Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for Drosophila research

Abstract

Expression of activated Ras, RasV12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here we used lineage restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type specific genes and the expected alignment with single cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.

Full text

Coleman- Gosser, Hu, Raghuvanshi etal. eLife 2023;12:e85814. DOI: https://doi.org/10.7554/eLife.85814 1 of 27
Continuous muscle, glial, epithelial,
neuronal, and hemocyte cell lines for
Drosophilaresearch
Nikki Coleman- Gosser1†, Yanhui Hu2†, Shiva Raghuvanshi1†, Shane Stitzinger1†,
Weihang Chen2, Arthur Luhur3, Daniel Mariyappa3, Molly Josifov1,
Andrew Zelhof3, Stephanie E Mohr2, Norbert Perrimon2,4*, Amanda Simcox1,5*
1Department of Molecular Genetics, Ohio State University, Columbus, United
States; 2Drosophila RNAi Screening Center and Department of Genetics, Harvard
Medical School, Boston, United States; 3Drosophila Genomics Resource Center and
Department of Biology, Indiana University, Bloomington, United States; 4Howard
Hughes Medical Institute, Chevy Chase, United States; 5National Science Foundation,
Alexandria, United States
Abstract Expression of activated Ras, RasV12, provides Drosophila cultured cells with a prolifer-
ation and survival advantage that simplifies the generation of continuous cell lines. Here, we used
lineage- restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithe-
lial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by
cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone
ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced
dendritic features in neuronal- like lines. Transcriptome analysis showed expression of key cell- type-
specific genes and the expected alignment with single- cell sequencing and in situ data. Overall, the
technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve,
and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource
Center.
Editor's evaluation
This valuable work describes the establishment and characterization of new cell lines derived from
specific tissues of the fruit fly Drosophila. The evidence supporting the claims of the authors is
convincing, with rigorous characterization of the cell lines and incorporation of their transcriptomes
into Drosophila Gene Expression Tool website for user- friendly access. These lines will be a valuable
resource that complements in vivo Drosophila genetics, improving biochemistry and facilitating high-
throughput screening.
Introduction
The use of cell cultures has been important for studying biological processes that are not easily acces-
sible in whole organisms (Klein et al., 2022). A number of advances in mammalian cell cultures,
for instance, development of 3D/organoid cultures (Rossi etal., 2018), improved genome editing
tools to manipulate induced pluripotent stem cells (Hockemeyer and Jaenisch, 2016), and better
optimized media formulations for recombinant protein expression Ritacco etal., 2018 have further
enhanced the utility of mammalian cell culture systems. These advances are accompanied by the avail-
ability of several distinct mammalian cell lines derived from different tissue types. Similarly, the use of
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*For correspondence:
perrimon@genetics.med.
harvard.edu (NP);
simcox.1@osu.edu (AS)
†These authors contributed
equally to this work
Competing interest: The authors
declare that no competing
interests exist.
Funding: See page 20
Received: 28 December 2022
Preprinted: 19 January 2023
Accepted: 12 July 2023
Published: 20 July 2023
Reviewing Editor: Erika A Bach,
New York University School of
Medicine, United States
This is an open- access article,
free of all copyright, and may be
freely reproduced, distributed,
transmitted, modified, built
upon, or otherwise used by
anyone for any lawful purpose.
The work is made available under
the Creative Commons CC0
public domain dedication.

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insect cell lines also complements whole organismal studies and helped to illuminate many aspects
of insect cell biology (Luhur etal., 2019) including development (Sato and Siomi, 2020), immunity
(Goodman etal., 2021; Chen etal., 2021), host–pathogen relationships (Smagghe etal., 2009), in
addition to biotechnological applications (Hong etal., 2022).
Fruit fly (Drosophila melanogaster) cell cultures are among the most widely used invertebrate cell
cultures (Luhur etal., 2019). Drosophila cell lines are relatively homogenous, and highly scalable for
both biochemical and high- throughput functional genomic analyses (Debec etal., 2016, Baum and
Cherbas, 2008; Zirin et al., 2022; Mohr, 2014; Viswanatha etal., 2019). These features underlie
their status as an important workhorse for scientific discovery in organismal development and as
models for human disease. There are approximately 250 distinct Drosophila cell lines housed by the
Drosophila Genomics Resource Center (DGRC) (Luhur etal., 2019). The majority of these cell lines,
initially established by independent laboratories worldwide, were donated to the DGRC. A subset
of 25 of these lines was subjected to transcriptome analysis, with the results demonstrating that
approximately half of the transcripts expressed by each of these lines were unique such that even
cell lines derived from the same tissue had distinct transcriptomic profiles (Cherbas etal., 2011).
Furthermore, the transcriptional profiles of several imaginal disc lines analyzed were found to match
profiles of cells from distinct spatial locations in the respective discs (Cherbas etal., 2011). All lines
exhibited transcript profiles indicative of cell growth and cell division, and not cellular differentiation,
as expected for proliferating cells (Cherbas etal., 2011). Thus, the transcriptional profiles of several
Drosophila cell lines provided a platform for subsequent analyses. For instance, a few examples of the
impact of this work include research into better understanding crosstalk between signaling pathways
(Ammeux etal., 2016), exploring transcription factor networks (Rhee etal., 2014), establishing small
RNA diversity (Wen etal., 2014), characterizing signaling pathways (Neal etal., 2019), nucleosomal
organization (Martin etal., 2017) among multiple other utilities reviewed extensively (Cherbas and
Gong, 2014; Luhur etal., 2019).
Over two- thirds of the D. melanogaster cell lines listed in the DGRC catalog were derived from
whole embryos and the remainder are from various larval imaginal discs, the larval central nervous
system, larval hemocytes, or adult ovaries. The potential of cells from these different sources to
eLife digest Fruit flies are widely used in the life and biomedical sciences as models of animal
biology. They are small in size and easy to care for in a laboratory, making them ideal for studying how
the body works. There are, however, some experiments that are difficult to perform on whole flies
and it would be advantageous to use populations of fruit fly cells grown in the laboratory – known as
cell cultures – instead.
Unlike studies in humans and other mammals, which – for ethical and practical reasons –heavily
rely on cell cultures, few studies have used fruit fly cell cultures. Recent work has shown that having
an always active version of a gene called Ras in fruit fly cells helps the cells to survive and grow in
cultures, making it simpler to generate new fruit fly cell lines compared with traditional methods.
However, the methods used to express activated Ras result in cell lines that can be a mixture of many
different types of cell, which limits how useful they are for research.
Here, Coleman- Gosser, Hu, Raghuvanshi, Stitzinger et al. aimed to use Ras to generate a collection
of cell lines from specific types of fruit fly cells in the muscle, nervous system, blood and other parts
of the body. The experiments show that selectively expressing activated Ras in an individual type of
cell enables them to outcompete other cells in culture to generate a cell line consisting only of the
cell type of interest.
The new cell lines offer models for experiments that more closely reflect their counterparts in flies.
For example, the team were able to recapitulate how fly muscles develop by treating one of the cell
lines with a hormone called ecdysone, which triggered the cells to mature into active muscle cells that
spontaneously contract and relax.
In the future, the new cell lines could be used for various experiments including high throughput
genetic screening or testing the effects of new drugs and other compounds. The method used in this
work may also be used by other researchers to generate more fruit fly cell lines.

