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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.


Related in: MedlinePlus

Identification of genes that are downstream Lhx5. Microarray analysis of wild type and Lhx5fl∕fl mutant E10.5 mamillary neuroepithelium and qRT-PCR validation of the identified candidates; (A) “Heat map” showing the expression of the identified candidates in all samples. (B) Principal component analysis of the microarray samples. (C) “Heat map” showing the expression of the candidates for qPCR analysis in all samples. (D,E) The qPCR validation identified 12 candidates downregulated (p < 0.05) (D) and 3 candidates upregulated in the Lhx5fl∕fl mutant (p < 0.05) (E). Mean ± SD; n = 3 biological replicates.
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Figure 6: Identification of genes that are downstream Lhx5. Microarray analysis of wild type and Lhx5fl∕fl mutant E10.5 mamillary neuroepithelium and qRT-PCR validation of the identified candidates; (A) “Heat map” showing the expression of the identified candidates in all samples. (B) Principal component analysis of the microarray samples. (C) “Heat map” showing the expression of the candidates for qPCR analysis in all samples. (D,E) The qPCR validation identified 12 candidates downregulated (p < 0.05) (D) and 3 candidates upregulated in the Lhx5fl∕fl mutant (p < 0.05) (E). Mean ± SD; n = 3 biological replicates.

Mentions: To understand the molecular basis of the MBO hypoplasia observed in Lhx5fl∕fl embryos we performed comparative expression profiling using microarrays. We extracted total RNA from the caudal hypothalamus of wild type and Lhx5fl∕fl mouse brains. Since tissue loss in the mutant could bias the results, we collected the tissue at E10.5, before any reduction in MBO size is apparent in the mutant (Figure 4I). Unsupervised hierarchical clustering of genes that were at least 1.5-fold up- or downregulated with P < 0.05 yielded a heat map (Figure 6A) indicating that the global gene expression patterns in embryos of the same genotype clustered together and that wild type and mutant samples were clearly distinct. Likewise, principal component analysis showed that mutant and wild type samples separate well from each other (Figure 6B). In this way we detected 56 downregulated and 41 upregulated named genes (as opposed to not yet identified transcripts like RIKEN clones, etc.) (Supplementary Table 1). After qRT-PCR validation we selected 15 candidates for further analysis (Figures 6C–E) including the cell fate determinants Tbx3, Olig2, and Otp as well as Lmo1, an interaction partner of LDB, the obligate cofactor of LHX proteins (Bach, 2000). In order to visualize the changes in spatial expression patterns, in situ hybridization analysis on tissue sections of E12.5 Lhx5fl∕+ and Lhx5fl∕fl embryos was performed (Supplementary Figure 3). All candidate genes downregulated in the Lhx5fl∕fl mutant (Figures 6C–E) were expressed in the Lhx5fl∕+ mamillary region (either in the neuroepithelium or in the mantle layer) and appeared reduced or absent in the mutant (Supplementary Figure 3). Some downregulated candidates showed complete loss of expression: Foxb2 (Supplementary Figures 3E,F), Ntm (Supplementary Figures 3Q,R), Lypd1 (Supplementary Figures 3S,T) and Cx36 (Supplementary Figures 3U,V). Others showed a strong reduction in labeling: Otp (Supplementary Figures 3G,H), Barhl1 (Supplementary Figures 3K,L), Nkx2.4 (Supplementary Figures 3C,D), Olig2 (Supplementary Figures 3I,J). Finally, other downregulated candidates showed pattern changes, like Tbx3 (Supplementary Figures 3A,B). As for the upregulated, Wnt5a (Supplementary Figures 3C′,D′) and Lrtm1 (Supplementary Figures 3W,X) showed clear ectopic expression in the mutant MBO, while Gal (Supplementary Figures 3A′,B′) showed increase in intensity.


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)

Identification of genes that are downstream Lhx5. Microarray analysis of wild type and Lhx5fl∕fl mutant E10.5 mamillary neuroepithelium and qRT-PCR validation of the identified candidates; (A) “Heat map” showing the expression of the identified candidates in all samples. (B) Principal component analysis of the microarray samples. (C) “Heat map” showing the expression of the candidates for qPCR analysis in all samples. (D,E) The qPCR validation identified 12 candidates downregulated (p < 0.05) (D) and 3 candidates upregulated in the Lhx5fl∕fl mutant (p < 0.05) (E). Mean ± SD; n = 3 biological replicates.
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Figure 6: Identification of genes that are downstream Lhx5. Microarray analysis of wild type and Lhx5fl∕fl mutant E10.5 mamillary neuroepithelium and qRT-PCR validation of the identified candidates; (A) “Heat map” showing the expression of the identified candidates in all samples. (B) Principal component analysis of the microarray samples. (C) “Heat map” showing the expression of the candidates for qPCR analysis in all samples. (D,E) The qPCR validation identified 12 candidates downregulated (p < 0.05) (D) and 3 candidates upregulated in the Lhx5fl∕fl mutant (p < 0.05) (E). Mean ± SD; n = 3 biological replicates.
Mentions: To understand the molecular basis of the MBO hypoplasia observed in Lhx5fl∕fl embryos we performed comparative expression profiling using microarrays. We extracted total RNA from the caudal hypothalamus of wild type and Lhx5fl∕fl mouse brains. Since tissue loss in the mutant could bias the results, we collected the tissue at E10.5, before any reduction in MBO size is apparent in the mutant (Figure 4I). Unsupervised hierarchical clustering of genes that were at least 1.5-fold up- or downregulated with P < 0.05 yielded a heat map (Figure 6A) indicating that the global gene expression patterns in embryos of the same genotype clustered together and that wild type and mutant samples were clearly distinct. Likewise, principal component analysis showed that mutant and wild type samples separate well from each other (Figure 6B). In this way we detected 56 downregulated and 41 upregulated named genes (as opposed to not yet identified transcripts like RIKEN clones, etc.) (Supplementary Table 1). After qRT-PCR validation we selected 15 candidates for further analysis (Figures 6C–E) including the cell fate determinants Tbx3, Olig2, and Otp as well as Lmo1, an interaction partner of LDB, the obligate cofactor of LHX proteins (Bach, 2000). In order to visualize the changes in spatial expression patterns, in situ hybridization analysis on tissue sections of E12.5 Lhx5fl∕+ and Lhx5fl∕fl embryos was performed (Supplementary Figure 3). All candidate genes downregulated in the Lhx5fl∕fl mutant (Figures 6C–E) were expressed in the Lhx5fl∕+ mamillary region (either in the neuroepithelium or in the mantle layer) and appeared reduced or absent in the mutant (Supplementary Figure 3). Some downregulated candidates showed complete loss of expression: Foxb2 (Supplementary Figures 3E,F), Ntm (Supplementary Figures 3Q,R), Lypd1 (Supplementary Figures 3S,T) and Cx36 (Supplementary Figures 3U,V). Others showed a strong reduction in labeling: Otp (Supplementary Figures 3G,H), Barhl1 (Supplementary Figures 3K,L), Nkx2.4 (Supplementary Figures 3C,D), Olig2 (Supplementary Figures 3I,J). Finally, other downregulated candidates showed pattern changes, like Tbx3 (Supplementary Figures 3A,B). As for the upregulated, Wnt5a (Supplementary Figures 3C′,D′) and Lrtm1 (Supplementary Figures 3W,X) showed clear ectopic expression in the mutant MBO, while Gal (Supplementary Figures 3A′,B′) showed increase in intensity.

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.


Related in: MedlinePlus