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Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye

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Abstract and Figures

Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools pose challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L² mutant and GMR-hid, GMR-GAL4 eye. L² exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly newt genes significantly restore missing photoreceptor cells both in L² and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.
Newt genes promote cell proliferation and downregulate cell death to promote rescue of L 2 mutant phenotype of loss-of-ventral eye (A-E) PH-3 staining (blue), as a marker to calculate cell proliferation in the developing eye imaginal disc of (A) L 2 mutant (red dotted boundary marking eye boundary), (B) L 2 mutant background where vna4 gene is misexpressed (L 2 ; ey>vna4), (C) ey-GAL4 driver, (D) misexpression of newt gene in ey-GAL4 domain (ey>vna4) (E) and wild type (CantonS). (A) Red dotted boundary represents the loss-of-ventral-eye area, and (B) white dotted boundary represents the rescue of loss-of-ventral eye phenotype where vna4 gene is misexpressed. (A 0 -E 0 ) single channel confocal images demonstrating PH-3 stained nuclei. The region of interest used to count the number of dividing cells is marked in yellow dotted boundaries. (F) Bar graph shows comparative increase in the rate of cell division. There is no significant change in cell proliferation rate among the (A and A 0 ) L 2 ; ey-GAL4, (C and C 0 ) ey-GAL4, (D and D 0 ) ey>vna4, (E and E 0 ) Canton-S (wild-type) eye disc. (A, A 0 , B, B 0 , and F) However, there is robust increase in the cell proliferation rate in (B and B 0 ) L 2 ; ey>vna4 background in comparison to (A and A 0 ) L 2 mutant (L 2 ; ey-GAL4). (G-K) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining serves as a marker for cell death assay. TUNEL staining (shown in green) marks the dying cells nuclei in (G and G 0 ) L 2 ; ey-GAL4, (H and H 0 ) L 2 ; ey>vna4, (I and I 0 ) ey-GAL4, (J and J 0 ) ey>vna4, (K) and wild type (Canton-S) eye imaginal disc. (G) Red dotted boundary represents the loss-of-ventral eye region, and (H) white dotted boundary represents the rescued lossof-ventral eye region. (G 0 -K 0 ) The TUNEL positive cells are counted within the zone of interest marked within the yellow dotted boundary as shown in single filter confocal images. (L) Bar graph shows decrease in the cell death when a newt gene is misexpressed in the L 2 -mutant background compared to only L 2 -mutant. The downregulation in cell death is comparable to the cell death occurring in the normal wild-type background. Statistical analysis was performed using the student's t-test for independent samples. Sample size used for the calculation was five in number (n = 5). Statistical significance was determined with 95% confidence (p < 0.05). All bar graphs show change in cell proliferation and cell death as average between 5 samples. Error bars show standard deviation (mean G SD), and symbols above the error bar * signify p value < 0.05, ** signify p value < 0.005, *** signify p value < 0.0005, and ns signify p value >0.05 respectively. All the images are displayed in the same polarity as dorsa domain-towards top, and ventral domain-towards bottom. Scale bar = 100 mm. See also Figures S5, S6, S7, S8, and S9.
… 
Newt gene regulates Wg both in the wild type, and in the L 2 -mutant background (A-H) Misexpression of wg in the Drosophila developing eye. (A, B) Wild type background (+/+; ey>wg-GFP). (C and D) Newt gene and Wg are misexpressed in the wild type background (+/+; ey>wg-GFP; vna4) exhibits increase in the eye size compared to (+/+; ey>wg-GFP) as seen in (A and C) bright field adult eye picture, and (B and D) third instar eyeantennal imaginal disc confocal image. (E, F) Misexpression of wg in the L 2 -mutant background (L 2 /+; ey>wg-GFP) increases the severity of small-eye phenotype of L 2 -mutant. (G and H) Misexpression of the newt gene and wg in the L 2 -mutant background (L 2 /+; ey>wg-GFP; vna4) exhibits a significant rescue of the reduced eye phenotype as seen in (E and G) bright field adult eye picture, and (F and H) third instar eye-antennal imaginal disc confocal image. The GFP reporter (green) marks ectopic misexpression of wg in the developing eye, Elav a proneural marker is shown in red. (I) The bar graph (I) represents the relative fold change in phenotype strength (eye size) compared between ey>wg-GFP and ey>wg-GFP; vna4, and between L 2 /+; ey>wg-GFP and L 2 /+; ey>wg-GFP; vna4. (J) Similarly bar graph (J) represents relative fold change in the ratio of Wg -GFP intensity per area of GFP expression between respective samples. Statistical analysis was performed using the student's t-test for independent samples. Statistical significance was determined with 95% confidence (p < 0.05). All bar graphs show values as the average between5 samples. Error bars show standard deviation (mean G SD), and symbols above the error bar signify as: ns is non-significant p value, ** signifies p value < 0.005, *** signifies p value < 0.0005 respectively. All the images are displayed in the same polarity as dorsal domain-towards top, and ventral domain-towards the bottom. Scale bar = 100 mm. See also Figure S12.
… 
Modulating positive and negative regulators of Wg signaling pathway affects the L mutant phenotype (A) Schematic presentation of the Wg signaling pathway showing various members of the canonical pathway. The positive regulators are in red, and negative regulators of the Wg pathway are in green. (B-E) Activating Wg signaling in the L 2 mutant eye background by misexpression of (B and C) arm alone (L 2 /+; ey>arm), (D and E) arm and vna4 (L 2 /+; ey>arm; vna4). Note that vna4 misexpression with arm (L 2 /+; ey>arm; vna4) in the eye rescues the L 2 /+; ey>arm reduced eye phenotype. (F-M) Blocking Wg signaling in L 2 mutant eye background by misexpression of (F and G) sgg alone (L 2 /+; ey>sgg), (J and K) dTCF DN alone (L 2 /+; ey> dTCF DN ) results in rescue of L 2 loss-of-ventral eye phenotype. Similarly, in L 2 mutant background misexpression of vna4 along with (H and I) sgg (L 2 /+; ey>sgg; vna4) and (L and M) dTCF DN (L 2 /+; ey> dTCF DN ; vna4) significantly rescue the partial loss-of-ventral eye phenotype. (N-Q) Blocking transport of Wg morphogen in L 2 mutant eye background by misexpression of (N and O) porc RNAi alone (L 2 /+; ey> porc RNAi ), (P and Q) porc RNAi and vna4 (L 2 /+; ey> porc RNAi ; vna4). Note that L 2 /+; ey> porc RNAi exhibits weak rescue of loss-of-ventral eye phenotype whereas vna4 misexpression with porc RNAi (L 2 /+; ey> porc RNAi ; vna4) enhances the phenotype strength. All the images are displayed in same polarity as dorsal domaintowards the top, and ventral domain-towards the bottom. Scale bar = 100 mm. See also Figures S13 and S14.
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iScience
Article
Newt regeneration genes regulate Wingless
signaling to restore patterning in Drosophila eye
Abijeet Singh
Mehta, Prajakta
Deshpande,
Anuradha
Venkatakrishnan
Chimata,
Panagiotis A.
