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Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans.

Doitsidou M, Flames N, Lee AC, Boyanov A, Hobert O - Nat. Methods (2008)

Bottom Line: We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs.We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression.This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York 10032, USA. md2398@columbia.edu

ABSTRACT
We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

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Screening for dopaminergic cell fate mutants(a, b) Transgenic strains used for the worm sorter screen. Scale bar represents 100 μm. (c) Experimental procedure of the manual screen. (d) Experimental procedure of the COPAS worm sorter screen. (e)-(j) Relative fluorescence intensity plots between red and green channels of sorted worms. Scale is defined by the sorting parameters (see Supplementary Methods). Each dot corresponds to a single worm. (e) Profile of a non-mutagenized population of the sorting strain. (f) Example profile of a mutagenized population of worms. Red arrows indicate individual animals that fall into the manually-set sorting window. (g-j) Profiles of homogenous populations of retrieved mutants: (g) ot344 (0 out of 8 cells expressing gfp), (h) ot337 (2-4 out of 8 cells expressing gfp), (i) ot406 (4-6 out of 8 cells expressing gfp), and (j) ot346 (7-9 out of 8 cells expressing gfp). The triangle area at the bottom right of each panel is an example of a sorting region chosen to demonstrate the profile differences between various populations. The actual sorting region used in the screening process was usually larger, to minimize the possibility of loosing mutants.
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Figure 1: Screening for dopaminergic cell fate mutants(a, b) Transgenic strains used for the worm sorter screen. Scale bar represents 100 μm. (c) Experimental procedure of the manual screen. (d) Experimental procedure of the COPAS worm sorter screen. (e)-(j) Relative fluorescence intensity plots between red and green channels of sorted worms. Scale is defined by the sorting parameters (see Supplementary Methods). Each dot corresponds to a single worm. (e) Profile of a non-mutagenized population of the sorting strain. (f) Example profile of a mutagenized population of worms. Red arrows indicate individual animals that fall into the manually-set sorting window. (g-j) Profiles of homogenous populations of retrieved mutants: (g) ot344 (0 out of 8 cells expressing gfp), (h) ot337 (2-4 out of 8 cells expressing gfp), (i) ot406 (4-6 out of 8 cells expressing gfp), and (j) ot346 (7-9 out of 8 cells expressing gfp). The triangle area at the bottom right of each panel is an example of a sorting region chosen to demonstrate the profile differences between various populations. The actual sorting region used in the screening process was usually larger, to minimize the possibility of loosing mutants.

Mentions: We describe here the use of the COPAS Biosort machine in a forward genetic screen for mutants defective in executing the dopaminergic cell fate. Dopaminergic neurons fulfill important functions across phylogeny, yet the genetic pathways that control dopaminergic cell fate are poorly understood to date 5. A gfp reporter fusion to the promoter of the dopamine neuron-specific dopamine re-uptake transporter dat-1 exclusively labels all dopaminergic neurons in vtIs1 [dat-1∷gfp], a transgenic strain that expresses gfp exclusively in all 8 dopaminergic neurons of the worm 146 (Fig. 1a). As there are only eight dopaminergic neurons in the ~1000 cell-containing C.elegans hermaphrodite, the use of dat-1∷gfp provides a challenging test system for the sensitivity of the worm sorter. We performed test runs to determine whether these eight cells provide enough signal strength for the worm sorter. We first ran a sorting test in which the transgenic strain carrying dat-1∷gfp was mixed with non-transgenic, i.e. non-gfp expressing worms. Non-transgenic animals could indeed be sorted out efficiently (Supplementary Table 1). To control for the variability in fluorescent intensities among transgenic individuals, we introduced a chromosomally integrated, broadly expressed rfp transgene (vsIs33 [dop-3∷rfp])7 in the background of our screening strain as an internal reference of fluorescence intensity. This strain expresses rfp in some body-wall muscles and in various head neurons, ventral nerve cord neurons, tail neurons, and two mechanosensory neurons -the PVDs- but not in dopaminergic neurons (Fig. 1b). The sorter was set to compare fluorescence between green and red channels and plot their ratio. The sorting region was set to isolate individuals with reduced green to red ratio of fluorescence. This substantially increased the efficiency of sorting gfp negative animals (Supplementary Table 1).


Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans.

