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Lhx5 controls mamillary differentiation in the developing hypothalamus of the mouse.

Heide M, Zhang Y, Zhou X, Zhao T, Miquelajáuregui A, Varela-Echavarría A, Alvarez-Bolado G - Front Neuroanat (2015)

Bottom Line: Microarray analysis and chromatin immunoprecipitation indicated that Lhx5 appears to be involved in Shh downregulation through Tbx3 and activates several MBO-specific regulator and effector genes.Finally, by tracing the caudal hypothalamic cell lineage we show that, in the Lhx5 mutant, at least some MBO cells are present but lack characteristic marker expression.Our work shows how the Lhx5 locus contributes to integrate regional specification pathways with downstream acquisition of neuronal identity in the MBO.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, University of Heidelberg Heidelberg, Germany.

ABSTRACT
Acquisition of specific neuronal identity by individual brain nuclei is a key step in brain development. However, how the mechanisms that confer neuronal identity are integrated with upstream regional specification networks is still mysterious. Expression of Sonic hedgehog (Shh), is required for hypothalamic specification and is later downregulated by Tbx3 to allow for the differentiation of the tubero-mamillary region. In this region, the mamillary body (MBO), is a large neuronal aggregate essential for memory formation. To clarify how MBO identity is acquired after regional specification, we investigated Lhx5, a transcription factor with restricted MBO expression. We first generated a hypomorph allele of Lhx5-in homozygotes, the MBO disappears after initial specification. Intriguingly, in these mutants, Tbx3 was downregulated and the Shh expression domain abnormally extended. Microarray analysis and chromatin immunoprecipitation indicated that Lhx5 appears to be involved in Shh downregulation through Tbx3 and activates several MBO-specific regulator and effector genes. Finally, by tracing the caudal hypothalamic cell lineage we show that, in the Lhx5 mutant, at least some MBO cells are present but lack characteristic marker expression. Our work shows how the Lhx5 locus contributes to integrate regional specification pathways with downstream acquisition of neuronal identity in the MBO.

No MeSH data available.


The tuberal and prethalamic regions seem unaffected in the Lhx5fl∕fl mutant. In situ hybridization detection of marker gene expression (as indicated) on sections of E12.5 embryos of in Lhx5fl∕+ and Lhx5fl∕fl mice (as indicated). Arrows indicate comparable points in the expression domains of controls and mutants. (A–F)Pitx2, Th, and Pitx3 are midbrain markers. (G–J)Arx and Olig2 are prethalamus markers. (K,L)Tbr1 is a marker of the thalamic eminence. (M–R)SF-1, Lef1 and Pomc are tuberal markers. See Results Section for details. Scale bars: 500 μm (in A, for sagittals; in G, for transverse sections).
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Figure 11: The tuberal and prethalamic regions seem unaffected in the Lhx5fl∕fl mutant. In situ hybridization detection of marker gene expression (as indicated) on sections of E12.5 embryos of in Lhx5fl∕+ and Lhx5fl∕fl mice (as indicated). Arrows indicate comparable points in the expression domains of controls and mutants. (A–F)Pitx2, Th, and Pitx3 are midbrain markers. (G–J)Arx and Olig2 are prethalamus markers. (K,L)Tbr1 is a marker of the thalamic eminence. (M–R)SF-1, Lef1 and Pomc are tuberal markers. See Results Section for details. Scale bars: 500 μm (in A, for sagittals; in G, for transverse sections).

Mentions: To learn more about the extension of the changes observed in the Lhx5 mutant, we performed a general analysis with markers for adjacent regions as well as markers of the tuberal region. Hypothetically, Lhx5 might act in posterior hypothalamic progenitors to repress midbrain identity and at the same time promoting expression of MBO markers. Therefore, in the mutants, the rostral end of the ventral midbrain would abnormally extend into the mamillary region of the hypothalamus. We tested this hypothesis by detecting three genetic markers of the ventral midbrain whose expression patterns inform about the rostral extension of the ventral midbrain: Pitx2, tyrosine hydroxylase (Th), and Pitx3 (Figures 11A–F). The domains of these three markers were essentially unaltered in the mutants (see arrows in Figures 11A–F), which belied any expansion of the midbrain domain into the Lhx5-deficient hypothalamus. Arx and Olig2 are markers of the prethalamus, an Lhx5-expressing region adjacent to the hypothalamus. Expression of both genes was maintained in the mutants (Figures 11G–J). The expression of Tbr1, a marker of the thalamic eminence, was not affected in the mutants either (Figures 11K,L). Finally, we explored the expression of several markers of the tuberal region. The expression of genes specific for some important hypothalamic nuclei, like SF-1 (Nr5a1) (marker of the ventromedial nucleus of the hypothalamus) and Pomc (marker of the arcuate nucleus) was not changed in the Lhx5 mutants (Figures 11M–P). Lef1, a marker of the boundary between the mammillary and tuberal regions, was also essentially unchanged in the mutants (Figures 11Q,R). Additionally, we performed apoptosis (caspase 3) and proliferation (Ki67) analyses on the hypothalamic tuberal region as well as the prethalamus, which showed no change in the mutant (not shown).