Tsonis, Amit Singh
asingh1@udayton.edu
Highlights
Newt proteins regulate
wingless/Wnt pathway to
rescue eye mutant(s) in
Drosophila
These proteins non-cell-
autonomously rescue
missing tissue in
Drosophila eye
Promotes cell
proliferation and
downregulates cell death
in Drosophila eye
mutant(s)
These newt genes may
have significant bearing
on our understanding of
regeneration
Mehta et al., iScience 24,
103166
October 22, 2021 ª2021 The
Author(s).
https://doi.org/10.1016/
j.isci.2021.103166
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iScience
Article
Newt regeneration genes regulate Wingless signaling
to restore patterning in Drosophila eye
Abijeet Singh Mehta,
1
Prajakta Deshpande,
1
Anuradha Venkatakrishnan Chimata,
1
Panagiotis A. Tsonis,
1,6
and Amit Singh
1,2,3,4,5,7,
*
SUMMARY
Newts utilize their unique genes to restore missing parts by strategic regulation
of conserved signaling pathways. Lack of genetic tools poses challenges to deter-
mine the function of such genes. Therefore, we used the Drosophila eye model to
demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s),
viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L
2
mutant
and GMR-hid, GMR-GAL4 eye. L
2
exhibits the loss of ventral half of early eye and
head involution defective (hid) triggers cell-death during later eye development.
Surprisingly, newt genes significantly restore missing photoreceptor cells both in
L
2
and GMR>hid background by upregulating cell-proliferation and blocking cell-
death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway
and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts down-
stream of newt genes. Our data highlights that unique newt proteins can regulate
conserved pathways to trigger a robust restoration of missing photoreceptor
cells in Drosophila eye model with weak restoration capability.
INTRODUCTION
A urodele amphibian, newts belong to the Salamandridae family (Weisrock et al., 2006). The newts are the
only four-legged vertebrates that exhibit a remarkable capability to regenerate tissues like limbs, tail,
heart, lens, spinal cord, brain, and retina throughout its lifetime( Mehtaand Singh, 2019a;Sanchez Alvarado
and Tsonis, 2006). This ability of the newt to regrow organs is because of their ability to reprogram and
dedifferentiate the terminally differentiated cells to trigger regeneration response (Mehta and Singh,
2019a). Such an exceptional regeneration ability of newts has been attributed to unique gene(s) that
may have evolved from forming the regeneration tool box (Bryant et al., 2017;Casco-Robles et al., 2018;
Elewa et al., 2017;Evans et al., 2018;Keinath et al., 2015;Kumaretal.,2007;Matsunami et al., 2019;Mehta
and Singh, 2019a;Nowoshilow et al., 2018;Sanor et al., 2020;Smith et al., 2009,2019). Earlier, a newt gene
Prod1, which encodes a transmembrane receptor, was found to be critical for maintaining proximodistal
identity (pattern memory) during newt limb regeneration (da Silva et al., 2002;Echeverri and Tanaka,
2005;Kumar et al., 2007;Mehta and Singh, 2019a). Homologs of Prod1 are yet to be identified outside
of salamanders (Garza-Garcia et al., 2009). Similarly, the transcript of a newt gene newtic1 was found to
be significantly enriched in a subset of erythrocyte s which formed an aggregate structure called erythrocyte
clump. These newtic1 expressing erythrocytes promote limb regeneration (Casco-Robles et al., 2018). Sur-
prisingly, our understanding of the underlying molecular genetic mechanism(s) responsible for promoting
lifelong regeneration of missing structures and/or rescue of pattern defects in newts is far from complete.
Notophthalmus viridescens (Newts) have enormous genome size (c310
10
bases), a long reproductive
cycle, and have limited genetic tools that makes it difficult to use this model to determine the underlying
molecular mechanism(s) behind the function of its unique regeneration genes. Some strategies to deter-
mine their regeneration potential are to introduce these unique genes in genetically tractable model(s)
that lack regeneration potential exhibited by newts such as mammals, Drosophila etc. Since the genetic
machinery is conserved across the species, the Drosophila model has been successfully used for such cross
species studies to model human disease and determine their underlying molecular genetic mechanism(s)
(Hughes et al., 2012;Sarkar et al., 2016,2018;Singh and Irvine, 2012;Tare et al., 2011).
Previously, a family of five protein members was identified in a comprehensive transcriptomic analysis from
Notophthalmus viridescens (red-spotted newt). These proteins are expressed in adult tissues and are
1
Department of Biology,
University of Dayton, Dayton,
OH 45469, USA
2
Premedical Program,
University of Dayton, Dayton,
USA
3
Center for Tissue
Regeneration and
Engineering at Dayton
(TREND), University of
Dayton, Dayton, USA
4
The Integrative Science and
Engineering Center,
University of Dayton, Dayton,
OH 45469, USA
5
Center for Genomic
Advocacy (TCGA), Indiana
State University, Terre Haute,
IN, USA
6
Deceased
7
Lead contact
*Correspondence:
asingh1@udayton.edu
https://doi.org/10.1016/j.isci.
2021.103166
iScience 24, 103166, October 22, 2021 ª2021 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1
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modulated during lens regeneration (Looso et al., 2013). The three-dimensional structure models of these
newt proteins by ab initio methods suggested that these proteins could act as ion transporters and/or
involved in signaling (Mehta et al., 2019). These unique newt genes were introduced in genetically tractable
Drosophila melanogaster (fruit fly) model by transgenic approaches to test their regeneration potential.
Such transgenic approaches were not feasible in N.viridescens, therefore, Drosophila served as a suitable
system. The transcriptomic analysis was performed in the transgenic Drosophila that are expressing newt
genes. Based on this study, graded expression of 2775 transcripts was reported in transgenic flies. In each
transgenic fly one of the five newly identified newt genes was ectopically expressed using the GAL4/UAS
binary target system (Brand and Perrimon, 1993;Mehta et al., 2019). Among 2775 transcripts, genes
involved in the fundamental developmental processes like cell cycle, apoptosis, and immune response
were highly enriched suggesting that these foreign genes from newt were able to modulate the expression
of Drosophila gene(s) (Mehta et al., 2019;Mehta and Singh, 2019b).
Drosophila, a hemimetabolous insect, serves as a versatile model organism to study development and
disease because of presence of fully sequenced and less redundant genome, presence of homologs or
orthologs for human disease related genes, a shorter life cycle and economy to maintain fly cultures
(Bier, 2005;Lenz et al., 2013;Singh and Irvine, 2012). Drosophila eye has been extensively used to under-
stand the fundamental process of development and model neurodegenerative disorders (Gogia et al.,
2020a;Kumar, 2018;Sarkar et al., 2016,2018;Singh and Irvine, 2012;Tare et al., 2011;Yeates et al.,
2019). The rich repository of genetic tools available in fly models, and the fact that eye is dispensable for
survival, makes Drosophila eye suitable for screening genes from other animals that can promote growth
and regeneration. The compound eye of the adult fly develops from a monolayer epithelium called the eye-
antennal imaginal disc, which develops from an embryonic ectoderm (Cohen, 1993;Held, 2002). The imag-
inal disc, a sac-like structure present inside the larva, comprise of two different layers: the peripodial mem-
brane (PM) and the disc proper (DP) (Kumar, 2020b). The DP develops into retina where as the PM of the eye-
antennal imaginal disc contributes to the adult head structures (Haynie and Bryant, 1986;Kango-Singh
et al., 2003;Kumar, 2020b;Milner et al., 1983). In third instar larval eye imaginal disc, a wave of synchronous
retinal differentiation moves anteriorly from the posterior margin of the eye disc and is referred to as
Morphogenetic Furrow (MF) (Kumar, 2013,2020a;Ready et al., 1976). The progression of MF transforms
the undifferentiated cells into differentiated photoreceptor cells. The adult compound eye comprises of
800 unit eyes or ommatidia (Kumar, 2013,2018,2020a;Ready et al., 1976). The ommatidial cells in the com-
pound eye are grouped together into two chiral forms, which are arranged in mirror image symmetry along
the DorsoVentral (DV) midline called the equator. Ventral domain is the default state of the entire early eye
imaginal primordium (Singh and Choi, 2003), and onset of the expression of the dorsal selector gene
pannier (pnr), establishes the dorsoventral (DV) lineage in the eye (Maurel-Zaffran and Treisman, 2000;
Singh and Choi, 2003;Tare et al., 2013). It has been reported that Lobe (L) is a cell survival gene required
for ventral eye development and growth, and acts upstream to the Wg signaling pathway (Singh et al.,
2006). During eye development Lpromotes cell survival by preventing ectopic induction of Wingless
(Wg) and JNK-Signaling pathway in the ventral eye or in the entire early eye disc before the establishment
of DV compartments (Singh et al., 2006,2012a). L
2
mutant exhibits a dominant, consistent phenotype of
preferential loss of the ventral eye pattern from early larval eye imaginal disc to the adult eye (Singh
et al., 2005).Linhibits the JNK signaling by downregulating Wg signaling and thereby prevent caspase-
dependent and caspase-independent cell death (Singh et al., 2006). Thus, Lplays an important role in
cell survival during eye development.
