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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

Normal proliferation and apoptosis in the Lhx5fl∕fl mutant. (A–D) Immunohistochemistry against Ki67 (red) and ßIII-Tubulin (green) in sections through the ventricular zone of the mamillary region of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos, DAPI (blue) as counterstaining. As expected, in the Lhx5fl∕+(A,B) cells in the ventricular zone were Ki67-positive (A) and mantle cells (young post-mitotic neurons) were ß-III-Tubulin positive (B). Similar results were found in Lhx5fl∕fl mutant embryos (C,D). (E,F) Immunohistochemistry to detect IddU (green) and BrdU/IddU (red) in sections of E12.5 Lhx5fl∕+ and mutant embryos, TO-PRO-III (blue) as counterstaining. IddU and BrdU/IddU could be detected in cells of the Lhx5fl∕+ as well as in cells of the mutant neuroepithelium. The signal could be clearly differentiated between Iddu positive (green) and IddU/BrdU double positive (yellow) cells (E,F). (G, H) Immunohistochemistry for the detection of active Caspase 3 (green) in section of E12.5 Lhx5fl∕+ and mutant embryos, DAPI (blue) as counterstaining. (I,J) Neither the duration of the S-phase (I) nor that of the whole cell cycle (J) in the mamillary ventricular zone showed differences between Lhx5fl∕+ and Lhx5fl∕fl mutant E10.5 through E12.5 embryos. (K) The number of apoptotic cells in the caudal hypothalamus was not increased in Lhx5fl∕fl mutant E11.5 through E14.5 embryos. Abbreviations: V: ventricle, MBO: mamillary body; Arrows in (G,H) indicate active Caspase 3 positive cells; Scale bars: 100 μm; n.s. not significant, *p < 0.05; mean ± SEM, n = 3 embryos.
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Figure 5: Normal proliferation and apoptosis in the Lhx5fl∕fl mutant. (A–D) Immunohistochemistry against Ki67 (red) and ßIII-Tubulin (green) in sections through the ventricular zone of the mamillary region of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos, DAPI (blue) as counterstaining. As expected, in the Lhx5fl∕+(A,B) cells in the ventricular zone were Ki67-positive (A) and mantle cells (young post-mitotic neurons) were ß-III-Tubulin positive (B). Similar results were found in Lhx5fl∕fl mutant embryos (C,D). (E,F) Immunohistochemistry to detect IddU (green) and BrdU/IddU (red) in sections of E12.5 Lhx5fl∕+ and mutant embryos, TO-PRO-III (blue) as counterstaining. IddU and BrdU/IddU could be detected in cells of the Lhx5fl∕+ as well as in cells of the mutant neuroepithelium. The signal could be clearly differentiated between Iddu positive (green) and IddU/BrdU double positive (yellow) cells (E,F). (G, H) Immunohistochemistry for the detection of active Caspase 3 (green) in section of E12.5 Lhx5fl∕+ and mutant embryos, DAPI (blue) as counterstaining. (I,J) Neither the duration of the S-phase (I) nor that of the whole cell cycle (J) in the mamillary ventricular zone showed differences between Lhx5fl∕+ and Lhx5fl∕fl mutant E10.5 through E12.5 embryos. (K) The number of apoptotic cells in the caudal hypothalamus was not increased in Lhx5fl∕fl mutant E11.5 through E14.5 embryos. Abbreviations: V: ventricle, MBO: mamillary body; Arrows in (G,H) indicate active Caspase 3 positive cells; Scale bars: 100 μm; n.s. not significant, *p < 0.05; mean ± SEM, n = 3 embryos.

Mentions: Next we asked if either a defect in proliferation or increased apoptosis were responsible for the reduction in MBO size in the Lhx5-deficient hypothalamus. An initial analysis of mitotic and post-mitotic compartments in the early mamillary region using antibodies against the proliferation marker Ki67 (Starborg et al., 1996) and the neuronal marker beta-III-tubulin revealed no difference between Lhx5fl∕+ and Lhx5fl∕fl (Figures 5A–D). We then applied the IddU/BrdU method (Nowakowski et al., 1989; Martynoga et al., 2005) to analyze cell cycle length in the Lhx5fl∕+ and Lhx5fl∕fl mamillary neuroepithelium of E10.5–E12.5 embryos (Figures 5E,F). This method allows for calculation of the length of the S-phase of the cell cycle. One proliferation marker, IddU, is injected in pregnant mice at one time-point. After a known interval (90 min), a second proliferation marker, BrdU (which can be independently detected with specific antibodies) is injected. Both label the DNA synthesized during the S-phase. After 30 min, the embryonic brains are collected and the cells labeled either only by IddU or by both IddU + BrdU are counted. From the ratio between both numbers of cells, and since we know the interval during which cells can incorporate IddU but not BrdU (90 min.), the length of the S-phase can be easily calculated (see Martynoga et al., 2005 for details).


