LIM homeobox protein 5 (Lhx5) is essential for mamillary body development

Abstract

The mamillary body (MM) is a group of hypothalamic nuclei related to memory and spatial navigation that interconnects hippocampal, thalamic, and tegmental regions. Here we demonstrate that Lhx5, a LIM-HD domain transcription factor expressed early in the developing posterior hypothalamus, is required for the generation of the MM and its derived tracts. The MM markers Foxb1, Sim2, and Lhx1 are absent in Lhx5 knock-out mice from early embryonic stages, suggesting abnormal specification of this region. This was supported by the absence of Nkx2.1 and expansion of Shh in the prospective mamillary area. Interestingly, we also found an ectopic domain expressing Lhx2 and Lhx9 along the anterio-posterior hypothalamic axis. Our results suggest that Lhx5 controls early aspects of hypothalamic development by regulating gene expression and cellular specification in the prospective MM.

Keywords: diencephalon; embryonic development; hypothalamus; mouse; transcription factor.

FIGURE 1

Expression patterns of Lhx5 in the developing hypothalamus. In situ hybridization for Lhx5 in whole-mount brain preparations of E10.5 (A), E11.5 (B), and E12.5 (C) embryos and in a section indicated in (C) by the dashed line (C’). Whole hybridized brains were hemisected along the sagittal plane and lateral views are shown (anterior is to the left). Abbreviations: Th, thalamus; MM, mamillary body; p, prosomere; POA, preoptic area; PT, pretectum; PTh, prethalamus; RM, retromamillary; SPa, subparaventricular area; Tel, telencephalon; Tg, tegmentum; Tu, tuberal. Scale bars: (A–C) 400 μm, (C’) 200 μm.

FIGURE 2

MM is absent in Lhx5 mutants. (A,B) Nissl staining in sagittal sections of control mice at E18.5 reveals the MM (arrowhead) that appears absent in Lhx5 mutants. (C,D) Comparable sections processed for Sevier–Munger silver staining reveal the fx (empty arrow), pm (filled arrow), mtt, (empty arrowhead), and mteg (filled arrowhead) tracts, in control but not in mutant littermates (anterior is to the left). (E–H) In situ hybridization for Foxb1 and Lhx1 in E18.5 coronal sections stained the MM in controls (arrowhead) and showed no expression in Lhx5 mutants. Note the presence of Lhx1 expression in the ZI (white arrow). Abbreviations: fx, fornix; MM, mamillary; mteg, mammillotegmental; mtt, mammillothalamic; PG, pontine gray; pm, principal mamillary; ZI, zona incerta. Scale bar: 400 μm.

FIGURE 3

Comparative analysis of MM markers in the developing embryo. Genetic expression profile of Nkx2.1, Foxb1, Sim2, and Lhx1 in the prospective MM in E12.5 preparations of whole-mount (A–H, anterior is to the left) and coronal sections (A’–H’) of corresponding Lhx5 mutant and control embryos, as indicated. Empty arrowheads point to the approximate location of MM in Lhx5 mutants. Dashed lines in (A–H) indicate the approximate plane of sections shown in (A’–H’). Brackets in (A,B) demarcate the extent of Nkx2.1 expression along the anterio-posterior axis. Abbreviations: Th, thalamus; EMT, eminentia thalami; IDv, ventral intrahypothalamic diagonal; Mb, midbrain; MM, mamillary; PVN, paraventricular nucleus; PT, pretectum; PTh, prethalamus; Tu, tuberal; ZLI, zona limitans intrathalamica. Scale bars: (A–H) 400 μm, (A’–H’) 200 μm.

FIGURE 4

Shh expression in Lhx5 mutants. Comparison of Shh expression on whole-mount ISH in control and mutant embryos at E11.5 (A,B), E12.5 (C,D) and E13.5 (E,F). Note that the posterior expression domain (empty arrowheads) is present at all stages analyzed (A–F) but appears expanded anteriorly in the mutant at E11.5 (brackets in A,B). (A–D) The ZLI is present at E11.5 and E12.5, but its dorsal extent (indicated with arrows) is shorter in Lhx5 mutants at E12.5. (A–F). Anterior is to the left. Abbreviations: ant., anterior; Th, thalamus; PT, pretectum; PTh, prethalamus; post., posterior; ZLI, zona limitans intrathalamica. Scale bars: (A,B) 200 μm, (C-F) 400 μm.

FIGURE 5

Lxh2 and Lhx9 misexpression in the Lhx5^-/- posterior hypothalamus. Whole-mount ISH for Lhx2 (A,B) and Lhx9 (C,D) in control and Lhx5 mutant E12.5 embryos (anterior is to the left), showing ectopic expression of both markers in the posterior hypothalamus of Lhx5 mutants (asterisks in B,D). (A,B) The Lhx2-expressing region in the AH was present in both conditions, but the tuberal expression domain appeared shortened along the anterior–posterior axis (brackets in A,B). (C,D) Lhx9 is expressed at low levels in the anterior hypothalamus in both controls and Lhx5 mutants, in a region that might correspond to the IDv domain (empty arrowheads). Note that expression of both genes in the Th seems unaltered in Lhx5 mutants. Abbreviations: AH, anterior hypothalamus; Th, thalamus; IDv, ventral intrahypothalamic diagonal; Mb, midbrain; PT, pretectum; PTh, prethalamus; Tu, tuberal. Scale bar: 200 μm.