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differentiate into adult cell types is not known. However, temporal transcriptional profiling of the
Ecdysone response of 41 cell lines (Stoiber etal., 2016) provided evidence that cell lines exhibited
varying levels of ecdysone sensitivity and potential for cellular differentiation, suggesting the possi-
bility of developing cell- type- specific cell lines with the capacity to differentiate.
As well as having unknown cellular origins, most Drosophila cell lines arose spontaneously, and
the time needed to develop a continuous cell line was often protracted. In contrast, expression of
activated Ras, RasV12, using the Gal4- UAS system, resulted in the rapid and reproducible generation
of continuous cell lines from primary embryonic cultures (Simcox etal., 2008b). The Ras method was
used to develop an array of mutant cell lines by using appropriate genotypes to establish the primary
cultures (Simcox etal., 2008a, Lee etal., 2015; Kahn etal., 2014; Lim etal., 2016; Nakato etal.,
2019). To date all lines have been generated using ubiquitous expression of UAS- Ras with Act5C- Gal4
and therefore the cell type in a given line is unknown.
Here, we describe a second- generation version of the Ras method in which RasV12 expression is
restricted to a lineage by using tissue- specific Gal4 drivers. This genetic ‘dissection’ provides only the
targeted cells with the survival and proliferation advantage conferred by RasV12 expression (Simcox
etal., 2008b). As we show, the approach has been successful and resulted in the generation of cell
lines with glial, epithelial, and muscle characteristics. Lines generated by broad RasV12 expression
should also include those of specific cell types and by using single- cell cloning and cell type char-
acterization (marker gene expression and RNAseq) we identified lines with neuronal and hemocyte
characteristics. Collectively, these cell lines provide in vitro models for five different cell types and are
expected to be a valuable resource for high- throughput and biochemical approaches, which rely on
large numbers of homogeneous cells.
Results
Primary cultures were established from embryos in which UAS- RasV12 expression was restricted to glial,
tracheal epithelial, and mesodermal cells using lineage- specific Gal4 drivers (Table1, Supplementary
file 1). A subset of continuous cell lines derived from each type of primary culture was analyzed with
regard to cell morphology, the presence of proteins characteristic of specific cell types, and other
attributes (Table1, Supplementary file 1, Supplementary file 2; Figure1). We also analyzed lines
with neuronal- or hemocyte- like characteristics that were cloned from parental lines resulting from
ubiquitous expression of UAS- RasV12 (Table1, Supplementary file 1, Supplementary file 2; Figure1).
Table 1. Cell lines analyzed.
Tissue- type
alignment Genotype Lines analyzed* DGRC stock name and number RRID
Glial Repo- Gal4; RasV12; bratdsRNA Rbr6 (parental)
Rbr6- 2
Rbr6- 4
Rbr6- F9
repo>Ras bratdsRNA- L6, 282
repo>Ras bratdsRNA- L6- Clone2, 326
repo>Ras bratdsRNA- L6- Clone4, 327
repo>Ras bratdsRNA- L6- CloneF9, 328
RRID:CVCL_XF57
RRID:CVCL_C7G9
RRID:CVCL_C7GA
RRID:CVCL_C7GB
Epithelial btl- Gal4; UAS- P35; UAS- RasV12 Btl3 (parental) btl>Ras attP- L3, 332 RRID:CVCL_B3N7
btl- Gal4; UAS- P35; attP, UAS- RasV12 Btl7 (parental)
Btl8 (parental)
btl>Ras attP- L7, 285
btl>Ras attP- L8, 286
RRID:CVCL_XF53
RRID:CVCL_XF54
Muscle 24B- Gal4; attP, UAS- RasV12 24B5 (parental)
24B5- B8
24B5- D8
24B>Ras attP- L5, 284
24B>Ras attP- L5- CloneB8, 323
RRID:CVCL_XF52
RRID:CVCL_C7G6
24B- Gal4; UAS- GFP; attP, UAS- RasV12 24BG1 (parental)
24BG1- F3†
24BG1- G1†
24B>Ras attP GFP- L1, 283
24B>Ras attP- G1- CloneF3, 325
24B>Ras attP- G1- CloneG1, 324
RRID:CVCL_XF51
RRID:CVCL_C7G8
RRID:CVCL_C7G7
Neuronal Act5C- GeneSwitch- Gal4; UAS- GFP;
attP, UAS- RasV12
ActGSB- 6‡
ActGSI- 2
Act5C- GS>Ras attP- LB- Clone6, 329
Act5C- GS>Ras attP- GFP- LI- Clone2, 330
RRID:CVCL_C7GC
RRID:CVCL_C7GD
Blood Act5C- GeneSwitch- Gal4; UAS- GFP;
attP, UAS- RasV12
ActGSI- 3 Act5C- GS>Ras attP- GFP- LI- Clone3, 331 RRID:CVCL_C7GE
*Clones unless indicated.
†Do not differentiate into active muscle.
‡These cells do not express GFP, the reason for this is not known.

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Figure 1. Morphology of cells. (A–C) Glial- lineage clones. The cells have an elongated morphology with variable lengths from approximately 20 to
>50µm (red arrowheads). (D–F) Tracheal- lineage cells. Btl3 and Btl7 cells form squamous epithelial sheets. Btl8 are closely associated but do not abut
each other to form a sheet. (G–J) Mesodermal- lineage cells. The cells have a bipolar morphology. Multinucleate cells are frequently found in 24BGI- F3
and 24BG1- GI clones (red arrowheads). (K, L) Neuronal- like clones. ActGSB- 6 cells are mainly bipolar; however, some have asymmetric processes or thin
Figure 1 continued on next page

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We further analyzed the cell lines by RNAseq to determine the transcriptome and signaling path-
ways (Figure2 and Figure2—figure supplements 1–3). The gene expression values (Fragments Per
Kilobase per Million mapped fragments, FKPM) are provided in Supplementary file 3. The dataset
(Ras cell lines) has been imported into the Drosophila Gene Expression Tool (DGET) database (https://
www.flyrnai.org/tools/dget/web/), which is the bulk RNAseq data portal at Drosophila RNAi Screening
Center (DRSC) (Hu etal., 2017). The TM4 package was used for making the plot in Figure2 (Wang
etal., 2017). As expected, the transcriptomes of the new cell lines are distinct from those of existing
cell lines (Cherbas etal., 2011; Figure2—figure supplement 1) and new cell lines derived from the
same Gal4 driver cluster with one another (Figure2—figure supplement 2). Moreover, comparison
of differentially expressed (DE) genes with RNAseq data from single- cell RNAseq data (Li etal., 2022;
Table2) or with known cell type- associated transcription factors (Figure2—figure supplement 3)
reveals that these cells express genes characteristic of specific cell types. The results of our detailed
characterization are described according to cell type in the sections below.
Glial-lineage cell lines
Repo is expressed exclusively in glial cells (Xiong etal., 1994). A repo- Gal4 driver that recapitulates
Repo expression was used to express UAS- RasV12 (Ogienko etal., 2020; Sepp et al., 2001). This
led to robust production of primary cultures however these failed to survive beyond early passages
(Supplementary file 1). To counter potential cell death or modulate growth signaling, additional
genotypes were tested including co- expression of UAS- transgenes encoding the P35 baculovirus cell
survival factor, dsRNAs targeting tumor suppressors, or the Gal4 inhibitor Gal80ts (Supplementary file
1). Co- expression of a UAS- bratdsRNA or expression of tub- Gal80ts each produced a single line of cells
that could be propagated for extended passages however the latter line was difficult to maintain and
eventually lost (Supplementary file 1). The repo- Gal4: UAS- bratdsRNA; UAS- RasV12 (Rbr6) line has been
passaged more than 50 times. The parental Rbr6 line and three clonal derivatives (Rbr6- 2, Rbr6- 4,
and Rbr6- F9) have an elongated morphology and stained positive for Repo (Table1; Figures1 and
3, and Figure3—figure supplement 1). A few cells expressed neuronal markers (Figure3—figure
supplement 1; Supplementary file 2). To induce differentiation, we gave cells two 24hr ecdysone
treatments separated by 24hr to approximate the pulses of ecdysone during the larval to pupal tran-
sition. Cells from each of the clones survived treatment with ecdysone suggesting they are of adult
type, two clones showed morphological changes and formed a network, and all continued to express
Repo (Figure3 and Figure3—figure supplement 2).
The results of RNAseq analysis revealed that the three Rbr6 clones have very similar expression
patterns (Figure 2—figure supplement 2). In addition, their DE gene signatures are also a close
match to gene signatures of glial cells as identified by single- cell RNAseq (Table 2) and to glial-
associated genes reported in the literature. For example, zydeco (zyd), which encodes a potassium-
dependent sodium/calcium exchanger, is upregulated in all three clones, consistent with the literature
(Zwarts etal., 2015; Featherstone, 2011), and gcm2, a transcription factor, is upregulated in two
clones (Figure2—figure supplement 3). These data suggest the Rbr6 clones will be a useful in vitro
source of glial cells.
Tracheal epithelium-lineage cell lines
Breathless is expressed in the tracheal epithelium and a btl- Gal4 driver was used to express UAS-
RasV12 (Shiga etal., 1996). Patches of cells with epithelial morphology proliferated in primary cultures
and several continuous lines were generated (Table1, Supplementary file 1). We were unable to
derive clones of these using dilution or selection methods, which were successful for other cell types.
Correspondingly, three parental lines were examined: Btl3, Btl7, and Btl8 (Table 1). All showed
expression of the epithelial marker Shotgun/E- Cadherin (Shg/Ecad) and two grew in a squamous
epithelial sheet with Ecad expression at the cell periphery (Figures1 and 4, and Figure4—figure
supplement 1). In comparison S2 did not show peripheral expression of Ecad (Figure4). Treatment of
processes (red arrowheads). ActGSI- 2 are bipolar. (M) Hemocyte- like clone ActGSI- 3. The cells form oating clusters that increase in cell number as they
proliferate. Individual cells have a round morphology. (N) Schneider’s S2 cells. The cells are thought to be of hemocyte type and grow as single round
cells in suspension. Scale bar = 10µm.
Figure 1 continued

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the squamous epithelial cells (Btl3 and Btl7) with
ecdysone caused aggregation and formation of
large multicellular clusters (Figure4, Figure4—
figure supplement 2).
RNAseq data analysis comparing the top
upregulated genes in the Btl cell lines with
scRNAseq datasets revealed that the lines closely
match the signatures of the adult trachea, a
network of epithelial tubules (Table 2) and Btl3
expresses trachealess (trh) a master regulator of
tracheal identity (Wilk et al., 1996; Figure 2—
figure supplement 3). Overall, the morpholog-
ical and molecular characteristics of the lines are
consistent with an epithelial cell type of tracheal
origin.
Mesodermal-lineage cell lines
The 24B- Gal4 driver is an insertion in held out
wings (how) and is expressed in mesoderm and
muscle cells (Brand and Perrimon, 1993; Zaffran
etal., 1997). Expression of UAS- RasV12 with 24B-
Gal4 readily produced continuous lines (Table1,
Supplementary file 1). Four clones (24B5- B8,
24B5- D8, 24BG1- F3, and 24BG1- G1) derived
from two parental lines (24B5 and 25BG1) were
analyzed in more detail (Table1). The cells had a
bipolar shape and expressed mesoderm markers
including Twist and Mef2 (Figures1 and 5, and
Figure5—figure supplement 1). When treated
with ecdysone, cells from both parental lines and
clones 24B5- B8 and 24B5- D8 elongated, fused
as indicated by multinucleate cells, formed a
network, and expressed Myosin heavy chain (Mhc)
(Figure5 and Figure5—figure supplement 2).
There was also extensive cell lysis. Beginning 2
days after the second ecdysone treatment, the
cells began to contract spontaneously. Contrac-
tion of cells from the 24B5 parental line and the
two derivative clones (24B5- B8 and 24B5- D8)
was visible in real time (Videos1 and 2), whereas
contraction of parental line 24BG1 cells was much
slower and visualized more clearly in time- lapse
(Videos 3 and 4). The clones 24BG1- F3 and
24BG1- G1 underwent morphological change
but did not express Mhc or contract (Figure5—
figure supplements 2 and 3). In later passages,
Figure 2. Expression levels of ligands and receptors
for major signaling pathways. The ligand and receptor
annotation for major signaling pathways was obtained
from FlyPhoneDB (https://www.yrnai.org/tools/
y_phone/web/). The expression levels of ligands
and receptors are represented as a heatmap of FPKM
values.
The online version of this article includes the following
gure supplement(s) for gure 2:
Figure supplement 1. Comparison of lineage-
restricted Ras cell lines with previously isolated
Drosophila cell lines.
Figure 2 continued on next page
Figure supplement 2. Principal component analysis
(PCA) of RNAseq data from the lineage- restricted Ras
cell lines.
Figure supplement 3. Relative expression of
transcription factors associated in the literature with
specic tissue lineages in the lineage- restricted Ras cell
lines.
Figure 2 continued