The wg gene, a Drosophila homolog of Wnt, serves as a ligand, and encodes a secreted signaling protein
that acts as a morphogen (Baker, 1987;Bejsovec, 2013;Swarup and Verheyen, 2012). The transcriptional
effector of the Wg/b-catenin signaling is Drosophila T cell Factor (dTCF), which upon activation by
Armadillo (Arm, human homolog b-catenin) leads to the transcription of Wg target genes. In the absence
of ligand Wg, a destruction complex composed of Adenomatous polyposis coli (Apc), and Axin degrades
Arm and thus prevents it to form a complex with dTCF. The pathway is activated by binding of Wg ligand to
its co-receptors Frizzled (Fz) and Arrow (Arr). Upon activation, Fz binds to Disheveled (Dsh) and Arr,
which binds to Axin and thus inactivating the destruction complex. This results in the translocation of
Arm (b-catenin) from the cytosol to the nucleus where Arm binds to dTCF and activates transcription
of Wg target genes (Bejsovec, 2006). During eye development, Wg plays different roles along the
spatiotemporal axis. During early eye development, Wg is required for growth. In the eye-antennal disc,
Wg acts as a negative regulator of eye development and inhibits morphogenetic furrow progression
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Figure 1. Newt regeneration genes rescues L
2
mutant’s loss-of-ventral eye phenotype
(A and B) Schematic representation of generating transgenic Drosophila carrying these newt (Notophthalmus viridescens)
candidate genes (vna1,vna2,vna3,vna4,andvna5).
(C) Bright field image of a (C) wild type adult eye (ey-GAL4),
(D–H) adult eye phenotype when ey-GAL4 drives expression of newt transgenes (D) UAS-vna1 (ey>vna1), (E) UAS-vna2
(ey>vna2), (F) UAS-vna3 (ey>vna3), (G) UAS-vna4 (ey>vna4), and (H) UAS-vna5 (ey>vna5).
(I) The bar graph shows there is no significant (ns) change in eye size between control and where ey-GAL4 drives
expression of newt transgenes (ey>vna1-vna5).
(J)Brightfieldimageofa(J)L
2
mutant (L
2
;ey-GAL4) adult eye where there is preferential loss-of- ventral eye phenotype,
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(Ma and Moses, 1995;Treisman and Rubin, 1995). In the early third instar eye-antennal imaginal disc, wg
expresses on the ventral margin. Ectopic expression of Wg triggers cell death of photoreceptor cells
causing eye defects (Baker, 1987;Cavodeassi et al., 1999;Singh, 2012;Singh et al., 2006). During pupal
development, Wg is involved in programmed cell death to eliminate extra cells (Cordero et al., 2004;Lin
et al., 2004;Swarup and Verheyen, 2012). Wg is also part of the dorsal eye gene hierarchy (Gogia et al.,
2020a;Tare et al., 2013). . Previously it has been shown that Wg pathway was upregulated in the developing
eye of L
2
background. This perturbation in Wg pathway causes loss of ventral eye in 100% of flies of L
2
/+
background (Gogia et al., 2020a;Singh et al., 2006,2011). Therefore, timing and level of wg expression
in this region is crucial for formation of a normally patterned eye.
Here, we demonstrate the potential of five newly identified newt genes in significantly rescuing the missing
photoreceptor cells in the ventral half of the eye of Drosophila L
2
mutant eye. In addition, these genes can
also rescue the reduced eye phenotype of GMR-hid, GMR-GAL4 flies, where activation of head involution
defective (hid) triggers cell death.These two eye defect phenotypes represent the two different devel-
opmental time points. Our data suggests that these genes are effective in rescuing eye defects all along
the developmental stages viz.,early(L
2
mutant) as well as later (GMR-hid,GMR-GAL4) time points of eye
development. Ectopic expression of these genes restores eye mutant phenotypes by promoting cell
proliferation, and downregulating cell death. Furthermore, these rescues by newt genes are non-cell-
autonomous in nature. Finally, the misexpression of these newt genes in Drosophila downregulates Wg
in L
2
mutant background, resulting in significant rescue of missing photoreceptor cells in the developing
Drosophila eye.
RESULTS
We employed Drosophila eye model to test the regeneration po tential of the five unique newt genes, which
have been named as: viropana1 (vna1), viropana2 (vna2), viropana3 (vna3), viropana4 (vna4), and viropana5
(vna5)(Mehta et al., 2021). All these genes consist of open reading frame (ORF), and additional 50and -30
untranslated regions (UTR) (Figure S1)(Looso et al., 2013). They all share common signal peptides, which
indicate that these proteins could be secreted (Petersen et al., 2011). Further, they might belong to the
same family, which is defined by the common motif (L-x(1,3)-C-L-x(2)-[AL]-L-x(3)-[AL]-[AET]-x(2)-[LV]-x-
[AS]-[ILV]-x-[DQ]-[LV]-[LV]-C-[AC]-[FIV]-x(3)-[DN]-[EP]-[AIV]-[EK]-x-K-[EN]-x-L) (Figure S1A). All five family
members showed high similarity to the proteins from the other newt species- pleurodeles waltl)(Fig-
ure S1B). Previously, it has been reported that these five genes are highly expressed in the tail of the
newt (Notophthalmus viridescens)(Looso et al., 2013). Therefore, in our study we isolated the total RNA
from the newt tail, generated cDNA to amplify these newt genes, and used this cDNA to generate trans-
genic flies containing these five genes (Figure 1A). We employed GAL4/UAS system (Brand and Perrimon,
1993) to allow targeted misexpression of these newt transgenes in the fly tissues (Figure 1B). To confirm if
targeted misexpression approach can translate newt proteins in Drosophila (Brand and Perrimon, 1993), we
tested localization of the fusion protein. As there are no antibodies available against these newt proteins,
we therefore used V5 epitope tagged transgenes to generate another set of transgenic flies (Wang et al.,
2019). The V5 tag sequence of about 42 bp long is fused toward the 30end of the 501 bp long ORF. We
tested all these transgenes using the antibody against V5 tag. Here we present the expression of one of
the transgenes viz.,vna4 using V5 tag (Figure S2). To detect if misexpression of these newt genes in
Drosophila can generate any developmental defects, we used tubulin –GAL4(tub –GAL4) to ubiquitously
misexpress newt genes in flies in the wild-type background (Figure 1B) and found no defects.
Figure 1. Continued
(K–O) L
2
mutant (L
2
;ey-GAL4) background where newt transgene (K) UAS-vna1 (L
2
;ey >vna1), (L) UAS-vna2 (L
2
;ey >vna2),
(M) UAS-vna3 (L
2
;ey >vna3), (N) UAS-vna4 (L
2
;ey>vna4), and (O) UAS-vna5 (L
2
;ey>vna5) are expressed in the developing
eye using ey-GAL4.
