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

lim-4 is expressed in and acts in the SMB neurons and its expression is autoregulated.(A) GFP expression of oyIs35 animals carrying an integrated lim-4p::gfp reporter is overlapped with expression of the flp-12p::mCherry reporter (the SMB marker) but not with expression of the ceh-17p::dsRed reporter (the SIA marker). Images are derived from z-stacks of confocal microscopy images while images in the upper-left boxed regions are single focal plane confocal microscopy images. Anterior is to the left. Scale bar: 50 μm. (B) In lim-4 mutants, expression of the lim-4p::gfp reporter is abolished in the cell bodies and processes of the SMB neurons in adult but not L1 larval stage animals. GFP expression of the lim-4p::gfp reporter is shown in wild-type (left column) or lim-4(lsk5) mutant (right column) animals at L1 larval (top) or adult stage (bottom). Note that the lim-4p::gfp reporter is not expressed in the AWB neurons of lim-4 mutants [14]. Images are derived from z-stacks of confocal microscopy images. Quantitative analysis of these phenotypes is shown in Table 1. Anterior is to the left. Scale bar: 20 μm. (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. Heat shocks were treated to L4 larval stage animals at 33°C twice for 30 minutes and after 14 hours, phenotypes were analyzed. Over two independent transgenic lines were tested. n≥50 for each. (D) The average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates significantly different between indicated animals at p<0.001 (one-way ANOVA test followed by the Tukey post-hoc test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. (E-F) Shown are representative pictures of lim-4 mutants containing the hsp::lim-4cDNA transgene with no heat shock or after heat shock treatment. Images are derived from z-stacks of confocal microscopy images (E: Scale bar: 50 μm) and derived from a light microscopy image (F: Scale bar: 0.5 mm). (G) lim-4 is required to maintain function of the SMB neurons. The average wave width was analyzed in lim-4(ky403);Ex[hsp::lim-4cDNA] at 1 day after, 3 day after or 6 day after heat shock treatment. n≥30 for each. Error bars are the SEM. *** Significantly different between no heat shock and heat shock treatment conditions at p<0.001(one-way ANOVA test followed by the Tukey post-hoc test).
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pgen.1005480.g004: lim-4 is expressed in and acts in the SMB neurons and its expression is autoregulated.(A) GFP expression of oyIs35 animals carrying an integrated lim-4p::gfp reporter is overlapped with expression of the flp-12p::mCherry reporter (the SMB marker) but not with expression of the ceh-17p::dsRed reporter (the SIA marker). Images are derived from z-stacks of confocal microscopy images while images in the upper-left boxed regions are single focal plane confocal microscopy images. Anterior is to the left. Scale bar: 50 μm. (B) In lim-4 mutants, expression of the lim-4p::gfp reporter is abolished in the cell bodies and processes of the SMB neurons in adult but not L1 larval stage animals. GFP expression of the lim-4p::gfp reporter is shown in wild-type (left column) or lim-4(lsk5) mutant (right column) animals at L1 larval (top) or adult stage (bottom). Note that the lim-4p::gfp reporter is not expressed in the AWB neurons of lim-4 mutants [14]. Images are derived from z-stacks of confocal microscopy images. Quantitative analysis of these phenotypes is shown in Table 1. Anterior is to the left. Scale bar: 20 μm. (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. Heat shocks were treated to L4 larval stage animals at 33°C twice for 30 minutes and after 14 hours, phenotypes were analyzed. Over two independent transgenic lines were tested. n≥50 for each. (D) The average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates significantly different between indicated animals at p<0.001 (one-way ANOVA test followed by the Tukey post-hoc test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. (E-F) Shown are representative pictures of lim-4 mutants containing the hsp::lim-4cDNA transgene with no heat shock or after heat shock treatment. Images are derived from z-stacks of confocal microscopy images (E: Scale bar: 50 μm) and derived from a light microscopy image (F: Scale bar: 0.5 mm). (G) lim-4 is required to maintain function of the SMB neurons. The average wave width was analyzed in lim-4(ky403);Ex[hsp::lim-4cDNA] at 1 day after, 3 day after or 6 day after heat shock treatment. n≥30 for each. Error bars are the SEM. *** Significantly different between no heat shock and heat shock treatment conditions at p<0.001(one-way ANOVA test followed by the Tukey post-hoc test).

Mentions: lim-4 has previously been shown to be expressed in several neuronal types in the head of postembryonic animals; these neurons include the AWB, SIA, SAA, RID, RIV, and RMD neurons, but not the SMB neurons [14]. Like the SMB neurons, the SIA neurons project their processes into the sub-lateral nerve cords and their cell morphology and position are similar to those of the SMB neurons [11]. To determine whether lim-4 expression was mis-identified in the SIA neurons, we examined the expression pattern of lim-4p::gfp transgene (oyIs35) that includes 3.6 kb of upstream sequence [14, 26] and compared it to that of ceh-17p::dsRed, a SIA marker [21] or flp-12p::mCherry, a SMB marker [13], respectively (Fig 4A). We observed co-localization of lim-4 expression with that of flp-12 but not of ceh-17, indicating that lim-4 is expressed in the SMB neurons rather than the SIA neurons. In support of this re-assignment, the expression of other SMB markers such as odr-2p::gfp or trp-1p::gfp was completely abolished in the SMB neurons of lim-4 mutants, whereas expression of ceh-17 was not affected (Fig 2B; S4 Fig).