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the 24BG1 parental line also lost expression of Mhc and the ability to contract (Figure5—figure
supplement 2). This highlights the importance of using early passage cells and avoiding extended
passaging that could alter the phenotypic (and genotypic) characteristics of the cells.
We also attempted to derive lines from Mef2- Gal4 because Mef2 regulates muscle development
and is expressed in muscle progenitors and differentiated muscle suggesting Mef2- Gal4 would
be a good candidate for deriving cell lines (Bour et al., 1995; Gossett et al., 1989; Lilly etal.,
1995; Ranganayakulu etal., 1995). However, only rare primary cultures had some proliferating cell
patches, and none progressed to continuous lines (Supplementary file 1; Figure5—figure supple-
ment 4). Analysis of larvae from the cross (Mef2- Gal4/+; UAS- GFP/UAS- RasV12) and control larvae
(Mef2- Gal4/+; UAS- GFP/+) showed that RasV12 expression disrupted muscle development, suggesting
that the prevalent amorphous GFP- positive cells observed in primary cultures were abnormal muscle
cells (Figure5—figure supplement 4).
The RNAseq analysis for 24B- Gal4- derived cell lines, identified the cells as muscle (Table 2).
24B5- B8 cells express high levels of the transcription factors nautilus (nau) and twist (twi) (Figure2—
figure supplement 3; Figure5—figure supplement 1; Table2), and high levels of myoblast city
(mbo), which encodes an unconventional bipartite GEF with a role in myoblast fusion (Erickson etal.,
1997). The capacity of these mesoderm- derived cell lines to differentiate into active muscle shows
that the cells are muscle precursors and thus should be a useful reagent to analyze muscle physiology
and development.
Neuronal-like cell lines
To target neuronal cells, we expressed UAS- RasV12 with the pan- neural drivers scratch- Gal4 and
elav- Gal4, however none of the primary cultures resulted in continuous cell lines (Supplementary file
1; Figure6—figure supplement 1). In previous work, we made primary cultures from embryos with
ubiquitous expression of UAS- RasV12 using the Act5C- Gal4 driver (Simcox etal., 2008b). The cells
growing in these cultures included neuronal cells (Simcox etal., 2008b). Here, we used an Act5C-
GeneSwitch- Gal4 driver to express UAS- RasV12. GeneSwitch- Gal4 is only active in the presence of the
drug, RU486/mifepristone, which provides the advantage of being able to regulate RasV12 expression
Table 2. RNAseq data analysis.
Tissue
type Cell line Cell cluster scRNAseq
Enrichment
p value
scRNAseq
scRNAseq
dataset
Cell type
based on in
situ data
Enrichment
p value in
situ
Glial Rbr6- 2
Adult reticular neuropil-
associated glial cell 8.13E−05 Whole body Glia 4.84E−05
Rbr6- 4 Cell body glial cell 7.56E−04 Whole body
Rbr6- F9 Adult glial cell 8.13E−05 Whole body Glia 3.42E−02
Epithelial Btl3 Adult tracheal cell 2.61E−06 Whole body Tracheal 1.08E−01
Btl7 Adult tracheal cell 8.81E−04 Oenocyte
Btl8 Adult tracheal cell 2.72E−02 Body Tracheal 2.05E−02
Muscle 24B5- B8 Muscle cell 2.93E−6
Male reprod
glands
24BG1- F3 Muscle cell 1.66E−04 Antenna
24BG1- G1 Muscle 8.83E−02
Neuronal ActGSI- 2
leg muscle motor neuron
system 5.79E−03 Whole body Neuron 6.68E−02
ActGSB- 6 adult ventral nervous 7.56E−04 Whole body Neuron 5.71E−02
Blood ActGSI- 3 hemocyte 1.00E−25 Whole body
Circulatory
system 1.29E−01
Analysis using the Drosophila RNAi Screening Center’s single- cell DataBase (DRscDB), all datasets used are from
FCA 10x Sequencing (https://ycellatlas.org/). The in situ data were from the BDGP (https://insitu.fruity.org/cgi-
bin/ex/insitu.pl) and the enrichment p value was calculated by a hypergeometric test.

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(Nicholson etal., 2008; Osterwalder etal., 2001). Several continuous lines were generated (Supple-
mentary file 1). Clones derived from two of these (ActGSB- 6 and ActGSI- 2) (Table1) were positive
for the neuronal marker, HRP (horseradish peroxidase) (Figure6, Figure6—figure supplements 2
and 3). After differentiation with ecdysone, expression of Futsch/MAPB1 (Hummel etal., 2000) and
Fas2 (Mao and Freeman, 2009) was enhanced and revealed axonal- like outgrowths from the cells
(Figure6 and Figure6—figure supplement 3). Differentiated cells also showed enhanced expression
of Elav, which is commonly used as a marker for postmitotic neurons (Figure6 and Figure6—figure
supplement 3; Robinow and White, 1991). Elav is also expressed transiently in glial cells and prolif-
erating neuroblasts Berger etal., 2007; however, the cells were negative for the glial marker Repo
(Supplementary file 2).
RNAseq analysis revealed that many neuronal genes are upregulated in these cell lines, including
Glutamic acid decarboxylase 1 (Gad1), slowpoke (slo), 5- hydroxytryptamine (serotonin) receptor
1A (5- HT1A), Protein C kinase 53E (Pkc53E), Diuretic hormone 31 Receptor (Dh31- R), and straight-
jacket (stj). In addition, comparison of the top upregulated genes in these cells to marker genes from
scRNAseq data identifies a cell type of neuronal origin as the best match (Table2). The cells should
be a useful source of neuronal cells.
Hemocyte-like cell line
Cells of clone ActGSI- 3 derived from the ActGSI parental line (UAS- RasV12 expression with Act5C-
GeneSwitch- Gal4; Table1, Supplementary file 1) show characteristics of hemocytes and express the
hemocyte marker Hemese (Figure7; Kurucz et al., 2003). They are also positive for HRP, but not
other neuronal markers (Figure7—figure supplement 1). ActGSI- 3 cells divide in floating clusters,
contrasting with S2 cells, which are also thought to be hemocytes, that grow as single cells (Figures1
and 7).
RNAseq analysis demonstrated that many hemocyte genes are upregulated in these cells, including
serpent (srp), Hemese (He), eater, u- shaped (ush), Cecropin A2 (CecA2), and Cecropin C (CecC).
Figure 3. Glial clone Rbr6- 2 cells express Repo. Cells were grown in plain medium (A, C) or treated with ecdysone
(B, D). (A, B) After ecdysone treatment, cells make a lace- like network. (C, D) Cells express Repo with or without
ecdysone treatment. Inset: DAPI (4′,6- diamidino- 2- phenylindole), DNA.
The online version of this article includes the following gure supplement(s) for gure 3:
Figure supplement 1. Marker gene expression in glial- lineage clones.
Figure supplement 2. Glial cell morphology with and without ecdysone treatment.
Figure supplement 3. Gross karyotypes of glial cell clones.

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Comparison of top upregulated genes with scRNAseq data showed that the cells have a strong match
to the top marker genes of hemocytes (Table2).
Growth, karyotype, and transfection efficiency of cell lines
We determined the cell density at confluence for the cell lines (Table3). The cells in each line grow to
confluence attached to the tissue- culture surface, except ActGSI- 3, which grow as floating cell clusters
(Figure8). The cells are not contact inhibited and cell clusters are formed allowing cells to grow to
higher density (Figure8). We determined the doubling time of 13 cell lines and clones using growth
curves (Table3; Figure8—figure supplement 1). Most had doubling times within a range of approx-
imately 20–40hr (Table3). The hemocyte- like clone ActGSI- 3 was an outlier with a longer doubling
time of 70hr (Table3). In cells from clones ActGSB- 6, ActGSI- 2, and ActGSI- 3, expression of RasV12 is
dependent on GeneSwitch Gal4, which is active only in the presence of mifepristone. In the absence
of the drug the cells become quiescent (Figure8—figure supplement 1).
We determined the gross karyotype of 13 cell lines and clones. In keeping with previous findings
for RasV12 expressing cell lines, most (8) were diploid, or near diploid (Simcox etal., 2008b; Table3;
Figure 4. Tracheal- lineage cells of line Btl3 express the epithelial cadherin Ecad/Shotgun. All panels show Btl3
cells except (B) that shows S2 cells. Cells were grown in plain medium (A–C, E) or treated with ecdysone (D, F).
(A) Btl3 cells form a squamous epithelial sheet and express Ecad/Shotgun at cell peripheries. (B) S2 cells grow as
single cells and Ecad expression is diffuse. (C) Btl3 cells form a sheet with small cell clusters and expressed Ecad at
the cell boundaries (E). (D) Ecdysone- treated cells form large multicellular clusters that expressed Ecad (F). Insets in
E and F show nuclei with DAPI.
The online version of this article includes the following gure supplement(s) for gure 4:
Figure supplement 1. Marker gene expression in tracheal- lineage lines.
Figure supplement 2. Morphology of tracheal epithelial parental lines after ecdysone treatment.
Figure supplement 3. Gross karyotypes of tracheal epithelial parental cell lines.

