Limits...
The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

Kim J, Yeon J, Choi SK, Huh YH, Fang Z, Park SJ, Kim MO, Ryoo ZY, Kang K, Kweon HS, Jeon WB, Li C, Kim K - PLoS Genet. (2015)

Bottom Line: Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans.Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines.Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Korea.

ABSTRACT
The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

No MeSH data available.


Related in: MedlinePlus

C. elegans LIM-4 or human LHX6 induces expression of cholinergic makers and neuronal characteristics in human neuroblastoma cells.(A) Shown is the sequence alignment of the homeodomain of C. elegans LIM-4 and human LHX6 and LHX8. Identical residues in at least two proteins are shown in red. (B) Average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates different between heat shock and no heat shock conditions at p<0.001 (student t-test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4D. (C) Percentage of animals of the indicated genotypes expressing stably integrated flp-12p::gfp reporter (ynIs25) is shown. Strong, weak or off expression is defined as GFP expression observed at 400x magnification in both cell bodies and processes, in only cell bodies, or not observed either in cell bodies or processes, respectively. Over two independent transgenic lines were tested. n≥50 for each. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4C. (D) Confocal images of SH-SY5Y human neuroblastoma cell line transfected by empty vector, C. elegans lim-4 or human LHX6 and immunostained with VAChT antibodies. Scale bar: 50 μm. Note that cells expressing either LHX6-GFP or lim-4-GFP exhibit spiky protrusions. (E) Ultrastructural analysis of LIM-4 or LHX6 transfected SH-SY5Y cell lines. Images from transmission electron microscope for untransfected (control), empty vector, lim-4-GFP, or LHX6-GFP transfected cells are shown. Left two columns are from peri-nuclear region and right two columns are from protruded region (far right images are higher magnification of the boxed area). Nu: nucleus, G: Golgi apparatus, M: mitochondria, ER: endoplasmic reticulum, DV: dense core vesicle, SV: synaptic vesicle. Scale bars: 1 μm. Confocal images of these control and transfected SH-SY5Y cells grown on the MatTek culture dish are shown in S14 Fig.
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pgen.1005480.g007: C. elegans LIM-4 or human LHX6 induces expression of cholinergic makers and neuronal characteristics in human neuroblastoma cells.(A) Shown is the sequence alignment of the homeodomain of C. elegans LIM-4 and human LHX6 and LHX8. Identical residues in at least two proteins are shown in red. (B) Average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates different between heat shock and no heat shock conditions at p<0.001 (student t-test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4D. (C) Percentage of animals of the indicated genotypes expressing stably integrated flp-12p::gfp reporter (ynIs25) is shown. Strong, weak or off expression is defined as GFP expression observed at 400x magnification in both cell bodies and processes, in only cell bodies, or not observed either in cell bodies or processes, respectively. Over two independent transgenic lines were tested. n≥50 for each. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4C. (D) Confocal images of SH-SY5Y human neuroblastoma cell line transfected by empty vector, C. elegans lim-4 or human LHX6 and immunostained with VAChT antibodies. Scale bar: 50 μm. Note that cells expressing either LHX6-GFP or lim-4-GFP exhibit spiky protrusions. (E) Ultrastructural analysis of LIM-4 or LHX6 transfected SH-SY5Y cell lines. Images from transmission electron microscope for untransfected (control), empty vector, lim-4-GFP, or LHX6-GFP transfected cells are shown. Left two columns are from peri-nuclear region and right two columns are from protruded region (far right images are higher magnification of the boxed area). Nu: nucleus, G: Golgi apparatus, M: mitochondria, ER: endoplasmic reticulum, DV: dense core vesicle, SV: synaptic vesicle. Scale bars: 1 μm. Confocal images of these control and transfected SH-SY5Y cells grown on the MatTek culture dish are shown in S14 Fig.

Mentions: The mammalian genome contains two LIM-4 orthologs, LHX6 and LHX8. In mice, these genes are largely expressed in the developing and adult striatum and orchestrate specification of interneuron identities; specifically, LHX6 and LHX8 are required to determine GABAergic/peptidergic and cholinergic interneuronal cell fate, respectively. In addition, these genes have redundant function to regulate expression of shh in MGE neurons [34]. LIM-4 exhibits a high degree of protein sequence homology to LHX6 and LHX8 (in particular, 60% identical in its homeodomain) (Fig 7A) [14], suggesting a functional conservation of these proteins.