(P) L
2
mutant adult eye is half the adult eye size of the ey-GAL4 which serves as a control. The bar graph clearly displays the
significant increase in the adult eye size of L
2
-mutant background where unique newt genes were misexpressed
compared to the adult eye size of the L
2
-mutant only (L
2
;ey-GAL4).Eyesizewasmeasuredusing ImageJ software tools
(http://rsb.info.nih.gov/ij/). Sample size used for statistical analysis was five (n = 5). Statistical analysis was performed
using the student’s t test for independent samples. Statistical significance was determined with 95% confidence (p < 0.05).
Error bars show standard deviation (mean GSD), and symbols above the error bar signify as: ns is non-significant p value,
* signifies p value < 0.05, ** signifies p value < 0.005, *** signifies p value < 0.0005, respectively. In this study ey is used as a
driver to misexpress newt genes in the developing Drosophila eye. All the images are displayed in the same polarity as
dorsal domain-towards top and ventral domain-towards bottom. Scale bar = 100 mm. See also Figures S1, S2, S3, and S4.
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Figure 2. Misexpression of newt genes exhibits non-cell-autonomous rescue
(A, B, B0,andB
00) Misexpression of vna4 in the dorsal half using DE-GAL4 driver (A) Adult eye and (B, B0,B
00) developing
third instar larval eye imaginal disc Note that Green fluorescence protein (GFP) (green) reporter marks the DE-GAL4 driver
domain and proneural marker Embryonic lethal abnormal visual system (Elav) (red) marks the neuronal fate. (B0)isasingle
channel confocal image showing GFP expression, and (B00) is a single channel confocal image showing Elav expression.
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Newt genes can rescue Drosophila eye mutants
We used the Drosophila eye model to evaluate the function of unique newt genes as the eye is not required
for the viability of the fly and it is easy to score the phenotypes in eye. We used ey-GAL4 driver (Hazelett
et al., 1998) to misexpress these newt genes in the developing eye imaginal disc, which develops into an
adult eye. The phenotypes of UAS-transgene alone wer e tested to rule out any contribution(s) in phenotype
due to insertion of the transgene. In comparison to the control, ey-GAL4 (Figure 1C), misexpression of
these transgenes ey-GAL4>UAS-vna1 (ey>vna1,Figure 1D), ey-GAL4>UAS-vna2 (ey>vna2,Figure 1E),
ey-GAL4>UAS-vna3 (ey>vna3,Figure 1F), ey-GAL4>UAS-vna4 (ey>vna4,Figure 1Gandey-GAL4>UAS-
vna5 (ey>vna5,Figure 1H), did not affect the eye size (Figure 1I). To investigate the regeneration potential
of these genes, we used eye mutant(s) such as L
2
mutant, which exhibits selective loss of ventral half of the
eye (Figure 1J). L
2
mutant exhibits loss-of-ventral-eye in 100% flies. Interestingly, targeted misexpression of
the newt gene(s) in L
2
;ey-GAL4 background [(L
2
;ey>vna1;Figure 1K), (L
2
;ey>vna2;Figure 1L), (L
2
;ey>vna3;
Figure 1M), (L
2
;ey>vna4;Figure 1N), (L
2
;ey>vna5;Figure 1O)] exhibit significant rescue of the loss-of-
ventral eye phenotype (Figure 1P). For each cross, three independent cultures were established and two
hundred flies for each set (triplicate, 600 flies) were c ounted to determine phenotype frequency. The rescue
frequency was about 40.83% in vna1, 37.7% in vna2, 49% in vna3, 58.2% in vna4, and 38.7% in vna5(Fig-
ure S3). Note that the strongest rescues (phenotype strength as well as rescue frequency %) were observed
with vna4 transgene (Figures 1PandS3;Table S1).
Because the L
2
eye mutant represents developmental defects in the genetic machinery involved in early eye
development, it raises a question whether the regeneration potential of these newt genes is restricted to
the early time window or even later stages of eye development. The GMR- GAL4 driver was used to drive
expression of transgenes in the differentiating retinal neurons of the larval eye imaginal disc, which con-
tinues all along to the adult eye (Moses and Rubin, 1991). Gain-of-function of head involution defective
(hid), triggers cell death (Bergmann et al., 1998;Grether et al., 1995;Hay et al., 1995;White et al., 1994).
Misexpression of hid using GMR-GAL4 (GMR>hid) results in a ‘‘No-eye’’ or highly reduced eye phenotype
(Figure S4B). However, misexpression of newt transgenes along with hid in the eye (GMR-GAL4>hid+vna4)
results in a significant rescue of the ‘‘No-eye’’ phenotype (Figure S4C). Thus, confirming that the restoration
potential of newt genes is not restricted to the early time window but can even extend to the later stages of
eye development.
Ectopic expression of newt genes exhibits non-cell-autonomous rescue
All 5 unique newt genes have signal peptides (Looso et al., 2013), so it is possible that these genes may have
non-cell-autonomous effects. To determine any such possibility, we misexpressed these genes within a
subset of retinal neuron population in the developing eye field of L
2
mutant and assay their regeneration
effect. Misexpression of vna4 using dorsal-eye - GAL4 (DE - GAL4) (Figure 2) directs its expression in the
dorsal half of the developing eye (Morrison and Halder, 2010)(Figures 2Aand2E).The rationale of this
experiment was to misexpress vna4 in the dorsal half of the eye and test its effect on an eye mutant, which
results in loss of the ventral half of the developing eye. Misexpression of both GFP (reporter) and newt vna4
in the dorsal domain of the developing eye (+/+; DE>GFP+vna4)(Figures 2A, 2B, 2B0,and2B
00), exhibits a
near wild-type eye.As a negative control, we tested GFP expression alone in L
2
-mutant background (L
2
/+;
DE>GFP)(Figures 2C, 2D, 2D0,and2D
00).We found that GFP expression was restricted to the dorsal half as
Figure 2. Continued
(C,D,D
0,andD
00)L
2
mutant eye where there is no misexpression of newt genes (L
2
/+; DE>GFP) (as negative control). (C)
Adult eye bright field image and (D, D0,D
00) third instar eye disc of L
2
mutant (L
2
/+; DE>GFP) exhibiting loss-of-ventral eye
phenotype and red dotted line marks the boundary of eye. GFP reporter (green) marks domain of DE-GAL4 expression
domain in L
2
mutant background.
(E, F, F0,andF
00) Misexpression of vna4 in dorsal eye domain of L
2
mutant (L
2
/+; DE>GFP; vna4) exhibits non-cell
autonomous rescue of loss-of-ventral eye phenotype in (E) Adult eye (white dotted boundary) and (F, F0,F
00)thirdinstar
eye disc. White dotted boundary marks the significantly rescued loss-of-ventral eye phenotype.
(G–J) Genetic mosaic ‘‘Flp out’’ somatic clones of (G, G0,andG
00) newt gene vna4 in the developing eye disc wild-type, (H,
H0,andH
00)GFPinL
2
background (L
2
/+; hsflp>GFP, served as the negative control) and (I, I0,I00,J,J
0,andJ
00) newt gene
vna4 clones in L
2
mutant (L
2
/+; hsflp>GFP;vna4) background. All clones are marked by GFP reporter (green). Note that
misexpressing vna4 rescue missing photoreceptor cells in a non-cell-autonomous manner, as photoreceptor cells rescue
marked by Elav (in red) extends outside the boundary of the clone marked by GFP (in green). (J, J0,andJ
00) exhibits the
magnified view of the vna4 misexpressing clone (green) in L
2
/+; hsflp>GFP; vna4 background. All the images are
displayed in the same polarity as dorsal domain-towards top, and ventral domain-towards bottom. Scale bar = 100mm,
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seen in the wild-type control (Figures 2B, 2B0,and2B
00). Furthermore, we did not see any rescue of L
2
mutant
phenotype of ventral eye loss (marked by red d otted boundary, Figures 2D, 2D0,and2D
00). Misexpression of
both GFP+vna4 inthedorsalhalfoftheL
2
mutant eye (L
2
/+; DE>GFP+vna4) exhibits a significant rescue of
the loss-of-ventral eye phenotype as seen in the adult eye, and the eye imaginal disc (marked by white-
dotted boundary, Figures 2E, 2F, 2F0,and2F
00). Even though the targeted misexpression of vna4is
restricted to the dorsal half of the developing eye, it was able to rescue the loss-of-ventral eye phenotype
suggesting that newt genes can show non-cell-autonomous rescue of Drosophila eye mutants.