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Figure3—figure supplement 3; Figure4—figure supplement 3; Figure5—figure supplement 5;
Figure6—figure supplement 4; Figure7—figure supplement 2). Related clones had similar karyo-
types, which likely indicates that parental lines may also be clonal as a result of selective pressure for
cells that grow well in culture. Some lines were polyploid and common aneuploid conditions include
loss of an X chromosome and varying numbers of chromosome 4 (Table3).
Nine parental and clonal lines were transfected with an Act5C- EGFP plasmid and the fraction of
GFP- positive cells was determined after 48hr. Cells from all lines tested could be transfected. The
range of efficiency was from 16% to 34% with most lines showing transfection of approximately one
quarter of the cells (Table3). Similarly treated, cells from the S2 line showed an efficiency of 53%.
Figure 5. Mesodermal- lineage cells of Clone 24B5- B8 express Myosin heavy chain after differentiation. Cells were
grown in plain medium (A, C) or treated with ecdysone (B, D–F). (A) Cells have a bipolar shape. (B) Ecdysone-
treated cells elongate and contract. (C) Control cells do not express Mhc. (D) Ecdysone- treated cells express
the muscle marker Mhc. Inset: DAPI, DNA. (E, F) Differentiated 24B5- B8 fuse to form muscle bers that contain
multiple nuclei (white arrowheads), some differentiate without fusing with other cells and have single nucleus (blue
arrowhead), and some fail to differentiate and remain spherical with a single nucleus (red arrowhead).
The online version of this article includes the following gure supplement(s) for gure 5:
Figure supplement 1. Marker gene expression in mesodermal- lineage clones.
Figure supplement 2. Immunostaining of mesodermal- lineage cells for Myosin heavy chain.
Figure supplement 3. Mesodermal cells showed altered morphology after ecdysone treatment.
Figure supplement 4. Mef2- Gal4; UAS- GFP; UAS- RasV12 cultures.
Figure supplement 5. Gross karyotypes of mesodermal cell clones.

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Discussion
Expressing activated Ras, RasV12, in primary cells provides a growth and survival advantage and leads
to the rapid and reliable generation of continuous cell lines—the so- called Ras method (Simcox etal.,
2008b). In a second- generation version of the Ras method, we found that restricting RasV12 expression
with lineage- specific Gal4 drivers gave the targeted cells a competitive advantage and produced
continuous lines with expected cell- type- specific phenotypes. With this approach we produced glial,
epithelial, and muscle cell lines using the repo-, btl-, and 24B/how- Gal4 drivers, respectively.
In theory, the approach could be used to produce cell lines corresponding to any cell type for
which there is an appropriate Gal4 driver. We tried to derive lines with Mef2- Gal4, a muscle master
regulator gene, and the pan- neuronal driver elav- Gal4; however, no continuous lines were produced
(Supplementary file 1; Figure5—figure supplement 4 and Figure6—figure supplement 1). In
both cases, RasV12 expression appeared to disrupt growth of the targeted cell type. In the case of the
muscle lineage, 24B/how- Gal4 was efficient at producing cell lines. The success with one and not the
other muscle driver shows that in practice, it may be necessary to test multiple Gal4 lines for a given
lineage. Drivers with very specific expression patterns may prove useful, including those generated by
the Split Gal4 system (Luan etal., 2006). As with any tissue- culture system, the unnatural conditions
of growing in vitro may select for ‘generic’ cells that survive well in culture and lose their lineage iden-
tities. This means that characterizing cell lines after generation for a battery of features (morpholog-
ical, physiological, and molecular) is an essential step in assessing whether cells represent the tissue
of origin expected for a given Gal4 driver.
repo- Gal4 is a pan- glial driver and many primary cultures expressing RasV12 with this driver reached
confluence and could be passaged several times
Video 1. 24B- Gal4B5- B8 cells contract spontaneously
after differentiation with ecdysone.
https://elifesciences.org/articles/85814/gures#video1
Video 2. 24B- Gal4B5- B8 cells contract spontaneously
after differentiation with ecdysone.
https://elifesciences.org/articles/85814/gures#video2
Video 3. 24B- Gal4GI cells contract spontaneously after
differentiation with ecdysone. Time- lapse video, view
looping.
https://elifesciences.org/articles/85814/gures#video3
Video 4. 24B- Gal4GI cells contract spontaneously after
differentiation with ecdysone. Time- lapse video, view
looping.
https://elifesciences.org/articles/85814/gures#video4

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Figure 6. Neuronal- like clone ActGSI- 2 expresses neuronal markers. ActGSI- 2 cells were grown in three conditions: RU486 (A, D, G, J, M); RU486 and
ecdysone (B, E, H, K, N), or with no additives (C, F, I, L, O). RU486/mifepristone is required for GeneSwtch- Gal4 activation, transgene expression,
and cell proliferation. (A) In the growing condition, cells reach conuence and continue to grow by piling up. (B) After ecdysone treatment cells
elongated and developed axonal- like outgrowths. (C) In the quiescent state (no RU), cells do not proliferate and fail to reach conuence. (D–F) Cells
in all conditions are positive for HRP. (G–I) Expression of Elav, is elevated after ecdysone treatment (H). (J–L) Expression of Futsch/MAP1B- like protein
(recognized by antibody 22C10) is elevated after ecdysone treatment (K). (M–O) Fas2 neural- adhesion protein. Cells show elevated expression after
ecdysone treatment (N). Insets: DAPI, DNA.
Figure 6 continued on next page

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but did not produce continuous lines (Supplementary file 1). We tested different genotypes to deter-
mine if the success rate could be improved by modulation of RasV12 expression (co- expression of the
Gal4 inhibitor Gal80ts), co- expression of the p35 baculovirus survival factor, or growth stimulation by
downregulation of tumor suppressors (dsRNA for warts or brat). One line, also harboring a Gal80ts
transgene, reached passage 25; however, the line was unstable and in early passages the cells vari-
ably lost Repo expression and changed morphologically. The one continuous glial line generated
expresses a transgene that targets the tumor suppressor, brat (repo- Gal4; UAS- RasV12; UAS- bratdsRNA).
Given a single success, it is not clear if downregulation of brat contributed to derivation of the line.
Moreover, there is no evidence that these genotypic variations enhanced cell line generation with
other drivers, as primary cultures expressing RasV12 without modulation or a survival factor produced
lines with similar success rates for the btl- Gal4 or 24B/how- Gal4 drivers (Supplementary file 1).
As with all types of tissue culture, best practices involve maintaining frozen aliquots of cell lines
at relatively low passage numbers. Aliquots of cells from the lines and clones described here, on
which RNAseq was performed, have been archived at similar passage numbers as those used for the
RNAseq analysis. This will allow users to start experimentation with the lines in a known state. The
importance of this is exemplified by line 24BG1, which lost the ability to contract and express the
muscle protein Mhc after multiple passages (Figure5—figure supplement 2).
The online version of this article includes the following gure supplement(s) for gure 6:
Figure supplement 1. elav- G4; UAS- GFP; UAS- RasV12 cultures.
Figure supplement 2. Marker gene expression in neuronal- like clones.
Figure supplement 3. Neuronal- like clone ActGSB- 6.
Figure supplement 4. Gross karyotypes of neuronal- like cell clones.
Figure 6 continued
Figure 7. Hemocyte- like Clone ActGSI- 3 morphology and marker expression. Cells were grown in three conditions: RU486 (A, D); RU486 and ecdysone
(B, E), or with no additives (C, F). (A) In the growing condition, cells formed oating clusters of multiple cells. (B) After ecdysone treatment cells formed
large aggregates and there was cell lysis. (C) In the quiescent state (no RU), individual round cells are seen. (D–F) Cells in all conditions express the
hemocyte cell marker Hemese, as recognized by the antibody H2. Inset: DAPI, DNA.
The online version of this article includes the following gure supplement(s) for gure 7:
Figure supplement 1. Marker gene expression in hemocyte- like clone.
Figure supplement 2. Gross karyotypes of hemocyte cell clone.