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)

Normal proliferation and apoptosis in the Lhx5fl∕fl mutant. (A–D) Immunohistochemistry against Ki67 (red) and ßIII-Tubulin (green) in sections through the ventricular zone of the mamillary region of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos, DAPI (blue) as counterstaining. As expected, in the Lhx5fl∕+(A,B) cells in the ventricular zone were Ki67-positive (A) and mantle cells (young post-mitotic neurons) were ß-III-Tubulin positive (B). Similar results were found in Lhx5fl∕fl mutant embryos (C,D). (E,F) Immunohistochemistry to detect IddU (green) and BrdU/IddU (red) in sections of E12.5 Lhx5fl∕+ and mutant embryos, TO-PRO-III (blue) as counterstaining. IddU and BrdU/IddU could be detected in cells of the Lhx5fl∕+ as well as in cells of the mutant neuroepithelium. The signal could be clearly differentiated between Iddu positive (green) and IddU/BrdU double positive (yellow) cells (E,F). (G, H) Immunohistochemistry for the detection of active Caspase 3 (green) in section of E12.5 Lhx5fl∕+ and mutant embryos, DAPI (blue) as counterstaining. (I,J) Neither the duration of the S-phase (I) nor that of the whole cell cycle (J) in the mamillary ventricular zone showed differences between Lhx5fl∕+ and Lhx5fl∕fl mutant E10.5 through E12.5 embryos. (K) The number of apoptotic cells in the caudal hypothalamus was not increased in Lhx5fl∕fl mutant E11.5 through E14.5 embryos. Abbreviations: V: ventricle, MBO: mamillary body; Arrows in (G,H) indicate active Caspase 3 positive cells; Scale bars: 100 μm; n.s. not significant, *p < 0.05; mean ± SEM, n = 3 embryos.
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Figure 5: Normal proliferation and apoptosis in the Lhx5fl∕fl mutant. (A–D) Immunohistochemistry against Ki67 (red) and ßIII-Tubulin (green) in sections through the ventricular zone of the mamillary region of E12.5 Lhx5fl∕+ and Lhx5fl∕fl mutant embryos, DAPI (blue) as counterstaining. As expected, in the Lhx5fl∕+(A,B) cells in the ventricular zone were Ki67-positive (A) and mantle cells (young post-mitotic neurons) were ß-III-Tubulin positive (B). Similar results were found in Lhx5fl∕fl mutant embryos (C,D). (E,F) Immunohistochemistry to detect IddU (green) and BrdU/IddU (red) in sections of E12.5 Lhx5fl∕+ and mutant embryos, TO-PRO-III (blue) as counterstaining. IddU and BrdU/IddU could be detected in cells of the Lhx5fl∕+ as well as in cells of the mutant neuroepithelium. The signal could be clearly differentiated between Iddu positive (green) and IddU/BrdU double positive (yellow) cells (E,F). (G, H) Immunohistochemistry for the detection of active Caspase 3 (green) in section of E12.5 Lhx5fl∕+ and mutant embryos, DAPI (blue) as counterstaining. (I,J) Neither the duration of the S-phase (I) nor that of the whole cell cycle (J) in the mamillary ventricular zone showed differences between Lhx5fl∕+ and Lhx5fl∕fl mutant E10.5 through E12.5 embryos. (K) The number of apoptotic cells in the caudal hypothalamus was not increased in Lhx5fl∕fl mutant E11.5 through E14.5 embryos. Abbreviations: V: ventricle, MBO: mamillary body; Arrows in (G,H) indicate active Caspase 3 positive cells; Scale bars: 100 μm; n.s. not significant, *p < 0.05; mean ± SEM, n = 3 embryos.
Mentions: Next we asked if either a defect in proliferation or increased apoptosis were responsible for the reduction in MBO size in the Lhx5-deficient hypothalamus. An initial analysis of mitotic and post-mitotic compartments in the early mamillary region using antibodies against the proliferation marker Ki67 (Starborg et al., 1996) and the neuronal marker beta-III-tubulin revealed no difference between Lhx5fl∕+ and Lhx5fl∕fl (Figures 5A–D). We then applied the IddU/BrdU method (Nowakowski et al., 1989; Martynoga et al., 2005) to analyze cell cycle length in the Lhx5fl∕+ and Lhx5fl∕fl mamillary neuroepithelium of E10.5–E12.5 embryos (Figures 5E,F). This method allows for calculation of the length of the S-phase of the cell cycle. One proliferation marker, IddU, is injected in pregnant mice at one time-point. After a known interval (90 min), a second proliferation marker, BrdU (which can be independently detected with specific antibodies) is injected. Both label the DNA synthesized during the S-phase. After 30 min, the embryonic brains are collected and the cells labeled either only by IddU or by both IddU + BrdU are counted. From the ratio between both numbers of cells, and since we know the interval during which cells can incorporate IddU but not BrdU (90 min.), the length of the S-phase can be easily calculated (see Martynoga et al., 2005 for details).

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