Lhx5 controls mamillary differentiation in the developing hypothalamus of the mouse.

Heide M, Zhang Y, Zhou X, Zhao T, Miquelajáuregui A, Varela-Echavarría A, Alvarez-Bolado G - Front Neuroanat (2015)

The tuberal and prethalamic regions seem unaffected in the Lhx5fl∕fl mutant. In situ hybridization detection of marker gene expression (as indicated) on sections of E12.5 embryos of in Lhx5fl∕+ and Lhx5fl∕fl mice (as indicated). Arrows indicate comparable points in the expression domains of controls and mutants. (A–F)Pitx2, Th, and Pitx3 are midbrain markers. (G–J)Arx and Olig2 are prethalamus markers. (K,L)Tbr1 is a marker of the thalamic eminence. (M–R)SF-1, Lef1 and Pomc are tuberal markers. See Results Section for details. Scale bars: 500 μm (in A, for sagittals; in G, for transverse sections).
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Related In: Results  -  Collection

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Figure 11: The tuberal and prethalamic regions seem unaffected in the Lhx5fl∕fl mutant. In situ hybridization detection of marker gene expression (as indicated) on sections of E12.5 embryos of in Lhx5fl∕+ and Lhx5fl∕fl mice (as indicated). Arrows indicate comparable points in the expression domains of controls and mutants. (A–F)Pitx2, Th, and Pitx3 are midbrain markers. (G–J)Arx and Olig2 are prethalamus markers. (K,L)Tbr1 is a marker of the thalamic eminence. (M–R)SF-1, Lef1 and Pomc are tuberal markers. See Results Section for details. Scale bars: 500 μm (in A, for sagittals; in G, for transverse sections).
Mentions: To learn more about the extension of the changes observed in the Lhx5 mutant, we performed a general analysis with markers for adjacent regions as well as markers of the tuberal region. Hypothetically, Lhx5 might act in posterior hypothalamic progenitors to repress midbrain identity and at the same time promoting expression of MBO markers. Therefore, in the mutants, the rostral end of the ventral midbrain would abnormally extend into the mamillary region of the hypothalamus. We tested this hypothesis by detecting three genetic markers of the ventral midbrain whose expression patterns inform about the rostral extension of the ventral midbrain: Pitx2, tyrosine hydroxylase (Th), and Pitx3 (Figures 11A–F). The domains of these three markers were essentially unaltered in the mutants (see arrows in Figures 11A–F), which belied any expansion of the midbrain domain into the Lhx5-deficient hypothalamus. Arx and Olig2 are markers of the prethalamus, an Lhx5-expressing region adjacent to the hypothalamus. Expression of both genes was maintained in the mutants (Figures 11G–J). The expression of Tbr1, a marker of the thalamic eminence, was not affected in the mutants either (Figures 11K,L). Finally, we explored the expression of several markers of the tuberal region. The expression of genes specific for some important hypothalamic nuclei, like SF-1 (Nr5a1) (marker of the ventromedial nucleus of the hypothalamus) and Pomc (marker of the arcuate nucleus) was not changed in the Lhx5 mutants (Figures 11M–P). Lef1, a marker of the boundary between the mammillary and tuberal regions, was also essentially unchanged in the mutants (Figures 11Q,R). Additionally, we performed apoptosis (caspase 3) and proliferation (Ki67) analyses on the hypothalamic tuberal region as well as the prethalamus, which showed no change in the mutant (not shown).

Bottom Line: Microarray analysis and chromatin immunoprecipitation indicated that Lhx5 appears to be involved in Shh downregulation through Tbx3 and activates several MBO-specific regulator and effector genes.Finally, by tracing the caudal hypothalamic cell lineage we show that, in the Lhx5 mutant, at least some MBO cells are present but lack characteristic marker expression.Our work shows how the Lhx5 locus contributes to integrate regional specification pathways with downstream acquisition of neuronal identity in the MBO.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, University of Heidelberg Heidelberg, Germany.

ABSTRACT
Acquisition of specific neuronal identity by individual brain nuclei is a key step in brain development. However, how the mechanisms that confer neuronal identity are integrated with upstream regional specification networks is still mysterious. Expression of Sonic hedgehog (Shh), is required for hypothalamic specification and is later downregulated by Tbx3 to allow for the differentiation of the tubero-mamillary region. In this region, the mamillary body (MBO), is a large neuronal aggregate essential for memory formation. To clarify how MBO identity is acquired after regional specification, we investigated Lhx5, a transcription factor with restricted MBO expression. We first generated a hypomorph allele of Lhx5-in homozygotes, the MBO disappears after initial specification. Intriguingly, in these mutants, Tbx3 was downregulated and the Shh expression domain abnormally extended. Microarray analysis and chromatin immunoprecipitation indicated that Lhx5 appears to be involved in Shh downregulation through Tbx3 and activates several MBO-specific regulator and effector genes. Finally, by tracing the caudal hypothalamic cell lineage we show that, in the Lhx5 mutant, at least some MBO cells are present but lack characteristic marker expression. Our work shows how the Lhx5 locus contributes to integrate regional specification pathways with downstream acquisition of neuronal identity in the MBO.

No MeSH data available.