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The mesodermal, neuronal, and glial cells represent in vitro counterparts of the tissues of origin
that can be used for studying development and physiology in an accessible and reproducible system.
The mesodermal cells that differentiate into active muscle will allow investigation of muscle fusion,
as the cells are multinucleate (Figure5), as well as muscle physiology and function. For example, the
cells contract spontaneously and in apparent waves (Videos1 and 2); however, the mechanism for
stimulation (if any) and regulation have not been investigated and may cast light on in vivo processes.
Given a variety of cell types, it will also be interesting to examine cell form and function in co- cultures,
for example, of glia and neurons.
The method and the cells will be useful for generating disease models. New lineage- specific lines
could be generated in the desired mutant background by establishing primary cultures from embryos
in which only the mutant genotype expresses RasV12 giving these cells a growth and survival advan-
tage (Simcox etal., 2008a). Derivative lines should include those of the desired cell type and geno-
type. Alternatively, the existing cell lines could be edited using CRISPR, or insertion of transgenes
using the attP site that most lines and clones contain (Supplementary file 1; Bateman etal., 2006;
Manivannan etal., 2015).
The cells with epithelial morphology derived from the tracheal lineage (Btl3 and Btl7) will provide
good models for investigating assay conditions that promote polarization and 3D cell interactions that
could allow the cells to manifest a more complex tissue architecture. In keeping with this possibility,
treating these cells with ecdysone to induce differentiation showed cell clumping suggestive of a
multicellular structure (Figure4—figure supplement 2).
RNAseq analysis of cells from the ActGSI- 3 cell clone showed a striking similarity to hemocytes,
and the cells may be a good model for studying immunity (Table2). The cells lyse after ecdysone
treatment suggesting they are of embryonic origin (Figure7). The cells grow as floating cell clumps
(Figures1 and 7) that may recapitulate subepidermal clusters of sessile hemocytes of the larva (Leitão
and Sucena, 2015; Márkus etal., 2009).
The most significantly upregulated marker genes in each cell line are significantly enriched for top
marker genes from expected cell types based on the single- cell RNAseq data from Fly Cell Atlas in
Table 3. Confluent density, growth, karyotype, and transfection efficiency of cell lines.
Tissue type Line
Confluent density
(×106)*
Doubling
time (hr) Karyotype
Transfection
efficiency (%)
Glial
Rbr6- 2 1.8 20 8, XY 24
Rbr6- 4 2.4 20 8, XY 28
Rbr6- F9 3.4 19 8, XY 22
Epithelial
Btl3 3.7 33 7, XY, –4 26
Btl7 2.6 37
Abnormal tetraploid, XX,
variable 4 34
Btl8 2.7 22 Abnormal tetraploid, XX, –4 16
Mesodermal
24B5- B8 1.4 29
Abnormal tetraploid, XXY,
variable 4 23
24B5- D8 5.1 23
Abnormal tetraploid, XX,
variable 4 27
24BG1- G1 2.8 21 8, XY (some –4) ND
24BG1- F3 2.7 35 8, XY (some –4) ND
Neuronal
ActGSB- 6 2.9 23 7, XO 29
ActGSI- 2 8.1 27 8, XX ND
Blood ActGSI- 3 1.9 70
Abnormal tetraploid, XX,
variable 4 ND
S2 6.2 ND ND 53
*Conuent density in one well of a 12- well plate, 3.5 cm2 surface area (average of three wells).

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Figure 8. Morphology of conuent cultures. (A–C) Glial- lineage clones. The cells grow in dense sheets and ridges with swirl patterns. (D–F) Tracheal-
lineage cells. Btl3 and Btl7 cells form squamous epithelial sheets with raised clusters of cells. Btl8 grow densely however individual cells remain separate.
(G–J) Mesodermal- lineage cells. The cells grow densely, and form raised clusters. (K, L) Neuronal- like clones. ActGSB- 6 cells grow densely and form
Figure 8 continued on next page

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most cases. This indicates the potential value of these cell lines as corresponding in vitro models for
studying these cell types. While the cells will prove to be valuable models, it should be noted that
even those showing a clear differentiated phenotype exhibit unexpected patterns of gene expression.
For example, some cells in the mesodermal clone, 24B5- B8, are positive for HRP (Figure5—figure
supplement 1; Supplementary file 2) and the two neuronal- like lines express a mesodermal marker,
Twist (Figure6—figure supplement 2; Supplementary file 2). This anomalous gene expression is
likely to be an effect of Ras activation on downstream pathways and genes. Ras/MAPK has a key role
in muscle cell determination (Buff etal., 1998; Carmena etal., 2002; Halfon etal., 2000) and acti-
vates downstream muscle determination genes. It will also be important to consider what genes are
not expressed by a given cell line, for example, glial cell missing (gcm) is not differentially expressed in
the three glial- lineage cell clones and gcm2 is differentially expressed in only two of the three clones.
Further, trachealess (trh) is only differentially expressed in one of the three tracheal- lineage cell lines.
Similarly, the muscle- specific transcription factors twist (twi), nautilus (nau), snail (sna), and Mef2 show
variable expression in the muscle- lineage cell clones. It should also be noted that the expression
patterns were determined for undifferentiated cells and expression levels could change after hormone
exposure.
The cells will have value for both low- and high- throughput approaches, including genetic or
compound screens for which screening in the relevant cell type will result in identifying targets that are
more likely to be of physiological relevance. Most of the cells have an attP- flanked cassette (Table1),
which makes them amenable to insertion of transgenes such as reporters by Recombination Mediated
Cassette Exchange (RMCE) (Bateman etal., 2006; Manivannan etal., 2015). Moreover, cells compe-
tent for RMCE can be modified by stable expression of Cas9 and then used for genome- wide CRISPR
pooled screening. With this approach, a library of single guide RNAs (sgRNAs) are integrated at RMCE
sites (Viswanatha etal., 2018; Viswanatha etal., 2019). This generates a pool of cells, each with a
different sgRNA, that can be subjected to a screen assay. Results are identified by PCR amplification
of inserted sgRNAs followed by next- generation sequencing to detect sgRNAs that are enriched or
depleted in the experimental cell pool as compared with a control. To date, pooled CRISPR screens in
Drosophila have only been performed in S2 cells, which have hemocyte- like features. The availability
of new cell lines with muscle, glial, and epithelial characteristics will enable screens designed to inter-
rogate biological processes specific to these cell types.
There are hundreds of Drosophila cell lines; however, the number corresponding to known cell
types is low. This is due in part to the lack of a method for generating cell lines from specific tissues.
We expect that the method described here, using restricted expression of RasV12, will be a tractable
approach for investigators to generate lines of cell types of interest. Single- cell cloning followed by
cell characterization (immunohistochemistry and RNAseq) also proved to be a useful method to iden-
tify cell- type- specific lines and this approach could identify additional valuable lines in the existing
collection at the DGRC. In summary, we show that lineage- restricted Ras expression and cell cloning
has produced a set of new cell lines that will be of immediate value for analyses in the five cell types
they represent.
Materials and methods
Fly stocks
The following fly stocks were used to create primary cell lines: Gal4 drivers: 24B/how- Gal4, w[*]; P(w[+
mW. hs]=GawB)how[24B] (BL 1767); repo- Gal4, P(GAL4)repo (BL 7415); btl- Gal4, P(GAL4- btl.S)3- 2 (BL
78328); Act5C- GeneSwitch- Gal4, P(UAS- GFP.S65T)Myo31DF[T2]; P(Act5C(- FRT)GAL4.Switch.PR)3
(BL 9431). Transgenes: UAS- RasV12 (3), P(w[+mC]=UAS- Ras85D.V12)TL1 (BL 64195); UAS- RasV12 (2),
P(w[+mC]=UAS- Ras85D.V12)2 (BL 64196); UAS- RasV12 with RMCE site (3), P(w[+mC]=UAS- Ras85D.
peaks and valleys. ActGSI- 2 cells grow densely with scattered raised clusters. (M) Hemocyte- like clone ActGSI- 3. The cells form oating clusters that
coalesce into large cell rafts. (N) Schneider’s S2 cells. The cells grow to high density in suspension. Scale bar = 200µm.
The online version of this article includes the following gure supplement(s) for gure 8:
Figure supplement 1. Growth curves.
Figure 8 continued