To rule out any domain specific restraint, or contribution of a DE-GAL4 background, we employed a ge-
netic mosaic approach. We generated gain-of-function clones using Flp out approach (Xu and Rubin,
1993), which result in clones of cells that misexpress high levels of the vna4 transgene and these clones
are marked by the GFP reporter (Figures 2G, 2H, 2I, and 2J). We found that the misexpression of these
vna transgenes rescues the loss-of-ventral eye phenotypes; however, these rescues extend beyond the
boundary of the clone. This data validates our prior observation that these newt genes exhibit non-cell-
autonomous rescue due to their secretory nature. Next, we wanted to investigate the mechanism by which
these newt genes can rescue loss-of-ventral eye phenotype.
Newt genes promote cell proliferation and downregulate cell death
To test the possibility of upregulation of cell proliferation resulting in the rescue of themissing tissue in the
L
2
mutant background, we compared number of dividing cells in the zone of interest between the L
2
mutant, and the L
2
mutant background where the newt gene was misexpressed (Figures 3A–3F). The prolif-
erating cells are marked by anti-phospho-histone H3 (PH3) marker (Figures 3A–3E, 3A0–3E0,S5A–S5H, and
S5A0–S5H0).Phosphorylation of a highly conserved Serine residue (Ser-10) in the histone H3 tail is consid-
ered a crucial event for the onset of mitosis and represents completion of cell cycle (Crosio et al., 2002;Kim
et al., 2017). Our results show robust increase in the number of dividing cells when vna4 is misexpressed in
the L
2
-mutant background (L
2
;ey >vna4) (shown in white dotted boundary Figure 3B) with respect to the
L
2
-mutant (L
2
;ey-GAL4) only (Figure 3F). Similar trend in the cell proliferation rate were observed with other
vna transgenes (Figures S5A–S5I). The L
2
mutant background, which served as control, did not show any
rescue of the loss-of-ventral eye phenotype due to lack of cell proliferation as evident from PH3 staining
(shown in red dotted boundary, Figure 3A). In addition, newt gene when misexpressed in the wild-type
background (ey > vna4)(Figures 3D, 3F, S5G, and S5I) did not show significant increase in cell proliferation
compared to its control (ey-GAL4) as well as wild-type fly (Canton-S) (Figures 3C, 3E, 3F, S5F, S5H, and
S5I).We calculated dividing cells in GMR-hid, GMR-GAL4 alone background, and as expected found upre-
gulation in cell proliferation when newt gene is misexpressed along with GMR-hid,GMR-GAL4(GMR-hid,
GMR-GAL4>vna4)(Figures S6B and S6C) compared to the GMR-hid, GMR-GAL4 background in which newt
gene has not been misexpressed (Figures S6A and S6C). These results clearly show that the newt gene(s)
caninducecellproliferationtorestorethemissingstructuresinL
2
-mutant and GMR-hid,GMR-GAL4eye
disc. These missing structures are t photoreceptor cells in our study .
During newt lens regeneration, both cell proliferation and apoptosis are observed (Tsonis et al., 2004). It has
been shown that the loss-of-ventral-eye phenotype observed in L
2
mutant is because of induction of both cas-
pase-dependent and caspase-independent cell death (Singh et al., 2006). Therefore, we did TUNEL (Terminal
deoxynucleotidyl transferase dUTP nick end labeling) assay to test if newt genes can block or downregulate
cell death to rescue L
2
mutant phenotype (Figures 3G–3L and S5J–S5R).TUNEL assay is based on the detec-
tion of fragmented DNA ends, which are characteristic of apoptotic cells (Sarkissian et al., 2014;White et al.,
2001). The number of dying cells are compared between the L
2
mutant, and the L
2
mutant background where
the newt gene was misexpressed. We found a significant reduction in the number of dying cells when newt
gene (vna4) is misexpressed in the L
2
mutant background (L
2
;ey >vna4)(Figures 3H, 3H0, and 3L) in compar-
ison to the L
2
-mutant alone (L
2
;ey-GAL4) (Figures 3G, 3G0, and 3L). Similarly,
ey-GAL4 (Figures 3I, 3I0, and 3L) exhibits signifiant downregulation in the rate of cell death with respect to
the L
2
mutant (L
2
;ey-GAL4) (Figures 3G, 3G0, and 3L). The value is significantly lower in comparison to the
L
2
mutant (L
2
;ey-GAL4), and almost equivalent to the L
2
mutant where the newt gene is misexpressed (L
2
;
ey >vna4) (Figure 3L). This suggests that newt gene(s) upon misexpression can rescue the L
2
mutant’s loss-
of-ventral eye phenotype by preventing excessive cell death of photoreceptor cells (marked in white dotted
boundary, Figures 3H and 3H0). We found a similar trend of reduction in cell death when other members of this
newt gene family were misexpressed (Figures S5J–S5R). However, when newt gene is misexpressed in the wild
type background (ey>vna4)(Figures 3J, 3L, S5P, and S5R) no significant change in cell death was observed in
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comparison to its control (ey-GAL4) as well as wild type fly (CantonS) (Figures 3I, 3K, 3L, S5O, S5Q, and S5R).
TUNEL assay was also performed in GMR-hid, GMR-GAL4 eye imaginal discs, and dying cells nuclei were
quantified. Our results report that misexpressing newt genes in GMR-hid, GMR-GAL4 (GMR-hid, GMR-
GAL4>vna4) robustly downregulates cell death (Figures S6E and S6F) in comparison to GMR-hid, GMR-
GAL4 strain alone (Figures S6D and S6F). We also tested the role of cell death using Dcp1 (Death caspase-
1) staining (Figure S7). Antibody against cleaved Dcp1 marks the dying cells in Drosophila (Sarkissian et al.,
2014). Dcp1 staining exhibits reduction in the number of dying cells when newt genes are misexpressed in
L
2
mutant background (Figures S7C–S7G), in comparison to the L
2
mutant alone (Figure S7B). These results
suggest that newt genes upregulate cell proliferation and downregulate cell death to significantly rescue
the L
2
mutant loss-of-ventral eye phenotype, and eye loss caused by misexpression of hid.
Figure 3. Newt genes promote cell proliferation and downregulate cell death to promote rescue of L
2
mutant phenotype of loss-of-ventral eye
(A–E) PH-3 staining (blue), as a marker to calculate cell proliferation in the developing eye imaginal disc of (A) L
2
mutant (red dotted boundary marking eye
boundary), (B) L
2
mutant background where vna4 gene is misexpressed (L
2
;ey>vna4), (C) ey-GAL4 driver, (D) misexpression of newt gene in ey-GAL4 domain
(ey>vna4) (E) and wild type (CantonS). (A) Red dotted boundary represents the loss-of-ventral-eye area, and (B) white dotted boundary represents the rescue
of loss-of-ventral eye phenotype where vna4 gene is misexpressed. (A0–E0) single channel confocal images demonstrating PH-3 stained nuclei. The region of
interest used to count the number of dividing cells is marked in yellow dotted boundaries.
