and in Sim1tlz/+;Sim2-/- (B) embryos, but are decreased in Sim1tlz/-;Sim2+/- embryos (C). No bundle was detected in Sim1tlz/-;Sim2-/- embryos (D). Note that axons progress in a domain that is stained. ß-galactosidase staining in the dorsal domain is weaker in C and D than in A and B because these thick sections (A,B) of early embryos are from a slightly different plane.

View larger version (95K): [in a new window]   Fig. 8. Loss of Foxb1 expression in E12.5 Sim1/Sim2 double mutants. Coronal sections through the MB of E12.5 Sim1+/+;Sim2+/+ (A,C,E,G) and Sim1-/-;Sim2-/- (B,D,F,H) embryos were hybridized either with a Sim1 (A,B), Lhx1 (C,D), Nkx2.1 (E,F) or Foxb1 (G,H) probe. The maintenance of Sim1, Lhx1 and Nkx2.1 expression in double mutants indicates that the loss of Sim1/Sim2 does not affect the early differentiation of MB neurons. Sim1/Sim2 are, however, required to maintain Foxb1 expression in the MB.

View larger version (101K): [in a new window]   Fig. 9. Mammillary body neurons survive until the end of gestation in Sim1/Sim2 double mutants. Coronal sections through the MB of E18.5 with various dosage of Sim1/Sim2, as indicated, hybridized with Sim1 (A,B), Lhx1 (C,D) and Foxb1 (E-H) probes. Sim1 and Lhx1 expression is maintained in the MB of Sim1/Sim2 double mutants. By contrast, Foxb1 expression is dramatically reduced in Sim1/Sim2 double mutants but not in embryos with at least one allele of Sim1/Sim2. Of note, the apparent smaller sizes of the MBs shown in G and H reflect different planes of section.

View larger version (97K): [in a new window]   Fig. 10. Sim1 and Sim2 repress Rig-1/Robo3 expression in the developing mammillary body. (A-T) Coronal sections through the MB of E12.5 Sim1+/-;Sim2+/+ and Sim1-/-;Sim2-/- embryos were hybridized either with a Slit1 (A-D), Slit2 (E-H), Robo1 (I-L), Rig-1/Robo3 (M-P) or Sim1 (Q-T) probe. Sections correspond either to the anterior or posterior aspect of the MB, as indicated. The sections hybridized with the Rig-1/Robo3 probe (M-P) are adjacent to those hybridized with the Sim1 probe (Q-T). Expression of Slit1, Slit2 and Robo1 is similar in Sim1+/-;Sim2+/+ and Sim1-/-;Sim2-/- embryos. In the anterior MB of Sim1+/-;Sim2+/+ embryos, Rig-1/Robo3 is expressed in a narrow region that includes a medial domain (yellow arrowhead) that is contained within the dorsal aspect of the MB Sim1 expression domain (M,Q). In Sim1-/-;Sim2-/- embryos, Rig-1/Robo3 expression in the prospective MB (arrow) occupies a larger area extending ventrally and laterally (N,R). Rig-1/Robo3 expression in the region dorsal to the MB (blue arrowhead) is decreased in Sim1-/-;Sim2-/- embryos (M,N). In the posterior MB, Rig-1/Robo3 expression is upregulated in Sim1-/-;Sim2-/- embryos (arrow) (O,P). (U-B') Coronal sections through the MB of E11.5 Sim1+/-;Sim2+/+ and Sim1-/-;Sim2-/- embryos were hybridized either with a Rig-1/Robo3 (U-X) or a Sim1 (Y-B') probe. Sections correspond either to the anterior or posterior aspect of the MB, as indicated. The sections hybridized with the Rig-1/Robo3 probe (U-X) are adjacent to those hybridized with the Sim1 probe (Y-B'). (U,V) Rig-1/Robo3 is ectopically expressed (V) in the MB (arrows). (W,X) Asterisks indicate a second region in which Rig-1/Robo3 expression is upregulated (X).