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V12)TL1, P(w[+mC]=attP.w[+].attP)JB89B (BL 64197); UAS- GFP nuclear, P( UAS- GFP. nls) 14 (BL 4775);
bratdsRNA, P(y[+t7.7] v[+t1.8]=TRiP.HMS01121)attP2 (BL 34646); UAS- p35 baculovirus death inhibitor,
P(w[+mC]=UAS- p35.H)BH1 (BL 5072) and Gal80ts, w[*]; P(w[+mC]=tubP- GAL80[ts])20 (BL 7019).
Setting up primary cultures
This follows a detailed method, which has additional information (Simcox, 2013), except that no
yeast paste is used on the egg collection plates. Yeast paste, even when sterilized, promotes contam-
ination in the cultures. Crosses were made between the Gal- 4 driver lines and UAS- RasV12 lines. Some
RasV12 stocks had additional alleles as noted in Supplementary file 1. Approximately 200 males
and 200 females of a cross were transferred into a laying cage, with a fluted Whatman 3MM paper
insert to increase surface area, and eggs were collected using 60- mm Petri dishes containing egg
laying medium. Egg collections were made during the day for 8hr at room temperature or 16hr
overnight at 17°C. After collection, approximately 3 ml of TXN (NaCl [0.7%], Triton X [0.02%] in
water) was added to the plate. Any hatched larvae, which rise to the surface, were removed and the
unhatched embryos were dislodged using a large soft paint brush to gently release them from the
surface. Embryos were tipped off with the liquid into a sieve. Additional rinsing and brushing were
used to ensure most embryos were dislodged and collected in the sieve. After thorough rinsing of
the embryos with TXN from a squirt bottle, the sieve was upended over a 15- ml Falcon tube and a
stream of TXN was used to transfer the embryos into the tube. Once the embryos settled, the TXN
was removed and replaced with 3 ml of 50% bleach (Clorox) in water. The tube was capped and
inverted three to five times and subsequently the embryos were treated using sterile techniques.
The embryos were allowed to settle at the bottom of the tube and the bleach was removed after
3–5min. The bleach dechorionates and surface sterilizes the embryos. The embryos were rinsed 2×
with 4ml of sterile TXN and transferred to a fresh tube of TXN to minimize bleach contamination.
After two additional TXN rinses the embryos were transferred to TXN in a 5- ml glass homogenizer
(with Teflon pestle). Embryos were rinsed in 3ml of water followed by a rinse in 1ml of Schneider’s
S2 medium (supplemented with 10% heat inactivated fetal bovine serum and 1× Pen- strep solution).
Embryos tend to clump in the Schneider’s S2 medium and stick to the sides of the homogenizer and
pipette and care is needed to remove the medium without disturbing the embryos. 3ml of fresh
Schneider’s S2 medium was added to the homogenizer and the embryos were disrupted by three
gentle strokes with the pestle. Care was taken to minimize bubbles by not withdrawing the pestle
beyond the surface of the liquid. The homogenate was allowed to settle for 2min and the super-
natant was transferred to a 15- ml Falcon tube leaving the large cell clumps and any whole embryos
in the bottom of the homogenizer. 3ml of fresh Schneider’s S2 medium was added to the homoge-
nizer and three more strokes, with a twist at the bottom, were used to disrupt remaining tissue and
embryos. The second homogenate was added to the Falcon tube. The tube was centrifuged in a
benchtop centrifuge at 1400 × g. The supernatant was discarded, and the pellet was resuspended in
3- ml Schneider’s S2 medium and centrifugation step and washing with Schneider’s S2 medium was
repeated twice more. The final pellet size was estimated and plated in 1 or more 12.5 cm2 T- flasks
with 2–3ml Schneider’s S2 medium. The number of flasks needed for a given pellet size can also be
estimated from the volume of packed embryos with approximately 30µl of packed embryos being
sufficient for one flask.
Culture conditions for new cell lines
Cells were grown in 25 cm2 T- flasks at 25°C in Schneider’s S2 medium and were passaged at between
90% and full confluence (Figure8) using trypsin to release cells from the tissue- culture surface. Trypsin
is needed as cells in all the lines are adherent except ActGSI3 cells that float freely (Figures1 and
8). Cells were pelleted and approximately 20–25% of the cells were plated in a new flask. Cells were
checked using an inverted microscope approximately every 5 days. The medium was changed on
cultures showing signs of poor cell health (extended processes, little growth). This was sometimes
necessary for cell types that are more metabolically active and acidify the medium, including the
mesodermal lines. Cells were passaged every 5–7 days. Cell freezing (Schneider’s S2 medium with
20% heat inactivated fetal bovine serum and 10% DMSO (Dimethyl sulfoxide)) was used to keep a
supply of frozen aliquots so that cells with similar passage numbers were used in experiments.

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Cell cloning
For puromycin selection, 2–6 × 105 cells in a 35- mM well were transfected with 0.4µg of DNA encoding
a puro resistance plasmid (pCoPURO, Addgene #17533) using Effectene Transfection Reagent
(QIAGEN). After 24hr, cells were selected with puromycin at 0.5–2.5µg/ml for 5 days. After 2–4
weeks, colonies were isolated and expanded. For dilution cloning, cells were seeded into a 96- well
plate at a concentration of 0.5–1 cell/well in 100µl conditioned media (Housden etal., 2015).
Hormone treatment
To simulate the major pulse of ecdysone at the larval to pupal transition, cells were treated with two
24hr doses of β-ecdysone (Sigma 5289- 74- 7) at 1µg/ml separated by 24hr in non- supplemented
medium.
Immunohistochemistry
Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15min or 3.5% form-
aldehyde (Sigma) for 30 min at room temperature, and then rinsed twice with 0.1% Tween- 20 in
phosphate- buffered saline (PBS- T). Cells were permeabilized (0.2% Triton X- 100 in PBS) for 10min
at room temperature. Cells were blocked (5% bovine serum albumin in PBS- T) for 30min at room
temperature and incubated with diluted primary antibodies overnight at 4°C. Cells were washed three
times with PBS- T and incubated with diluted secondary antibodies in blocking buffer for 1hr at room
temperature or overnight at 4°C. Cells were washed three times with PBS- T and mounted in Vecta-
Shield with DAPI (Vector Laboratories). For the Dcad2 antibody, cells were fixed and processed as
described in Oda etal., 1994. The following primary antibodies and dilutions were used: HRP (rabbit
polyclonal, Jackson ImmunoResearch 323- 005- 021, 1:500), 22C10 (mouse monoclonal anti- Futsch,
Developmental Studies Hybridoma Bank, DSHB, 1:100), ELAV (rat monoclonal, DSHB 7E8A10, 1:100),
Repo (mouse monoclonal, DSHB 8D12, 1:100), FasII (mouse monoclonal, DSHB 1D4, 1:100), Twist (a
gift from M. Levine, UC Berkeley, CA, guinea pig 1:500), MHC (mouse monoclonal, DSHB 3E8- 3D3,
1:100), Dcad2 (rat monoclonal, DSHB, 1:100), and DMef2 (a gift from J. R. Jacobs [Vanderploeg
etal., 2012], rabbit polyclonal, 1:500), H2 (mouse monoclonal, [Kurucz etal., 2003], 1:10). Cells were
incubated with the following secondary antibodies at the indicated dilutions: Cy3- conjugated goat
anti- mouse (Jackson ImmunoResearch 115- 165- 003, 1:1000), Cy3- conjugated goat anti- rat (Jackson
ImmunoResearch 112- 165- 003, 1:1000), Cy3- conjugated goat anti- guinea pig (Jackson ImmunoRe-
search 106- 165- 003, 1:1000), Cy3- conjugated goat anti- rabbit (Jackson ImmunoResearch 111- 165-
045, 1:1000), and Alexa Fluor 488- conjugated donkey anti- rabbit (Invitrogen A- 21206, 1:1000).
Growth curve analysis
1–2 × 105 cells were plated in a 12- well plate. Cells were counted from triplicate wells every 3 days
over a 9- day period. Doubling time was calculated using log2 cell numbers (Roth, 2006).
Karyotype analysis
Cells were grown to 50–90% confluence and incubated with 0.05µg/ml KaryoMAX (Gibco- Thermo
Fisher 15212012) for 3–18hr. Cells were processed for analysis using the method in Lee etal., 2014,
which uses 0.5% sodium citrate as a hypotonic solution and a 3:1 ice cold mix of methanol and acetic
acid as a fix. After dropping fixed cells, slides were air dried and mounted in VectaShield with DAPI
(Vector Laboratories) and viewed with an Olympus BX41 microscope.
Transfection
Cells in a 6- well plate (approximately 70% confluent) were transfected with 0.4µg of an Actin5C- EGFP
plasmid (pAc5.1B- EGFP, Addgene #21181) using Effectene Transfection Reagent (QIAGEN). The frac-
tion of GFP- positive cells was scored after 48hr.
RNA extraction and RNAseq
Cell cultures were grown and expanded in their respective media. All cell lines were cultured in
Schneiders Drosophila Medium (Gibco Cat # 21720001), supplemented with 10% fetal bovine serum
(Cytiva Hyclone Cat SH30070.03). For Act5C- GS>Ras attP- GFP- LI- Clone 2, Act5C- GS>Ras attP- GFP-
LI- Clone 3, and Act5C- GS>Ras attP- GFP- LB- Clone 6 cultures were grown in the same basal media

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Coleman- Gosser, Hu, Raghuvanshi etal. eLife 2023;12:e85814. DOI: https://doi.org/10.7554/eLife.85814 19 of 27
supplemented with 10nM of Mifepristone (Thermo Fisher Cat# H11001). Cultures were allowed to
grow in T- 25 flasks to become confluent before treatment with trypsin (Gibco Cat# 12604013) for
4min to dislodge the cell monolayer from the growth surface. The cells were resuspended in 4ml of
their respective media and 1ml of the cell suspension was collected for pelleting, followed by washing
in 1× PBS, and then flash- freezing in liquid nitrogen. All cell samples were processed in triplicates.
Total RNA was isolated from the pellets using the TRIzol reagent (Life Technologies [Ambion],
Cat#:15596018) as per the manufacturer’s instructions. The isolated total RNA was subjected to further
purification using the RNeasy Mini Kit (QIAGEN, Cat#74104) and the RNA post- cleanup was eluted
in RNase- free water. The eluted total RNA was confirmed to have a A260/A280 ratio >1.8 and RIN >7.
Upon passing the quality control parameters, Illumina TruSeq libraries were constructed using
TruSeq stranded mRNA HT kit (Illumina, Cat# RS- 122- 2103). Paired end sequencing was performed
on an Illumina NextSeq 500 with a 150- cycle high output kits (Illumina, Cat# FC- 404- 2002).
RNAseq data analysis
Raw data processing was performed using the STAR sequence aligner (https://github.com/alexdobin/
STAR; Dobin et al., 2013). Reads were aligned to the Drosophila genome and featureCounts were
used to get gene counts from all samples into a count matrix for downstream analysis. A principal
component analysis plot was produced using heatmaply. FPKM values were calculated using fpkm(-
DEseq2) using gene length output by featureCounts. The reference genome used was FB2022_05,
dmel_r6.48 (FlyBase) (Jenkins etal., 2022). Both raw sequencing reads and the count matrix were
deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number
GSE219105. The processed dataset has also been imported into DGET database for user to mine
gene(s) of interest or search for genes with similar expression pattern (https://www.flyrnai.org/tools/
dget/web/).
Each sample was compared against all other samples by using DESeq2 ( Love etal., 2014) to
determine differentially expressed genes (DE calling). The set of top DE genes for each cell line was
compared with the top 100 markers in single- cell RNAseq datasets corresponding to cell types in the
Fly Cell Atlas 10× datasets (Li etal., 2022). Enrichment analysis was conducted using the DRscDB tool
to identify the Fly Cell Atlas cell type that matched closely to each cell line (Hu etal., 2021). We also
compared the DE genes with the genes identify in various tissues in embryo and larval based on in situ
data (PMID: 24359758, 17645804, 12537577) and majority of the best matching tissues are consistent
with the analysis using scRNAseq datasets (Table2).
The RNAseq data for the cell lines described in this work were also compared with RNAseq datasets
determined previously for 24 other Drosophila cell lines (Cherbas etal., 2011). The comparison was
conducted by hierarchical clustering analysis using Pearson correlation coefficient scores. To survey
the activities of major signaling pathways in the cell lines, we specifically selected the ligands and
receptors annotated at FlyPhoneDB (PMID: 35100387) to plot their expression levels using heatmap.
Materials availability
All cell lines described here have been deposited to the Drosophila Genomics Resource Center
(DGRC) at Indiana University. The lines are available for distribution to the research community.
Acknowledgements
We thank M Levine, J R Jacobs, and D Hultmark for antibodies and the Bloomington Stock Center for
fly stocks. We thank Mikhail Kouzminov for help with data analysis. Funding This work is supported
by the National Institutes of Health (NIH Office of the Director R24 OD019847 to NP, SEM, and
AS, P40OD010949 to the DGRC, and NIH NIGMS P41 GM132087 to the DRSC- BTRR), the National
Science Foundation (IOS 1419535 to AS, and support while serving at the National Science Founda-
tion to AS), the Howard Hughes Medical Institute (NP), and a grant from Women & Philanthropy at
the Ohio State University (to AS).

