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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 MBO in Lhx5fl∕fl mutant embryos fails to increase in size. (A–C) Nissl staining of sagittal sections of E18.5 brains, rostral to the left, genotypes as indicated. (D,E) Immunohistochemistry against GFP (green; from the reporter gene included in the Foxb1Cre−eGFP∕+ line) and neurofilaments (red), counterstained with DAPI (blue). (The Lhx5fl∕fl mutant is not crossed with Foxb1Cre−eGFP∕+ and therefore has no eGFP reporter gene to be detected). (F–H) Antibody detection of LHX5 protein (red), counterstained with DAPI (blue). Arrows in (A,D) show the principal mamillary tract. The asterisks show the place where the MBO would be expected. Scale bar in (A): 100 μm. MBO, mamillary body; P, Pons. (I) MBO size quantitation during development; mean ±SD; n = 3 embryos per age and genotype; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 4: The MBO in Lhx5fl∕fl mutant embryos fails to increase in size. (A–C) Nissl staining of sagittal sections of E18.5 brains, rostral to the left, genotypes as indicated. (D,E) Immunohistochemistry against GFP (green; from the reporter gene included in the Foxb1Cre−eGFP∕+ line) and neurofilaments (red), counterstained with DAPI (blue). (The Lhx5fl∕fl mutant is not crossed with Foxb1Cre−eGFP∕+ and therefore has no eGFP reporter gene to be detected). (F–H) Antibody detection of LHX5 protein (red), counterstained with DAPI (blue). Arrows in (A,D) show the principal mamillary tract. The asterisks show the place where the MBO would be expected. Scale bar in (A): 100 μm. MBO, mamillary body; P, Pons. (I) MBO size quantitation during development; mean ±SD; n = 3 embryos per age and genotype; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: To analyze the role of Lhx5 in the development of the MBO, we crossed the Lhx5fl∕fl conditional line with the Foxb1Cre−eGFP∕+ line (Zhao et al., 2007). Since Foxb1 is a specific marker of the developing MBO (Alvarez-Bolado et al., 2000b), this crossing leads to a conditional inactivation of Lhx5 in the MBO. In Nissl-stained sagittal sections at E18.5 the MBO of Foxb1Cre−eGFP∕+ mice was visible as a compact mass of neurons giving rise to a characteristic axonal bundle (the principal mamillary tract) (Figure 4A). These structures were absent in Foxb1Cre−eGFP∕+;Lhx5fl∕fl brains (Figure 4B) as well as in Lhx5fl∕fl brains (Figure 4C). The absence of the MBO was confirmed by loss of GFP antibody detection (enhanced GFP reporter of the iCre-IRES-eGFP-cassette), confirming the histological result (Figures 4D,E; the non-recombined Lhx5fl∕fl brains lack of course the eGFP reporter). Finally, antibody detection of LHX5 protein (Figures 4F–H) showed that they are indeed lost in the non-recombined Lhx5fl∕fl brains as well as in the ones from Foxb1-Cre-eGFP+∕−;Lhx5fl∕fl crosses. The loss of the MBO in the non-recombined Lhx5fl∕fl embryos was indistinguishable from that in the Foxb1-Cre-eGFP+∕−;Lhx5fl∕fl embryos. To quantify the loss of the MBO we labeled sagittal sections of Foxb1-Cre-eGFP+∕−;Lhx5fl∕+and Foxb1-Cre-eGFP+∕−;Lhx5fl∕fl with anti-GFP antibodies and measured the MBO area (using ImageJ software) (Figure 4I), uncovering a failure of the mutant MBO to grow to a normal size from E13.5 on.


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 MBO in Lhx5fl∕fl mutant embryos fails to increase in size. (A–C) Nissl staining of sagittal sections of E18.5 brains, rostral to the left, genotypes as indicated. (D,E) Immunohistochemistry against GFP (green; from the reporter gene included in the Foxb1Cre−eGFP∕+ line) and neurofilaments (red), counterstained with DAPI (blue). (The Lhx5fl∕fl mutant is not crossed with Foxb1Cre−eGFP∕+ and therefore has no eGFP reporter gene to be detected). (F–H) Antibody detection of LHX5 protein (red), counterstained with DAPI (blue). Arrows in (A,D) show the principal mamillary tract. The asterisks show the place where the MBO would be expected. Scale bar in (A): 100 μm. MBO, mamillary body; P, Pons. (I) MBO size quantitation during development; mean ±SD; n = 3 embryos per age and genotype; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
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Related In: Results  -  Collection

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Figure 4: The MBO in Lhx5fl∕fl mutant embryos fails to increase in size. (A–C) Nissl staining of sagittal sections of E18.5 brains, rostral to the left, genotypes as indicated. (D,E) Immunohistochemistry against GFP (green; from the reporter gene included in the Foxb1Cre−eGFP∕+ line) and neurofilaments (red), counterstained with DAPI (blue). (The Lhx5fl∕fl mutant is not crossed with Foxb1Cre−eGFP∕+ and therefore has no eGFP reporter gene to be detected). (F–H) Antibody detection of LHX5 protein (red), counterstained with DAPI (blue). Arrows in (A,D) show the principal mamillary tract. The asterisks show the place where the MBO would be expected. Scale bar in (A): 100 μm. MBO, mamillary body; P, Pons. (I) MBO size quantitation during development; mean ±SD; n = 3 embryos per age and genotype; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: To analyze the role of Lhx5 in the development of the MBO, we crossed the Lhx5fl∕fl conditional line with the Foxb1Cre−eGFP∕+ line (Zhao et al., 2007). Since Foxb1 is a specific marker of the developing MBO (Alvarez-Bolado et al., 2000b), this crossing leads to a conditional inactivation of Lhx5 in the MBO. In Nissl-stained sagittal sections at E18.5 the MBO of Foxb1Cre−eGFP∕+ mice was visible as a compact mass of neurons giving rise to a characteristic axonal bundle (the principal mamillary tract) (Figure 4A). These structures were absent in Foxb1Cre−eGFP∕+;Lhx5fl∕fl brains (Figure 4B) as well as in Lhx5fl∕fl brains (Figure 4C). The absence of the MBO was confirmed by loss of GFP antibody detection (enhanced GFP reporter of the iCre-IRES-eGFP-cassette), confirming the histological result (Figures 4D,E; the non-recombined Lhx5fl∕fl brains lack of course the eGFP reporter). Finally, antibody detection of LHX5 protein (Figures 4F–H) showed that they are indeed lost in the non-recombined Lhx5fl∕fl brains as well as in the ones from Foxb1-Cre-eGFP+∕−;Lhx5fl∕fl crosses. The loss of the MBO in the non-recombined Lhx5fl∕fl embryos was indistinguishable from that in the Foxb1-Cre-eGFP+∕−;Lhx5fl∕fl embryos. To quantify the loss of the MBO we labeled sagittal sections of Foxb1-Cre-eGFP+∕−;Lhx5fl∕+and Foxb1-Cre-eGFP+∕−;Lhx5fl∕fl with anti-GFP antibodies and measured the MBO area (using ImageJ software) (Figure 4I), uncovering a failure of the mutant MBO to grow to a normal size from E13.5 on.

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.