Doitsidou M, Flames N, Lee AC, Boyanov A, Hobert O - Nat. Methods (2008)

Screening for dopaminergic cell fate mutants(a, b) Transgenic strains used for the worm sorter screen. Scale bar represents 100 μm. (c) Experimental procedure of the manual screen. (d) Experimental procedure of the COPAS worm sorter screen. (e)-(j) Relative fluorescence intensity plots between red and green channels of sorted worms. Scale is defined by the sorting parameters (see Supplementary Methods). Each dot corresponds to a single worm. (e) Profile of a non-mutagenized population of the sorting strain. (f) Example profile of a mutagenized population of worms. Red arrows indicate individual animals that fall into the manually-set sorting window. (g-j) Profiles of homogenous populations of retrieved mutants: (g) ot344 (0 out of 8 cells expressing gfp), (h) ot337 (2-4 out of 8 cells expressing gfp), (i) ot406 (4-6 out of 8 cells expressing gfp), and (j) ot346 (7-9 out of 8 cells expressing gfp). The triangle area at the bottom right of each panel is an example of a sorting region chosen to demonstrate the profile differences between various populations. The actual sorting region used in the screening process was usually larger, to minimize the possibility of loosing mutants.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2693092&req=5

Figure 1: Screening for dopaminergic cell fate mutants(a, b) Transgenic strains used for the worm sorter screen. Scale bar represents 100 μm. (c) Experimental procedure of the manual screen. (d) Experimental procedure of the COPAS worm sorter screen. (e)-(j) Relative fluorescence intensity plots between red and green channels of sorted worms. Scale is defined by the sorting parameters (see Supplementary Methods). Each dot corresponds to a single worm. (e) Profile of a non-mutagenized population of the sorting strain. (f) Example profile of a mutagenized population of worms. Red arrows indicate individual animals that fall into the manually-set sorting window. (g-j) Profiles of homogenous populations of retrieved mutants: (g) ot344 (0 out of 8 cells expressing gfp), (h) ot337 (2-4 out of 8 cells expressing gfp), (i) ot406 (4-6 out of 8 cells expressing gfp), and (j) ot346 (7-9 out of 8 cells expressing gfp). The triangle area at the bottom right of each panel is an example of a sorting region chosen to demonstrate the profile differences between various populations. The actual sorting region used in the screening process was usually larger, to minimize the possibility of loosing mutants.
Mentions: We describe here the use of the COPAS Biosort machine in a forward genetic screen for mutants defective in executing the dopaminergic cell fate. Dopaminergic neurons fulfill important functions across phylogeny, yet the genetic pathways that control dopaminergic cell fate are poorly understood to date 5. A gfp reporter fusion to the promoter of the dopamine neuron-specific dopamine re-uptake transporter dat-1 exclusively labels all dopaminergic neurons in vtIs1 [dat-1∷gfp], a transgenic strain that expresses gfp exclusively in all 8 dopaminergic neurons of the worm 146 (Fig. 1a). As there are only eight dopaminergic neurons in the ~1000 cell-containing C.elegans hermaphrodite, the use of dat-1∷gfp provides a challenging test system for the sensitivity of the worm sorter. We performed test runs to determine whether these eight cells provide enough signal strength for the worm sorter. We first ran a sorting test in which the transgenic strain carrying dat-1∷gfp was mixed with non-transgenic, i.e. non-gfp expressing worms. Non-transgenic animals could indeed be sorted out efficiently (Supplementary Table 1). To control for the variability in fluorescent intensities among transgenic individuals, we introduced a chromosomally integrated, broadly expressed rfp transgene (vsIs33 [dop-3∷rfp])7 in the background of our screening strain as an internal reference of fluorescence intensity. This strain expresses rfp in some body-wall muscles and in various head neurons, ventral nerve cord neurons, tail neurons, and two mechanosensory neurons -the PVDs- but not in dopaminergic neurons (Fig. 1b). The sorter was set to compare fluorescence between green and red channels and plot their ratio. The sorting region was set to isolate individuals with reduced green to red ratio of fluorescence. This substantially increased the efficiency of sorting gfp negative animals (Supplementary Table 1).

Bottom Line: We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs.We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression.This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York 10032, USA. md2398@columbia.edu

ABSTRACT
We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

Show MeSH
Related in: MedlinePlus