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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 Foxb1 lineage of the MBO is present in the Lhx5fl∕flmutant. (A,B) ß-galactosidase activity (blue) reveals the Foxb1 lineage in sagittal sections of E13.5 embryos of Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕+(A) and Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕fl(B). Arrows show the extension of the Foxb1 lineage in the caudal hypothalamus. (C–J)In situ hybridization detection of marker gene expression (as indicated) on sagittal sections of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos. Arrowheads mark the position of the MBO. Scale bars: 500 μm.
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Figure 10: The Foxb1 lineage of the MBO is present in the Lhx5fl∕flmutant. (A,B) ß-galactosidase activity (blue) reveals the Foxb1 lineage in sagittal sections of E13.5 embryos of Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕+(A) and Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕fl(B). Arrows show the extension of the Foxb1 lineage in the caudal hypothalamus. (C–J)In situ hybridization detection of marker gene expression (as indicated) on sagittal sections of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos. Arrowheads mark the position of the MBO. Scale bars: 500 μm.

Mentions: Other transcription factor genes downregulated in our Lhx5 mutant are Olig2 and Otp, known to be involved in MBO development (see Discussion), as well as Foxb2, Barhl1, Nkx2-4, and Arx. Since we did not detect changes in apoptosis or proliferation defects, it seems likely that the cells constituting the MBO primordium are present in the mutants but that they have lost their specific MBO identity. The subsequent loss of cell adhesion protein expression (Cx36-Gdj and Ntm) could underlie the loss of morphological appearance of the MBO and make it undetectable. Since Foxb1 is an early marker of the mamillary neuroepithelium and the developing MBO (Kaestner et al., 1996; Alvarez-Bolado et al., 2000b), we used β-galactosidase detection in Foxb1-Cre;ROSA26R mice to reveal the MBO lineage. In Foxb1-Cre+∕−;Lhx5fl∕+;Rosa26R+ a large caudal hypothalamic domain including the mamillary area was labeled in E13.5 embryos (Figure 10A). In Foxb1-Cre+∕−;Lhx5fl∕fl;Rosa26R+, however, there is only a restricted, round domain formed by cells of the Foxb1 lineage at E13.5 (Figure 10B), corresponding in appearance and position to the MBO. We assume that these are abnormally undifferentiated cells originally fated for the MBO. Expression analysis of specific MBO markers Foxb1, Lhx1, Sim1 and Sim2 (Figures 10C,E,G,I) showed strong downregulation in the mutant (Figures 10D,F,H,J). Additionally, the preserved expression of Lhx1 in regions other than the mamillary (Figures 10I,J) indicates that the result is specific. This result confirmed the presence of MBO cells with abnormal loss of identity in our mutant.


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 Foxb1 lineage of the MBO is present in the Lhx5fl∕flmutant. (A,B) ß-galactosidase activity (blue) reveals the Foxb1 lineage in sagittal sections of E13.5 embryos of Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕+(A) and Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕fl(B). Arrows show the extension of the Foxb1 lineage in the caudal hypothalamus. (C–J)In situ hybridization detection of marker gene expression (as indicated) on sagittal sections of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos. Arrowheads mark the position of the MBO. Scale bars: 500 μm.
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Related In: Results  -  Collection

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Figure 10: The Foxb1 lineage of the MBO is present in the Lhx5fl∕flmutant. (A,B) ß-galactosidase activity (blue) reveals the Foxb1 lineage in sagittal sections of E13.5 embryos of Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕+(A) and Foxb1Cre−eGFP∕+;ROSA26R;Lhx5fl∕fl(B). Arrows show the extension of the Foxb1 lineage in the caudal hypothalamus. (C–J)In situ hybridization detection of marker gene expression (as indicated) on sagittal sections of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos. Arrowheads mark the position of the MBO. Scale bars: 500 μm.
Mentions: Other transcription factor genes downregulated in our Lhx5 mutant are Olig2 and Otp, known to be involved in MBO development (see Discussion), as well as Foxb2, Barhl1, Nkx2-4, and Arx. Since we did not detect changes in apoptosis or proliferation defects, it seems likely that the cells constituting the MBO primordium are present in the mutants but that they have lost their specific MBO identity. The subsequent loss of cell adhesion protein expression (Cx36-Gdj and Ntm) could underlie the loss of morphological appearance of the MBO and make it undetectable. Since Foxb1 is an early marker of the mamillary neuroepithelium and the developing MBO (Kaestner et al., 1996; Alvarez-Bolado et al., 2000b), we used β-galactosidase detection in Foxb1-Cre;ROSA26R mice to reveal the MBO lineage. In Foxb1-Cre+∕−;Lhx5fl∕+;Rosa26R+ a large caudal hypothalamic domain including the mamillary area was labeled in E13.5 embryos (Figure 10A). In Foxb1-Cre+∕−;Lhx5fl∕fl;Rosa26R+, however, there is only a restricted, round domain formed by cells of the Foxb1 lineage at E13.5 (Figure 10B), corresponding in appearance and position to the MBO. We assume that these are abnormally undifferentiated cells originally fated for the MBO. Expression analysis of specific MBO markers Foxb1, Lhx1, Sim1 and Sim2 (Figures 10C,E,G,I) showed strong downregulation in the mutant (Figures 10D,F,H,J). Additionally, the preserved expression of Lhx1 in regions other than the mamillary (Figures 10I,J) indicates that the result is specific. This result confirmed the presence of MBO cells with abnormal loss of identity in our mutant.

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.