(F) Bar graph shows comparative increase in the rate of cell division. There is no significant change in cell proliferation rate among the (A and A0)L
2
;ey-GAL4,
(C and C0)ey-GAL4, (D and D0)ey>vna4,(EandE
0) Canton-S (wild-type) eye disc. (A, A0,B,B
0, and F) However, there is robust increase in the cell proliferation
rate in (B and B0)L
2
;ey>vna4 background in comparison to (A and A0)L
2
mutant (L
2
;ey-GAL4).
(G–K) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining serves as a marker for cell death assay. TUNEL staining
(shown in green) marks the dying cells nuclei in (G and G0)L
2
;ey-GAL4, (H and H0)L
2
;ey>vna4,(IandI
0)ey-GAL4, (J and J0)ey>vna4,(K)andwildtype
(Canton-S) eye imaginal disc. (G) Red dotted boundary represents the loss-of-ventral eye region, and (H) white dotted boundary represents the rescued loss-
of-ventral eye region. (G0–K0) The TUNEL positive cells are counted within the zone of interest marked within the yellow dotted boundary as shown in single
filter confocal images.
(L) Bar graph shows decrease in the cell death when a newt gene is misexpressed in the L
2
-mutant background compared to only L
2
-mutant. The
downregulation in cell death is comparable to the cell death occurring in the normal wild- type background. Statistical analysis was performed using the
student’s t-test for independent samples. Sample size used for the calculation was five in number (n = 5). Statistical significance was determined with 95%
confidence (p < 0.05). All bar graphs show change in cell proliferation and cell death as average between 5 samples. Error bars show standard deviation
(mean GSD), and symbols above the error bar * signify p value < 0.05, ** signify p value < 0.005, *** signify p value < 0.0005, and ns signify p value >0.05
respectively. All the images are displayed in the same polarity as dorsa domain-towards top, and ventral domain-towards bottom. Scale bar = 100 mm. See
also Figures S5, S6, S7, S8, and S9.
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We further validated the role of cell death by blocking caspase-dependent cell death using baculovirus p35
misexpression (Hay et al., 1994). Even though misexpression of p35 in L
2
mutant background (L
2
/+; ey >
p35) exhibits rescue of loss-of-ventral eye phenotype, the phenotype strength (relative eye size ratio of
1.42 G0.29 and rescue frequency of 22.7%) of L
2
/+; ey > p35 was significantly weaker than phenotype
strength (relative eye size ratio = 2.34 G0.51 and rescue frequency of 58.2%) of L
2
/+; ey > vna4 (Figures
S8C, S8D, S8F, and S8G). This data suggests that the newt genes can upregulate cell proliferation and
also block cell death, but cell proliferation may be playing significant role in rescuing the missing tissues
in the L
2
mutant eye. To validate this hypothesis, we compared rate of cell proliferation and cell death be-
tween L
2
mutant (L
2
;ey-GAL4), L2/+; ey > p35,andL
2
;ey >vna4 (Figure S9). As expected, there was a sig-
nificant downregulation in cell death (Figures S9A, S9A0,S9B,S9B
0, and S9D), and non-significant change in
cell proliferation (Figures S9E, S9E0, S9F, S9F0, and S9H) between L
2
;ey-GAL4 and L
2
/+; ey > p35. However,
an opposite trend is observed when comparing L
2
/+; ey > p35 and L
2
;ey >vna4. There is a non-significant
change in cell death (Figures S9B, S9B0,S9C,S9C
0, and S9D), but highly significant change in cell prolifer-
ation (Figures S9F, S9F0, S9G, S9G0, and S9H). Thus, it is clearly demonstrated that newt genes favor cell
proliferation more than cell death in order to restore the missing structures. Therefore, it becomes war-
ranted to understand what signaling pathway is triggered by these newt genes to promote the rescue.
Newt genes downregulate wg expression in developing eye
We screened for the signaling pathway(s) that are involved in the rescue of L
2
mutant loss-of-ventral eye
phenotype. We identified members of evolutionarily conserved Wg/Wnt signaling pathway in our high
throughput screen (RNA sequencing) where newt genes were misexpressed in the fly tissue (Figure S10).
It is reported that L
2
mutant phenotype can be rescued by downregulating wg (Singh et al., 2006). To
determine the involvement of wg in promoting rescue of L
2
mutant phenotype by unique newt genes,
we examined expression levels of Wg in larval eye-antennal imaginal disc (Figures 4A–4C, 4A0–4C0,and
4D). Wg expression is seen at the antero-lateral margins of the developing eye imaginal disc (Baker,
1987;Cavodeassi et al., 1999;Singh et al., 2006). In L
2
mutant, robust ectopic expression of Wg is seen
at the ventral eye margins (Figures 4Band4B
0)(Singh et al., 2006,2011). Misexpression of newt gene
(vna4)inL
2
mutant background downregulates the Wg expression at the ventral margin of eye-antennal
imaginal disc (Figures 4Cand4C
0), as evident from the signal intensity plots (integrative density) in various
backgrounds (Figure 4D). The semi-quantitative Western blot, showed that Wg levels increased 3 folds in L
2
mutant as compared to ey-GAL4 control whereas misexpressing vna4 in the L
2
mutant background
decreased the Wg expression by 1.6-fold as compared to L
2
mutant (L
2
;ey-GAL4) (Figures 4Eand4F).
Similar trends were seen with other newt genes (vna1, vna2, vna3, and vna5) when misexpressed in L
2
mutant background (Figures S11C–S11F, S11C0–S11F0, and S11G). Thus, the newt genes can downregulate
wg expression in Drosophila L
2
mutant to partially rescue ventral eye-loss-phenotype.
To further test our hypothesis, we misexpressed wg (the target identified in our screen) along with vna4
transgene in the L
2
mutant background. As control, we misexpressed wg in the developing eye (ey>wg),
which result in reduced eye phenotype (Figures 5Aand5B)(Lee and Treisman, 2001;Singh et al.,
2012a). Misexpressing vna4 in the wild-type background where wg is ectopically induced (+/+; ey>wg+
vna4), rescues the reduced eye phenotype (Figures 5C and 5D) at a frequency of 16.8% (Figure S12;Table
S1). Misexpression of wg in the L
2
/+ mutants eye background (L
2
/+; ey>wg) completely eliminate the eye
field as seen in the eye imaginal disc and the adult eye (Figures 5E and 5F) whereas, misexpressing vna4
along with wg in the L
2
/+ mutant eye disc (L
2
/+; ey>wg;vna4) partially rescues the loss-of-ventral eye
phenotype (Figures 5G and 5H) in 21% flies (Figure S12;Table S1). The phenotype strength shows the sig-
nificant rescue of the eye loss phenotype in both wild-type (+/+; ey>wg+vna4;Figures 5C and 5D) as well as
L
2
(L
2
/+; ey>wg;vna4;Figures 5G and 5H) background(s) where wg was ectopically co-expressed (Figure 5I).
The changes in the ectopically overexpressed wg-GFP levels were non-significant, suggesting that
newt genes can regulate endogenous Wg levels to promote rescue (Figure 4) and not the ectopically
overexpressed Wg levels (Figure 5J).
