Limits...
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 direct LHX5 targets and negative regulation of the LHX5 function by LMO1. (A) Position weight matrix of the binding motif enriched in the identified LHX5 binding sequences. (B) Luciferase assay validation of the identified LHX5 binding sites for Lmo1, Foxb2, and Tbx3 in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line. Mean ± SD, p < 0.001. (C) Model of the regulatory function of LMO1 on LHX5 that we propose (see the Results Section for details). (D) Luciferase assay in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line stably transfected with a vector expressing luciferase under the control of the LHX5 binding site found in Lmo1. Increasing amounts of an Lmo1 expression vector were cotransfected. Mean ± SD.
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Figure 7: Identification of direct LHX5 targets and negative regulation of the LHX5 function by LMO1. (A) Position weight matrix of the binding motif enriched in the identified LHX5 binding sequences. (B) Luciferase assay validation of the identified LHX5 binding sites for Lmo1, Foxb2, and Tbx3 in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line. Mean ± SD, p < 0.001. (C) Model of the regulatory function of LMO1 on LHX5 that we propose (see the Results Section for details). (D) Luciferase assay in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line stably transfected with a vector expressing luciferase under the control of the LHX5 binding site found in Lmo1. Increasing amounts of an Lmo1 expression vector were cotransfected. Mean ± SD.

Mentions: In order to elucidate whether the regulatory interactions observed above are direct we used chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) (Robertson et al., 2007). Performing this analysis on primary tissue would have been the best choice. This was however not possible since there is no ChIP-grade available antibody against LHX5 (and the only antibody against LHX5 known to us identifies also LHX1). For this reason we chose to transfect a construct expressing a fusion protein of Lhx5 plus FLAG tag (which can be reliably identified with antibodies) into a stable cell line known to express Lhx5. Then we used the Tet-On Advanced Inducible Gene Expression System to regulate the Lhx5 expression level so that it mimics the natural expression level (see Materials and Methods for details). We identified 546 possible LHX5 binding sites, which we assigned to corresponding genes using the nearest downstream method (Peak Annotation with Peak Analyzer). We then analyzed these binding sites for enriched motifs using the DREME software (Bailey, 2011) and found a motif (Figure 7A) that is enriched in 32.18% of the binding sites and corresponds to a predicted LHX5 binding motif (Berger et al., 2008). Of the loci corresponding to qPCR-validated microarray candidates, three showed this motif— Lmo1, Tbx3, and Foxb2. We then performed luciferase assays to test whether these binding sites can regulate expression of their downstream genes; the results indicated (Figure 7B) that Lmo1, Foxb2, and Tbx3 are possible direct targets of LHX5 (Figure 7B). LIM-domain-only (LMO) proteins (like LMO1) can negatively regulate the function of LIM-HD transcription factors by competing with them for binding to the dimer of their obligate co-factor LIM domain-binding protein (LDB) (Bach, 2000; Chen et al., 2010). When the two binding domains of the LDB dimer are occupied by two copies of a LIM-homeodomain protein (like LHX5), this protein is active as a transcriptional regulator. On the contrary, if one of the LIM copies is substituted by an LMO protein, the LIM-homeodomain transcription factor is not active anymore. The downregulation of Lmo1 that we observed in the Lhx5 mutant suggests that transcription of this negative LHX regulator, in turn, is activated by LHX5, thereby providing a negative feedback loop for Lhx5 (Figure 7C). We used luciferase assays to test this hypothesis and found that LMO1 exerts dose-dependent inhibition of transcriptional activation from the LHX5 binding site (Figure 7D). In summary, we showed that Tbx3, Foxb2, and Lmo1 are possible direct targets of LHX5 and, additionally, that LMO1 negatively regulates LHX5 via a negative feedback loop.


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 direct LHX5 targets and negative regulation of the LHX5 function by LMO1. (A) Position weight matrix of the binding motif enriched in the identified LHX5 binding sequences. (B) Luciferase assay validation of the identified LHX5 binding sites for Lmo1, Foxb2, and Tbx3 in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line. Mean ± SD, p < 0.001. (C) Model of the regulatory function of LMO1 on LHX5 that we propose (see the Results Section for details). (D) Luciferase assay in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line stably transfected with a vector expressing luciferase under the control of the LHX5 binding site found in Lmo1. Increasing amounts of an Lmo1 expression vector were cotransfected. Mean ± SD.
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Related In: Results  -  Collection

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Figure 7: Identification of direct LHX5 targets and negative regulation of the LHX5 function by LMO1. (A) Position weight matrix of the binding motif enriched in the identified LHX5 binding sequences. (B) Luciferase assay validation of the identified LHX5 binding sites for Lmo1, Foxb2, and Tbx3 in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line. Mean ± SD, p < 0.001. (C) Model of the regulatory function of LMO1 on LHX5 that we propose (see the Results Section for details). (D) Luciferase assay in the doxycyclin-dependent Lhx5-expressing Neuro2a cell line stably transfected with a vector expressing luciferase under the control of the LHX5 binding site found in Lmo1. Increasing amounts of an Lmo1 expression vector were cotransfected. Mean ± SD.
Mentions: In order to elucidate whether the regulatory interactions observed above are direct we used chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) (Robertson et al., 2007). Performing this analysis on primary tissue would have been the best choice. This was however not possible since there is no ChIP-grade available antibody against LHX5 (and the only antibody against LHX5 known to us identifies also LHX1). For this reason we chose to transfect a construct expressing a fusion protein of Lhx5 plus FLAG tag (which can be reliably identified with antibodies) into a stable cell line known to express Lhx5. Then we used the Tet-On Advanced Inducible Gene Expression System to regulate the Lhx5 expression level so that it mimics the natural expression level (see Materials and Methods for details). We identified 546 possible LHX5 binding sites, which we assigned to corresponding genes using the nearest downstream method (Peak Annotation with Peak Analyzer). We then analyzed these binding sites for enriched motifs using the DREME software (Bailey, 2011) and found a motif (Figure 7A) that is enriched in 32.18% of the binding sites and corresponds to a predicted LHX5 binding motif (Berger et al., 2008). Of the loci corresponding to qPCR-validated microarray candidates, three showed this motif— Lmo1, Tbx3, and Foxb2. We then performed luciferase assays to test whether these binding sites can regulate expression of their downstream genes; the results indicated (Figure 7B) that Lmo1, Foxb2, and Tbx3 are possible direct targets of LHX5 (Figure 7B). LIM-domain-only (LMO) proteins (like LMO1) can negatively regulate the function of LIM-HD transcription factors by competing with them for binding to the dimer of their obligate co-factor LIM domain-binding protein (LDB) (Bach, 2000; Chen et al., 2010). When the two binding domains of the LDB dimer are occupied by two copies of a LIM-homeodomain protein (like LHX5), this protein is active as a transcriptional regulator. On the contrary, if one of the LIM copies is substituted by an LMO protein, the LIM-homeodomain transcription factor is not active anymore. The downregulation of Lmo1 that we observed in the Lhx5 mutant suggests that transcription of this negative LHX regulator, in turn, is activated by LHX5, thereby providing a negative feedback loop for Lhx5 (Figure 7C). We used luciferase assays to test this hypothesis and found that LMO1 exerts dose-dependent inhibition of transcriptional activation from the LHX5 binding site (Figure 7D). In summary, we showed that Tbx3, Foxb2, and Lmo1 are possible direct targets of LHX5 and, additionally, that LMO1 negatively regulates LHX5 via a negative feedback loop.

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