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Additional information
Funding
Funder Grant reference number Author
National Institutes of
Health (NIH) Ofce of the
Director
R24 OD019847 Norbert Perrimon
Stephanie E Mohr
Amanda Simcox
National Institutes of
Health
P40OD010949 Andrew Zelhof
National Institutes of
Health
P41 GM132087 Norbert Perrimon
Stephanie E Mohr
National Science
Foundation
IOS 1419535 Amanda Simcox
Howard Hughes Medical
Institute
Norbert Perrimon
Women & Philanthropy at
The Ohio State University
Grant Amanda Simcox
National Science
Foundation
Support while serving
at the National Science
Foundation
Amanda Simcox
The funders had no role in study design, data collection, and interpretation, or the
decision to submit the work for publication. Any opinion, ndings, and conclusions
or recommendations expressed in this material are those of the authors and do not
necessarily reect the views of the National Science Foundation.
Author contributions
Nikki Coleman- Gosser, Shane Stitzinger, Formal analysis, Investigation, Writing – review and editing;
Yanhui Hu, Formal analysis, Writing – review and editing; Shiva Raghuvanshi, Molly Josifov, Investiga-
tion; Weihang Chen, Formal analysis; Arthur Luhur, Daniel Mariyappa, Investigation, Writing – review
and editing; Andrew Zelhof, Funding acquisition, Writing – review and editing; Stephanie E Mohr,
Norbert Perrimon, Supervision, Funding acquisition, Writing – review and editing; Amanda Simcox,
Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology,
Writing - original draft, Project administration, Writing – review and editing
Author ORCIDs
Daniel Mariyappa
http://orcid.org/0000-0003-4775-1656
Molly Josifov
http://orcid.org/0000-0002-2899-7186
Stephanie E Mohr
http://orcid.org/0000-0001-9639-7708
Norbert Perrimon
http://orcid.org/0000-0001-7542-472X
Amanda Simcox
http://orcid.org/0000-0002-5572-7042
Decision letter and Author response
Decision letter https://doi.org/10.7554/eLife.85814.sa1
Author response https://doi.org/10.7554/eLife.85814.sa2
Additional files
Supplementary files
• Supplementary file 1. Primary cultures and continuous lines produced from indicated genotypes.
Glial primary cultures grew well at first and could be passaged several times; however, only one
continuous line was produced. This line was cloned using single- cell dilution to produce three clonal
derivatives. Tracheal lines were produced readily. Cloning the parental lines was not successful with
either single- cell dilution or puro selection. Mesodermal lines were produced using expression of
RasV12 with 24B- Gal4 but not Mef2- Gal4. Cloning of the continuous lines was done using single-
cell dilution. Neuronal. Expression of RasV12 with neuronal Gal4 drivers (elav- Gal4 or scratch- Gal4)
did not give rise to continuous lines. Cloning of lines generated by broad expression of RasV12 with

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Act5C- GeneSwitch- Gal4 produced two clonal lines with neuronal characteristics and one with
hemocyte characteristics. These were cloned using puro selection.
• Supplementary file 2. Analysis of marker gene expression in parental lines and clones. Cell lines
and their clonal derivatives were stained with antibodies against the indicated markers. The fraction
of cells staining positive was determined. The intensity and cellular location of the signal are
indicated in cases when there was variation. The clones and parental lines highlighted were analyzed
by RNAseq.
• Supplementary file 3. Fragments Per Kilobase of transcript per Million mapped reads (FPKM).
FPKM values are shown for each of the clones and parental lines that were analyzed by RNAseq.
• MDAR checklist
Data availability
Sequencing data have been deposited in GEO under accession code GSE219105.
The following dataset was generated:
Author(s) Year Dataset title Dataset URL Database and Identifier
Mariyappa D, Luhur
A, Zelhof A, Hu Y,
Simcox A
2022 Continuous muscle, glial,
epithelial, neuronal, and
hemocyte cell lines for
Drosophila research
https://www. ncbi.
nlm. nih. gov/ geo/
query/ acc. cgi? acc=
GSE219105
NCBI Gene Expression
Omnibus, GSE219105
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Appendix 1
Appendix 1 Continued on next page
Appendix 1—key resources table
Reagent type
(species) or
resource Designation Source or reference Identifiers Additional information
Genetic reagent (D.
melanogaster)24B/how- Gal4 Bloomington Drosophila Stock Center
Stock # 1767;
FLYB:FBti0150063;
RRID:BDSC_1767
FlyBase symbol: P(w[+ mW. hs]=GawB)
how[24B]
Genetic reagent (D.
melanogaster)repo- Gal4 Bloomington Drosophila Stock Center
Stock # 7415;
FLYB:FBti0018692; RRID:BDSC_7415 FlyBase symbol: P(GAL4)repo
Genetic reagent (D.
melanogaster)btl- Gal4 Bloomington Drosophila Stock Center
Stock # 78328;
FLYB:FBti019793;
RRID:BDSC_78328 FlyBase symbol: P(GAL4- btl.S)3–2
Genetic reagent (D.
melanogaster)
Act5C- GeneSwitch-
Gal4 Bloomington Drosophila Stock Center
Stock # 9431;
FLYB:FBti0003040,FBti0076553;
RRID:BDSC_9431
FlyBase symbol: P(UAS- GFP.S65T)
Myo31DF[T2]; P(Act5C(- FRT)GAL4.Switch.
PR)3
Genetic reagent (D.
melanogaster)UAS- RasV12 (3) Bloomington Drosophila Stock Center
Stock # 64195;
FLYB:FBti0012505; RRID:BDSC_64195
FlyBase symbol: P(w[+mC]=UAS- Ras85D.
V12)TL1
Genetic reagent (D.
melanogaster)UAS- RasV12 (2) Bloomington Drosophila Stock Center
Stock # 64196;
FLYB:FBti0180323; RRID:BDSC_64196
FlyBase symbol: P(w[+mC]=UAS- Ras85D.
V12)2
Genetic reagent (D.
melanogaster)
UAS- RasV12 with RMCE
site (3) Bloomington Drosophila Stock Center
Stock # 64197;
FLYB: FBti0012505, FBti0102080;
RRID:BDSC_64197
FlyBase symbol: P(w[+mC]=UAS- Ras85D.
V12)TL1, P(w[+mC]=attP.w[+].attP)JB89B
Genetic reagent (D.
melanogaster)UAS- GFP nuclear Bloomington Drosophila Stock Center
Stock # 4775;
FLYB: FBti0012492;
RRID:BDSC_4775 FlyBase symbol: P( UAS- GFP. nls) 14
Genetic reagent (D.
melanogaster) bratdsRNA Bloomington Drosophila Stock Center
Stock # 34646;
FLYB:FBti0140815; RRID:BDSC_34646
FlyBase symbol: P(y[+t7.7] v[+t1.8]=TRiP.
HMS01121)attP2
Genetic reagent (D.
melanogaster)
UAS- p35 baculovirus
death inhibitor Bloomington Drosophila Stock Center
Stock # 5072;
FLYB:FBti0012594; RRID:BDSC_5072
FlyBase symbol: P(w[+mC]=UAS- p35.H)
BH1
Genetic reagent (D.
melanogaster) Gal80ts Bloomington Drosophila Stock Center
Stock # 7019;
FLYB:FBti0027796; RRID:BDSC_7019
FlyBase symbol: P(w[+mC]=tubP-
GAL80[ts])20
Cell line (D.
melanogaster) S2 Drosophila Genomics Resource Center
Stock # 181; FLYB:FBtc0000181;
RRID:CVCL_Z992
Cell line maintained in N. Perrimon lab;
FlyBase symbol: S2- DRSC.
Cell line (D.
melanogaster)24B5- B8 Drosophila Genomics Resource Center Stock # 323; RRID:CVCL_C7G6 24B>Ras attP- L5- CloneB8
Cell line (D.
melanogaster)24BG1- G1 Drosophila Genomics Resource Center Stock # 324; RRID:CVCL_C7G7 24B>Ras attP- G1- CloneG1
Cell line (D.
melanogaster)24BG1- F3 Drosophila Genomics Resource Center Stock # 325; RRID:CVCL_C7G8 24B>Ras attP- G1- CloneF3
Cell line (D.
melanogaster)Rbr6- 2 Drosophila Genomics Resource Center Stock # 326; RRID:CVCL_C7G9 repo>Ras bratdsRNA- L6- Clone2
Cell line (D.
melanogaster)Rbr6- 4 Drosophila Genomics Resource Center Stock # 327; RRID:CVCL_C7GA repo>Ras bratdsRNA- L6- Clone4
Cell line (D.
melanogaster)Rbr6- F9 Drosophila Genomics Resource Center Stock # 328; RRID:CVCL_C7GB repo>Ras bratdsRNA- L6- CloneF9
Cell line (D.
melanogaster)ActGSI- 2 Drosophila Genomics Resource Center Stock # 329; RRID:CVCL_C7GC Act5C- GS>Ras attP- LB- Clone6
Cell line (D.
melanogaster)ActGSI- 2 Drosophila Genomics Resource Center Stock # 330; RRID:CVCL_C7GD Act5C- GS>Ras attP- GFP- LI- Clone2
Cell line (D.
melanogaster)ActGSI- 3 Drosophila Genomics Resource Center Stock # 331; RRID:CVCL_C7GE Act5C- GS>Ras attP- GFP- LI- Clone3
Cell line (D.
melanogaster) Btl3 Drosophila Genomics Resource Center Stock # 332; RRID:CVCL_B3N7 btl>Ras attP- L3

