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

A cis-regulatory motif is necessary and sufficient to drive expression of terminally differentiated SMB markers.(A) Shown are the predicted binding site (top) for the homeodomain of LIM-4 from a web based tool, PreMoTF (http://stormo.wustl.edu/PreMoTF) [33] and the putative cis-regulatory SMB motif (bottom) identified from promoter analysis of flp-12, odr-2, and unc-17 genes. (B) The percentage of transgenic animals expressing flp-12p:: gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined except a flp-12 promoter (-2652) construct of which number was derived from one integrated line. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below. Analysis between -523 bp and -339 bp upstream of flp-12 promoter is shown in S10 Fig. (C) Insertion of the SMB motif into the non-SMB expressed flp-7 gene promoter induces flp-7 expression in the SMB neurons. The inserted cis-regulatory sequences identified from the flp-12 promoter analysis and the inserted site in the flp-7 promoter are indicated. GFP expression is overlapped with expression of the lim-4pΔ3::mCherry reporter in the SMB neurons of wild-type animals. 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 bars: 50 μm. (D) The percentage of transgenic animals expressing odr-2p::gfp or unc-17p::gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549117&req=5

pgen.1005480.g006: A cis-regulatory motif is necessary and sufficient to drive expression of terminally differentiated SMB markers.(A) Shown are the predicted binding site (top) for the homeodomain of LIM-4 from a web based tool, PreMoTF (http://stormo.wustl.edu/PreMoTF) [33] and the putative cis-regulatory SMB motif (bottom) identified from promoter analysis of flp-12, odr-2, and unc-17 genes. (B) The percentage of transgenic animals expressing flp-12p:: gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined except a flp-12 promoter (-2652) construct of which number was derived from one integrated line. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below. Analysis between -523 bp and -339 bp upstream of flp-12 promoter is shown in S10 Fig. (C) Insertion of the SMB motif into the non-SMB expressed flp-7 gene promoter induces flp-7 expression in the SMB neurons. The inserted cis-regulatory sequences identified from the flp-12 promoter analysis and the inserted site in the flp-7 promoter are indicated. GFP expression is overlapped with expression of the lim-4pΔ3::mCherry reporter in the SMB neurons of wild-type animals. 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 bars: 50 μm. (D) The percentage of transgenic animals expressing odr-2p::gfp or unc-17p::gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below.

Mentions: Homeodomain transcription factors generally bind well-defined DNA sequences to control transcription of target genes [31]. Systemic analysis of homeodomain DNA-binding specificities allowed prediction of the recognition motif of each homeodomain protein, and a cis-regulatory motif containing the consensus TAAT core DNA sequences was predicted to be the binding site for the homeodomain of LIM-4 and its mammalian and Drosophila homologs (LHX6/8 and Arrowhead, respectively) (Fig 6A; S9 Fig) [32, 33]. Indeed, LHX6 and LHX8 have been shown to directly bind to the predicted DNA sequences (ATAATCA) in the promoter regions of the Shh gene [34].


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)

A cis-regulatory motif is necessary and sufficient to drive expression of terminally differentiated SMB markers.(A) Shown are the predicted binding site (top) for the homeodomain of LIM-4 from a web based tool, PreMoTF (http://stormo.wustl.edu/PreMoTF) [33] and the putative cis-regulatory SMB motif (bottom) identified from promoter analysis of flp-12, odr-2, and unc-17 genes. (B) The percentage of transgenic animals expressing flp-12p:: gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined except a flp-12 promoter (-2652) construct of which number was derived from one integrated line. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below. Analysis between -523 bp and -339 bp upstream of flp-12 promoter is shown in S10 Fig. (C) Insertion of the SMB motif into the non-SMB expressed flp-7 gene promoter induces flp-7 expression in the SMB neurons. The inserted cis-regulatory sequences identified from the flp-12 promoter analysis and the inserted site in the flp-7 promoter are indicated. GFP expression is overlapped with expression of the lim-4pΔ3::mCherry reporter in the SMB neurons of wild-type animals. 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 bars: 50 μm. (D) The percentage of transgenic animals expressing odr-2p::gfp or unc-17p::gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549117&req=5

pgen.1005480.g006: A cis-regulatory motif is necessary and sufficient to drive expression of terminally differentiated SMB markers.(A) Shown are the predicted binding site (top) for the homeodomain of LIM-4 from a web based tool, PreMoTF (http://stormo.wustl.edu/PreMoTF) [33] and the putative cis-regulatory SMB motif (bottom) identified from promoter analysis of flp-12, odr-2, and unc-17 genes. (B) The percentage of transgenic animals expressing flp-12p:: gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined except a flp-12 promoter (-2652) construct of which number was derived from one integrated line. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below. Analysis between -523 bp and -339 bp upstream of flp-12 promoter is shown in S10 Fig. (C) Insertion of the SMB motif into the non-SMB expressed flp-7 gene promoter induces flp-7 expression in the SMB neurons. The inserted cis-regulatory sequences identified from the flp-12 promoter analysis and the inserted site in the flp-7 promoter are indicated. GFP expression is overlapped with expression of the lim-4pΔ3::mCherry reporter in the SMB neurons of wild-type animals. 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 bars: 50 μm. (D) The percentage of transgenic animals expressing odr-2p::gfp or unc-17p::gfp reporter construct in the indicated neurons is shown. Strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. At least two independent extrachromosomal lines for each construct were examined. n≥50 for each. Identified cis-regulatory sequences found in other Caenorhabditis species are shown below.
Mentions: Homeodomain transcription factors generally bind well-defined DNA sequences to control transcription of target genes [31]. Systemic analysis of homeodomain DNA-binding specificities allowed prediction of the recognition motif of each homeodomain protein, and a cis-regulatory motif containing the consensus TAAT core DNA sequences was predicted to be the binding site for the homeodomain of LIM-4 and its mammalian and Drosophila homologs (LHX6/8 and Arrowhead, respectively) (Fig 6A; S9 Fig) [32, 33]. Indeed, LHX6 and LHX8 have been shown to directly bind to the predicted DNA sequences (ATAATCA) in the promoter regions of the Shh gene [34].

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