TY - JOUR
T1 - Using single-cell RNA sequencing to generate predictive cell-type-specific split-GAL4 reagents throughout development
AU - Chen, Yu Chieh David
AU - Chen, Yen Chung
AU - Rajesh, Raghuvanshi
AU - Shoji, Nathalie
AU - Jacy, Maisha
AU - Lacin, Haluk
AU - Erclik, Ted
AU - Desplan, Claude
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Fengqiu Diao and Ben White for providing technical assistance on designing split-GAL4 triple donor constructs. We thank all the Desplan lab member for critical feedback on this work, especially Isabel Holguera, Bogdan Sieriebriennikov, Ryan Loker and Sromana Mukherjee. Stocks were obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537). This work was funded by grants from the NIH (R01EY013010 and R01EY13012 to C.D.). Y.-C.D.C. is supported by NIH NRSA (5F32EY032750). Y.-C.C. was supported by the MacCracken Program at New York University, by a NYSTEM institutional training grant (Contract #C322560GG), and by Scholarship to Study Abroad from the Ministry of Education, Taiwan. R.R. was supported by the MacCracken Program at New York University. All cartoon schematics of different cell types were created with BioRender.com.
Funding Information:
ACKNOWLEDGMENTS. We thank Fengqiu Diao and Ben White for providing technical assistance on designing split-GAL4 triple donor constructs. We thank all the Desplan lab member for critical feedback on this work, especially Isabel Holguera, Bogdan Sieriebriennikov, Ryan Loker and Sromana Mukherjee. Stocks were obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537). This work was funded by grants from the NIH (R01EY013010 and R01EY13012 to C.D.). Y.-C.D.C. is supported by NIH NRSA (5F32EY032750). Y.-C.C. was supported by the MacCracken Program at New York University, by a NYSTEM institutional training grant (Contract #C322560GG), and by Scholarship to Study Abroad from the Ministry of Education,Taiwan.R.R.was supported by the MacCracken Program at New York University. All cartoon schematics of different cell types were created with BioRender.com.
Publisher Copyright:
Copyright © 2023 the Author(s).
PY - 2023/8/8
Y1 - 2023/8/8
N2 - Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells and has been extremely useful in analyzing functional circuits in adults. However, these enhancer-based GAL4 lines often do not reflect the expression of nearby gene(s) as they only represent a small portion of the full gene regulatory elements. While genetic intersectional techniques such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resources to screen through combinations of enhancer expression patterns. Here, we use existing developmental single-cell RNA sequencing (scRNAseq) datasets to select gene pairs for split-GAL4 and provide a highly efficient and predictive pipeline (scMarco) to generate cell-type-specific split-GAL4 lines at any time during development, based on the native gene regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines or by CRISPR knock-in. We use the developing Drosophila visual system as a model to demonstrate the high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types, annotating clusters in scRNAseq datasets as well as in identifying novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to any other Drosophila tissue. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource for the Drosophila community.
AB - Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells and has been extremely useful in analyzing functional circuits in adults. However, these enhancer-based GAL4 lines often do not reflect the expression of nearby gene(s) as they only represent a small portion of the full gene regulatory elements. While genetic intersectional techniques such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resources to screen through combinations of enhancer expression patterns. Here, we use existing developmental single-cell RNA sequencing (scRNAseq) datasets to select gene pairs for split-GAL4 and provide a highly efficient and predictive pipeline (scMarco) to generate cell-type-specific split-GAL4 lines at any time during development, based on the native gene regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines or by CRISPR knock-in. We use the developing Drosophila visual system as a model to demonstrate the high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types, annotating clusters in scRNAseq datasets as well as in identifying novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to any other Drosophila tissue. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource for the Drosophila community.
KW - Drosophila visual system
KW - MiMIC/CRIMIC
KW - Single-cell RNA sequencing
KW - Split-GAL4
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U2 - 10.1073/pnas.2307451120
DO - 10.1073/pnas.2307451120
M3 - Article
C2 - 37523539
AN - SCOPUS:85167842957
SN - 0027-8424
VL - 120
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 32
M1 - e2307451120
ER -