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)

lim-4 is expressed in and acts in the SMB neurons and its expression is autoregulated.(A) GFP expression of oyIs35 animals carrying an integrated lim-4p::gfp reporter is overlapped with expression of the flp-12p::mCherry reporter (the SMB marker) but not with expression of the ceh-17p::dsRed reporter (the SIA marker). Images are derived from z-stacks of confocal microscopy images while images in the upper-left boxed regions are single focal plane confocal microscopy images. Anterior is to the left. Scale bar: 50 μm. (B) In lim-4 mutants, expression of the lim-4p::gfp reporter is abolished in the cell bodies and processes of the SMB neurons in adult but not L1 larval stage animals. GFP expression of the lim-4p::gfp reporter is shown in wild-type (left column) or lim-4(lsk5) mutant (right column) animals at L1 larval (top) or adult stage (bottom). Note that the lim-4p::gfp reporter is not expressed in the AWB neurons of lim-4 mutants [14]. Images are derived from z-stacks of confocal microscopy images. Quantitative analysis of these phenotypes is shown in Table 1. Anterior is to the left. Scale bar: 20 μm. (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. Heat shocks were treated to L4 larval stage animals at 33°C twice for 30 minutes and after 14 hours, phenotypes were analyzed. Over two independent transgenic lines were tested. n≥50 for each. (D) The average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates significantly different between indicated animals at p<0.001 (one-way ANOVA test followed by the Tukey post-hoc test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. (E-F) Shown are representative pictures of lim-4 mutants containing the hsp::lim-4cDNA transgene with no heat shock or after heat shock treatment. Images are derived from z-stacks of confocal microscopy images (E: Scale bar: 50 μm) and derived from a light microscopy image (F: Scale bar: 0.5 mm). (G) lim-4 is required to maintain function of the SMB neurons. The average wave width was analyzed in lim-4(ky403);Ex[hsp::lim-4cDNA] at 1 day after, 3 day after or 6 day after heat shock treatment. n≥30 for each. Error bars are the SEM. *** Significantly different between no heat shock and heat shock treatment conditions at p<0.001(one-way ANOVA test followed by the Tukey post-hoc test).
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pgen.1005480.g004: lim-4 is expressed in and acts in the SMB neurons and its expression is autoregulated.(A) GFP expression of oyIs35 animals carrying an integrated lim-4p::gfp reporter is overlapped with expression of the flp-12p::mCherry reporter (the SMB marker) but not with expression of the ceh-17p::dsRed reporter (the SIA marker). Images are derived from z-stacks of confocal microscopy images while images in the upper-left boxed regions are single focal plane confocal microscopy images. Anterior is to the left. Scale bar: 50 μm. (B) In lim-4 mutants, expression of the lim-4p::gfp reporter is abolished in the cell bodies and processes of the SMB neurons in adult but not L1 larval stage animals. GFP expression of the lim-4p::gfp reporter is shown in wild-type (left column) or lim-4(lsk5) mutant (right column) animals at L1 larval (top) or adult stage (bottom). Note that the lim-4p::gfp reporter is not expressed in the AWB neurons of lim-4 mutants [14]. Images are derived from z-stacks of confocal microscopy images. Quantitative analysis of these phenotypes is shown in Table 1. Anterior is to the left. Scale bar: 20 μm. (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. Heat shocks were treated to L4 larval stage animals at 33°C twice for 30 minutes and after 14 hours, phenotypes were analyzed. Over two independent transgenic lines were tested. n≥50 for each. (D) The average of wave width or wavelength of the indicated genotypes. n≥30 for each. Error bars are the SEM. *** indicates significantly different between indicated animals at p<0.001 (one-way ANOVA test followed by the Tukey post-hoc test). Heat shocks were applied to L4 larval stage animals at 33°C twice for 30 minutes and phenotypes were analyzed after 14 hours. (E-F) Shown are representative pictures of lim-4 mutants containing the hsp::lim-4cDNA transgene with no heat shock or after heat shock treatment. Images are derived from z-stacks of confocal microscopy images (E: Scale bar: 50 μm) and derived from a light microscopy image (F: Scale bar: 0.5 mm). (G) lim-4 is required to maintain function of the SMB neurons. The average wave width was analyzed in lim-4(ky403);Ex[hsp::lim-4cDNA] at 1 day after, 3 day after or 6 day after heat shock treatment. n≥30 for each. Error bars are the SEM. *** Significantly different between no heat shock and heat shock treatment conditions at p<0.001(one-way ANOVA test followed by the Tukey post-hoc test).
Mentions: lim-4 has previously been shown to be expressed in several neuronal types in the head of postembryonic animals; these neurons include the AWB, SIA, SAA, RID, RIV, and RMD neurons, but not the SMB neurons [14]. Like the SMB neurons, the SIA neurons project their processes into the sub-lateral nerve cords and their cell morphology and position are similar to those of the SMB neurons [11]. To determine whether lim-4 expression was mis-identified in the SIA neurons, we examined the expression pattern of lim-4p::gfp transgene (oyIs35) that includes 3.6 kb of upstream sequence [14, 26] and compared it to that of ceh-17p::dsRed, a SIA marker [21] or flp-12p::mCherry, a SMB marker [13], respectively (Fig 4A). We observed co-localization of lim-4 expression with that of flp-12 but not of ceh-17, indicating that lim-4 is expressed in the SMB neurons rather than the SIA neurons. In support of this re-assignment, the expression of other SMB markers such as odr-2p::gfp or trp-1p::gfp was completely abolished in the SMB neurons of lim-4 mutants, whereas expression of ceh-17 was not affected (Fig 2B; S4 Fig).

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