Newt genes negatively regulate Wg/Wnt signaling
To investigate if newt genes regulate expression of wg alone or they regulate the Wg signaling pa thway, we
tested the effector and inhibitor of the Wg-signaling pathway. The rationale was to modulate the levels of
Wg signaling and sample its effect on the L
2
mutant phenotype as well as the L
2
mutant background where
vna4 is misexpressed (L
2
/+; ey> vna4)(Figure 6). In creasing the levels of Arm (a vertebrate homolog of b-Cat-
enin, an effector of Wg signaling) in the wild-type eye (ey>arm)resultsinsmalleyes(Figures S13A and S13B)
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and in the L
2
/+ background, (L
2
/+; ey>arm) it results in elimination of the entire eye field (Figures 6Band6C).
Misexpressing vna4 along with arm in the L
2
/+ mutant background (L
2
/+; ey>arm;vna4) partially rescues the
loss-of-ventral eye phenotype (Figures 6 D and 6E) and the rescue frequency is 22.8% (Figure S14A; Table S1).
Similarly, misexpressing vna4 and arm in the wild-type background (+/+; ey>arm;vna4) partially rescues the
small eye phenotype defect caused by ey>arm phenotype (Figures S13C and S13D), and the rescue fre-
quency is 21% (Figure S14A; Table S1). This data suggests that newt gene can modulate the Wg signaling
pathway components to promote rescue of reduced eye phenotype both in L
2
-mutantaswellasthewild-
type background(s). However, the rescue frequency obtained by misexpressing newt gene in addition to
ectopically misexpressing arm (L
2
/+; ey>arm;vna4, 22.8%) and/or wg (L
2
/+; ey>wg;vna4, 21%) (Figures
S12 and S14A;Table S1) is lower than the rescue frequency obtained when only newt gene is misexpressed
in the L
2
-mutant background (L
2
/+; ey>arm;vna4, 58.2%). This could be because of severity of loss-of-ventral
eye phenotype caused by the combined effect of L
2
mutation and gain-of-function of wg,orarm.
Blocking Wg signaling by misexpressing shaggy (sgg), an inhibitor of Wg signaling, in the eye (ey>sgg,Fig-
ures S13E and S13F) (Hazelett et al., 1998;Singh et al., 2002,2006) or along with vna4 in the wild-type back-
ground (+/+; ey>sgg;vna4,Figures S13G and S13H) does not affect the eye size. Misexpression of sgg in
Figure 4. Newt genes downregulate Wg expression to rescue loss-of-ventral eye phenotype.
(A–D) Immunohistochemistry showing Wg modulation. (A–C) Eye disc showing Wg staining in green, and Elav in red.
(A0–C0) Single channel confocal images showing Wg expression. The green window is the area of interest that is utilized to
calculate (D and D0) integrative density (intensity plot). The bar graph represents the Wg intensity at the ventral margin
(within the green box) in (A and A0)wildtypefly(ey-GAL4), (B and B0)L
2
-mutant fly, (C and C0) Misexpression of newt gene
(vna4)intheL
2
mutation background. Note that vna4 misexpression downregulates Wg expression.
(E and F) Western blot (WB) to semiquantitative modulation of Wg expression levels. (E) The blot shows the expression of
Wg protein in the Drosophila eye. The samples were loaded in the following sequence: Lane 1- ey-GAL4, Lane 2- L
2
;
ey-GAL4, Lane 3- L
2
;ey>vna4. Alpha tubulin is used as a loading control. Molecular weight of Wg is 46kD, and alpha
tubulin is 55 kD. The samples were treated with anti-Wg antibody, and anti-atubulin antibody. (F) Bar graph representing
as a relative Wg level, is a measure of signal intensity of the bands, which clearly demonstrates that newt genes when
misexpressed in the L
2
-mutant background downregulates Wg levels. Statistical analysis was performed using the
student’s t-test for independent samples. Statistical significance was determined with 95% confidence (p < 0.05). All bar
graphs show integrative density as a scale to measure Wg intensity for each sample represented as the average between
5. Error bars show standard deviation (mean GSD), and symbols above the error bar * signify p value < 0.05, and ** signify
p value < 0.005 respectively. All the images are displayed in the same polarity as dorsal domain-towards top, and ventral
domain-towards bottom. Scale bar = 100 mm. See also Figures S10 and S11.
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the L
2
/+ mutants (L
2
/+; ey>sgg), significantly rescues the loss-of-ventral eye phenotype in 27.3% of flies
(Figures 6F, 6G, and S14B; Table S1)(Singh et al., 2006). Similarly, misexpressing vna4 and sgg in the
L
2
/+ mutant eye (L
2
/+; ey>sgg;vna4) rescues the loss-of-ventral eye phenotype (Figures 6Hand6I)and
rescue frequency increased dramatically from 27.3% to 71.5% (Figure S14B; Table S1). The transcription fac-
tor dTCF is the downstream target of Wg signaling, and is downregulated by misexpression of the N-ter-
minal deleted dominant-negative form of TCF (dTCF
DN
)(van de Wetering et al., 1997). Misexpression of
dTCF
DN
aloneintheeye(ey>dTCF
DN
,Figures S13I and S13J) or dTCF
DN
with vna4 (+/+; ey> dTCF
DN
;
vna4,Figures S13K and S13L) does not affect the eye size. Misexpressing dTCF
DN
in the eye of L
2
/+ mutants
Figure 5. Newt gene regulates Wg both in the wild type, and in the L
2
-mutant background
(A–H) Misexpression of wg in the Drosophila developing eye. (A, B) Wild type background (+/+; ey>wg-GFP). (C and D)
Newt gene and Wg are misexpressed in the wild type background (+/+; ey>wg-GFP;vna4) exhibits increase in the eye
size compared to (+/+; ey>wg-GFP) as seen in (A and C) bright field adult eye picture, and (B and D) third instar eye-
antennal imaginal disc confocal image. (E, F) Misexpression of wg in the L
2
–mutant background (L
2
/+; ey>wg-GFP)
increases the severity of small-eye phenotype of L
2
–mutant. (G and H) Misexpression of the newt gene and wg in the
L
2
-mutant background (L
2
/+; ey>wg-GFP;vna4) exhibits a significant rescue of the reduced eye phenotype as seen in
(E and G) bright field adult eye picture, and (F and H) third instar eye-antennal imaginal disc confocal image. The GFP
reporter (green) marks ectopic misexpression of wg in the developing eye, Elav a proneural marker is shown in red.
(I) The bar graph (I) represents the relative fold change in phenotype strength (eye size) compared between ey>wg-GFP
and ey>wg-GFP;vna4, and between L
2
/+; ey>wg-GFP and L
2
/+; ey>wg-GFP;vna4.
(J) Similarly bar graph (J) represents relative fold change in the ratio of Wg -GFP intensity per area of GFP expression
between respective samples. Statistical analysis was performed using the student’s t-test for independent samples.
Statistical significance was determined with 95% confidence (p < 0.05). All bar graphs show values as the average between5
samples. Error bars show standard deviation (mean GSD), and symbols above the error bar signify as: ns is non-significant
p value, ** signifies p value < 0.005, *** signifies p value < 0.0005 respectively. All the images are displayed in the same
polarity as dorsal domain-towards top, and ventral domain-towards the bottom. Scale bar = 100 mm. See also Figure S12.
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(L
2
/+; ey>dTCF
DN
) significantly rescues the loss-of-ventral eye phenotype in 21.8% of flies (Figures 6J, 6K,
and S14B; Table S1)(Singh et al., 2006). Similarly, misexpressing vna4 and dTCF
DN
in the eye of L
2
mutants
(L
2
/+; ey> dTCF
DN
;vna4) rescues the loss-of-ventral eye phenotype (Figures 6L and 6M). As expected,
rescue frequency again dramatically increased from 21.8% to 70.6% (Figure S14B; Table S1).