The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

Kim J, Yeon J, Choi SK, Huh YH, Fang Z, Park SJ, Kim MO, Ryoo ZY, Kang K, Kweon HS, Jeon WB, Li C, Kim K - PLoS Genet. (2015)

C. elegans LIM-4 or human LHX6 induces expression of cholinergic makers and neuronal characteristics in human neuroblastoma cells.(A) Shown is the sequence alignment of the homeodomain of C. elegans LIM-4 and human LHX6 and LHX8. Identical residues in at least two proteins are shown in red. (B) Average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates different between heat shock and no heat shock conditions at p<0.001 (student t-test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4D. (C) Percentage of animals of the indicated genotypes expressing stably integrated flp-12p::gfp reporter (ynIs25) is shown. Strong, weak or off expression is defined as GFP expression observed at 400x magnification in both cell bodies and processes, in only cell bodies, or not observed either in cell bodies or processes, respectively. Over two independent transgenic lines were tested. n≥50 for each. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4C. (D) Confocal images of SH-SY5Y human neuroblastoma cell line transfected by empty vector, C. elegans lim-4 or human LHX6 and immunostained with VAChT antibodies. Scale bar: 50 μm. Note that cells expressing either LHX6-GFP or lim-4-GFP exhibit spiky protrusions. (E) Ultrastructural analysis of LIM-4 or LHX6 transfected SH-SY5Y cell lines. Images from transmission electron microscope for untransfected (control), empty vector, lim-4-GFP, or LHX6-GFP transfected cells are shown. Left two columns are from peri-nuclear region and right two columns are from protruded region (far right images are higher magnification of the boxed area). Nu: nucleus, G: Golgi apparatus, M: mitochondria, ER: endoplasmic reticulum, DV: dense core vesicle, SV: synaptic vesicle. Scale bars: 1 μm. Confocal images of these control and transfected SH-SY5Y cells grown on the MatTek culture dish are shown in S14 Fig.
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pgen.1005480.g007: C. elegans LIM-4 or human LHX6 induces expression of cholinergic makers and neuronal characteristics in human neuroblastoma cells.(A) Shown is the sequence alignment of the homeodomain of C. elegans LIM-4 and human LHX6 and LHX8. Identical residues in at least two proteins are shown in red. (B) Average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates different between heat shock and no heat shock conditions at p<0.001 (student t-test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4D. (C) Percentage of animals of the indicated genotypes expressing stably integrated flp-12p::gfp reporter (ynIs25) is shown. Strong, weak or off expression is defined as GFP expression observed at 400x magnification in both cell bodies and processes, in only cell bodies, or not observed either in cell bodies or processes, respectively. Over two independent transgenic lines were tested. n≥50 for each. Data of WT,-, or Ex[hsp::lim-4cDNA] are from Fig 4C. (D) Confocal images of SH-SY5Y human neuroblastoma cell line transfected by empty vector, C. elegans lim-4 or human LHX6 and immunostained with VAChT antibodies. Scale bar: 50 μm. Note that cells expressing either LHX6-GFP or lim-4-GFP exhibit spiky protrusions. (E) Ultrastructural analysis of LIM-4 or LHX6 transfected SH-SY5Y cell lines. Images from transmission electron microscope for untransfected (control), empty vector, lim-4-GFP, or LHX6-GFP transfected cells are shown. Left two columns are from peri-nuclear region and right two columns are from protruded region (far right images are higher magnification of the boxed area). Nu: nucleus, G: Golgi apparatus, M: mitochondria, ER: endoplasmic reticulum, DV: dense core vesicle, SV: synaptic vesicle. Scale bars: 1 μm. Confocal images of these control and transfected SH-SY5Y cells grown on the MatTek culture dish are shown in S14 Fig.
Mentions: The mammalian genome contains two LIM-4 orthologs, LHX6 and LHX8. In mice, these genes are largely expressed in the developing and adult striatum and orchestrate specification of interneuron identities; specifically, LHX6 and LHX8 are required to determine GABAergic/peptidergic and cholinergic interneuronal cell fate, respectively. In addition, these genes have redundant function to regulate expression of shh in MGE neurons [34]. LIM-4 exhibits a high degree of protein sequence homology to LHX6 and LHX8 (in particular, 60% identical in its homeodomain) (Fig 7A) [14], suggesting a functional conservation of these proteins.

Bottom Line: Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans.Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines.Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, Korea.

ABSTRACT
The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

No MeSH data available.


Related in: MedlinePlus