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Coleman- Gosser, Hu, Raghuvanshi etal. eLife 2023;12:e85814. DOI: https://doi.org/10.7554/eLife.85814 26 of 27
Reagent type
(species) or
resource Designation Source or reference Identifiers Additional information
Cell line (D.
melanogaster)OK6- 3 Drosophila Genomics Resource Center
Stock # 281;
RRID:CVCL_XF56 OK6>Ras attP- L3
Cell line (D.
melanogaster) Rbr6 Drosophila Genomics Resource Center Stock # 282; RRID:CVCL_XF57 repo>Ras bratdsRNA- L6
Cell line (D.
melanogaster) 24BG1 Drosophila Genomics Resource Center Stock # 283; RRID:CVCL_XF51 24B>Ras attP GFP- L1
Cell line (D.
melanogaster) 24B5 Drosophila Genomics Resource Center Stock # 284; RRID:CVCL_XF52 24B>Ras attP- L5
Cell line (D.
melanogaster) Btl7 Drosophila Genomics Resource Center Stock # 285; RRID:CVCL_XF53 btl>Ras attP- L7
Cell line (D.
melanogaster) Btl8 Drosophila Genomics Resource Center Stock # 286; RRID:CVCL_XF54 btl>Ras attP- L8
Cell line (D.
melanogaster)OK6- 2 Drosophila Genomics Resource Center
Stock # 287;
RRID:CVCL_XF55 OK6>Ras attP- L2
cell line (E. coli)DH5- alpha Thermo Fisher Cat. # 18265017
Subcloning efciency DH5- alpha
competent cells
Transfected
construct (D.
melanogaster)pAc5.1B- EGFP Addgene
Cat. # 21181; http://n2t.net/addgene:
21181; RRID:Addgene_21181
pAc5.1B- EGFP was a gift from Elisa
Izaurralde
Transfected
construct (D.
melanogaster) pCoPURO Addgene
Cat. # 17533; http://n2t.net/addgene:
17533; RRID:Addgene_17533
pCoPURO was a gift from Francis
Castellino
Antibody
AfniPure Rabbit Anti-
Horseradish Peroxidase
(Rabbit polyclonal) Jackson ImmunoResearch Cat. # 323- 005- 021; RRID: AB_2314648 Rabbit polyclonal; IF (1:500)
Antibody
22C10 (mouse
monoclonal)
Developmental Studies Hybridoma
Bank
Cat. # 22C10
RRID: AB_528403. FBgn0259108
22C10 was deposited to the DSHB by
Benzer, S./Colley, N.; mouse monoclonal;
IF (1:100)
Antibody
Rat- Elav- 7E8A10 anti-
elav (rat monoclonal)
Developmental Studies Hybridoma
Bank
Cat. # Rat- Elav- 7E8A10 anti- elav,
RRID:AB_528218
Rat- Elav- 7E8A10 anti- elav was deposited
to the DSHB by Rubin, G.M.; rat
monoclonal; IF (1:100)
Antibody
8D12 anti- Repo (mouse
monoclonal)
Developmental Studies Hybridoma
Bank Cat. # 8D12 anti- Repo, RRID:AB_528448
8D12 anti- Repo was deposited to
the DSHB by Goodman, C.; mouse
monoclonal; IF (1:100)
Antibody
1D4 anti- Fasciclin II
(mouse monoclonal)
Developmental Studies Hybridoma
Bank
Cat. # 1D4 anti- Fasciclin II,
RRID:AB_528235
1D4 anti- Fasciclin II was deposited to
the DSHB by Goodman, C.; mouse
monoclonal; IF (1:100)
Antibody
Guinea pig anti- Twist
(guinea pig polyclonal) M.Levine, UC Berkeley, CA
A gift from M. Levine, UC Berkeley, CA;
guinea pig polyclonal; IF (1:500)
Antibody
3E8- 3D3 (mouse
monoclonal)
Developmental Studies Hybridoma
Bank Cat. # 3E8- 3D3, RRID:AB_2721944
3E8- 3D3 was deposited to the DSHB by
Saide, J.D.; mouse monoclonal; IF (1:100)
Antibody
DCAD2 (rat
monoclonal)
Developmental Studies Hybridoma
Bank Cat. # DCAD2, RRID:AB_528120
DCAD2 was deposited to the DSHB by
Uemura, T.; rat, monoclonal; IF (1:100)
Antibody
Rabbit anti- DMef2
(rabbit polyclonal) doi:10.1101/gad.9.6.730
A gift from J. R. Jacobs; rabbit polyclonal;
IF (1:500)
Antibody
Mouse anti- H2 (mouse
monoclonal) doi:10.1073/pnas.0436940100 Kurucz etal., 2003; IF (1:10)
Antibody
Cy3 AfniPure Goat
Anti- Mouse IgG (H+L)
(Goat polyclonal) Jackson ImmunoResearch Cat. # 115- 165- 003; RRID: AB_2338680 Goat polyclonal; IF (1:1000)
Antibody
Cy3 AfniPure Goat
Anti- Rat IgG (H+L)
(Goat polyclonal) Jackson ImmunoResearch Cat. # 112- 165- 003; RRID: AB_2338240 Goat polyclonal; IF (1:1000)
Appendix 1 Continued
Appendix 1 Continued on next page

Tools and resources Cell Biology | Developmental Biology
Coleman- Gosser, Hu, Raghuvanshi etal. eLife 2023;12:e85814. DOI: https://doi.org/10.7554/eLife.85814 27 of 27
Reagent type
(species) or
resource Designation Source or reference Identifiers Additional information
Antibody
Cy3 AfniPure Goat
Anti- Guinea Pig IgG
(H+L) (Goat polyclonal) Jackson ImmunoResearch Cat. # 106- 165- 003; RRID: AB_2337423 Goat polyclonal; IF (1:1000)
Antibody
Cy3 AfniPure Goat
Anti- Rabbit IgG (H+L)
(Goat polyclonal) Jackson ImmunoResearch Cat. # 111- 165- 045; RRID: AB_2338003 Goat polyclonal; IF (1:1000)
Antibody
Donkey anti- Rabbit IgG
(H+L) Highly Cross-
Adsorbed Secondary
Antibody, Alexa Fluor
488 (donkey polyclonal) Thermo Fisher Cat. # A- 21206; RRID: AB_2535792 Donkey polyclonal; IF (1:1000)
Commercial assay
or kit
Effectene Transfection
Reagent QIAGEN Cat. # 301425
Commercial assay
or kit
NucleoSpin Plasmid Kit
(No Lid) Macherey- Nagel Cat. # 740499.250
Commercial assay
or kit
DNeasy Blood & Tissue
Kit QIAGEN Cat. # 69504
Chemical
compound, drug
KaryoMAX Colcemid
Solution in PBS Gibco Thermo Fisher Cat. # 15212–012
Chemical
compound, drug
Schneider′s Insect
Medium Sigma- Aldrich Cat. # S0146
Chemical
compound, drug FBS Gibco Thermo Fisher Cat. # 26140–079
Chemical
compound, drug
0.05% Trypsin–EDTA
(1×) Gibco Thermo Fisher Cat. # 25300–120
Chemical
compound, drug
Penicillin–streptomycin
(10,000U/ml) Gibco Thermo Fisher Cat. # 15140122
Chemical
compound, drug Mifepristone Invitrogen Thermo Fisher Cat. # H11001
Chemical
compound, drug 20- Hydroxyecdysone Sigma- Aldrich Cat. # H5142
Chemical
compound, drug
VECTASHIELD Antifade
Mounting Medium With
DAPI Vector Laboratories Cat. # H1200
Software, algorithm
GraphPad Prism version
9.5.1 https://www.graphpad.com/ RRID:SCR_002798
Software, algorithm Fiji doi:10.1038/nmeth.2019 RRID:SCR_002285
Appendix 1 Continued