Our results demonstrate that misexpression of newt genes when Wg signaling is downregulated (using in-
hibitors of Wg) in L
2
mutant background (L
2
/+; ey> sgg;vna4, 71.5%) or (L
2
/+; ey> dTCF
DN
;vna4, 70.6%)
significantly increased the rescue frequency of the L
2
mutant phenotype. The rescue frequency obtained
is greater than either misexpressing newt gene (L
2
/+; ey>vna4, 58.2%) alone or only misexpressing
negative regulators (L
2
/+; ey> sgg, 27.3%) or (L
2
/+; ey> dTCF
DN
, 21.8%). The converse phenotypes were
seen when Wg signaling was upregulated. This strongly suggests that newt gene(s) interact with the Wg
signaling pathway to promote rescue of L
2
mutant phenotype.
Porcupine (Porc) is involved in post-translational modification of Wg nascent protein in the producing cell
and thus facilitates its transport outside to the receiving cell. Wg ligand can bind to its receptor on the
Figure 6. Modulating positive and negative regulators of Wg signaling pathway affects the Lmutant phenotype
(A) Schematic presentation of the Wg signaling pathway showing various members of the canonical pathway. The positive regulators are in red, and negative
regulators of the Wg pathway are in green.
(B–E) Activating Wg signaling in the L
2
mutant eye background by misexpression of (B and C) arm alone (L
2
/+; ey>arm), (D and E) arm and vna4 (L
2
/+; ey>arm;
vna4). Note that vna4 misexpression with arm (L
2
/+; ey>arm;vna4) in the eye rescues the L
2
/+; ey>arm reduced eye phenotype.
(F–M) Blocking Wg signaling in L
2
mutant eye background by misexpression of (F and G) sgg alone (L
2
/+; ey>sgg), (J and K) dTCF
DN
alone (L
2
/+; ey>
dTCF
DN
) results in rescue of L
2
loss-of-ventral eye phenotype. Similarly, in L
2
mutant background misexpression of vna4 along with (H and I) sgg (L
2
/+;
ey>sgg;vna4)and(LandM)dTCF
DN
(L
2
/+; ey> dTCF
DN
;vna4) significantly rescue the partial loss-of-ventral eye phenotype.
(N–Q) Blocking transport of Wg morphogen in L
2
mutant eye background by misexpression of (N and O) porc
RNAi
alone (L
2
/+; ey> porc
RNAi
), (P and Q)
porc
RNAi
and vna4 (L
2
/+; ey> porc
RNAi
;vna4). Note that L
2
/+; ey> porc
RNAi
exhibits weak rescue of loss-of-ventral eye phenotype whereas vna4
misexpression with porc
RNAi
(L
2
/+; ey> porc
RNAi
;vna4) enhances the phenotype strength. All the images are displayed in same polarity as dorsal domain-
towards the top, and ventral domain-towards the bottom. Scale bar = 100 mm. See also Figures S13 and S14.
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12 iScience 24, 103166, October 22, 2021
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receiving cell (Manoukian et al., 1995;Swarup and Verheyen, 2012) thus modulating downstream pathway
components (Figure 6A). We blocked Wg transport from the producing cell using porc
RNAi
and sampled its
effect on L
2
mutant phenotype, and L
2
mutant where vna4 is misexpressed. Misexpression of porc
RNAi
alone (ey> porc
RNAi
)(Figures S13M and S13N), and along with vna4 in the wild-type background (+/+;
ey> porc
RNAi
;vna4)(Figures S13O and S13P) does not affect the eye size. Misexpressing porc
RNAi
in the
L
2
mutant eye (L
2
/+; ey> porc
RNAi
) significantly rescues the loss-of-ventral eye phenotype in 20.8% of flies
(Figures 6N, 6O, and S14B; Table S1). Similarly, vna4 misexpressed in the eye of L
2
mutants along with
porc
RNAi
(L
2
/+; ey> porc
RNAi
;vna4) rescued the loss-of-ventral eye phenotype (Figures 6P and 6Q). The
rescue frequency increased dramatically from 20.8% to 68.1% (Figure S14B; Table S1). Thus, confirming
that newt genes can modulate levels of Wg signaling to promote restoration response in L
2
eye mutants.
DISCUSSION
Notophthalmus viridescens have an enormous genome size (c310
10
bases), a long reproductive cycle,
and have limited genetic tools that makes it difficult to use this model to ascertain the molecular mecha-
nism(s) behind the function of its unique genes (Mehta and Singh, 2019a). One of the strategies can be
to introduce these genes into models with array of genetic tools and where regeneration potential is lower
as seen in models like Drosophila,Bombyx mori etc (Gopinathan et al., 1998;Kango-Singh et al., 2001;
Mehta and Singh, 2019a;Singh et al., 2007). The rationale is to test the efficacy of these newt genes in trig-
gering restoration response in the model systems with lower restoration potential. We and others have
introduced human, plant and other vertebrate genes by transgenic approaches in Drosophila (Deshpande
et al., 2019;Gogia et al., 2020b;Hughes et al., 2012;Sarkar et al., 2018;Tare et al., 2011). These transgenic
flies have been utilized for targeted expression of foreign genes in flies using a targeted misexpression
approach (Brand and Perrimon, 1993). The successful misexpression of the unique newt genes (vertebrate
genes) in Drosophila using the same targeted misexpression approach has been reported earlier (Mehta
et al., 2019;Mehta and Singh, 2019b).
To test theregeneration potential of these newt genes,we took a distinctiveapproach of usingDrosophila model
because the signaling pathways, which are involved in regeneration and/or tissue growth, patterning and devel-
opment are evolutionarily conserved across the species (Kango-Singh and Singh, 2009;Mehta and Singh, 2019a;
Wang and Hu, 2020). Even though humans (Homo sapiens), which evolved approx. 200,000 years ago (Stringer,
2016) and are 541 million years apart from Drosophila, still share 75% of genes with flies (Pandey and Nichols,
2011).Despite the gap of 250 million years between newts and Drosophila, our studies suggest that the proteins
encoded by unique newt genes are functional in Drosophila (Figures 1 and S2). Therefore, it is plausible that the
pathways that newt genes can modulate in Drosophila might share parallels with their mechanism of action in
newts. We found that the five newly identified genes from newt, can (i) rescue Lmutant eye phenotype (early
eye mutant) as well as GMR-hid GMR-GAL4 (late retinal degeneration) phenotype, (ii) promote cell proliferation,
and downregulate cell death to restore the loss-of-ventral-eye phenotype, and (iii) downregulate Wg signaling
(Figure 7). Ourstudies clearly demonstrate that the potential of these newt genes in triggering rescue of missing
photoreceptor cells in Drosophila eye model exists along the temporal axis from early eye development (L
2
mutant) to the later stages of eye development (GMR-hid GMR-GAL4).
Newt genes promote cell proliferation to restore Drosophila eye mutant phenotype
Regeneration involves the formation of a blastema, a proliferating zone of undifferentiated cells to restore the
missing structures (Brockes and Kintner, 1986;Mehta and Singh, 2019a;Sanchez Alvarado and Tsonis, 2006).
Furthermore, a network of newt gene(s) triggers proliferation response to promote lens regeneration (Eguchi
and Shingai, 1971;Grogg et al., 2005;Looso et al., 2013;Madhavan et al., 2006). A similar proliferating response
was observed when newt genes were misexpressed in the Drosophila L
2
mutant eye (Figures 3BandS5), and in
GMR-hid, GMR-GAL4 eye discs (Figures S6A–S6C). L
2
mutant flies exhibit a loss-of-ventral eye phenotype, and
misexpressing newt genes robustly increased the number of proliferating cells (Figures 3,7,andS5), which
clearly mimics the classic mechanism of epimorphic regeneration in the newt lens. Similarly, GMR-hid,GMR-
GAL4>vna4 eye discs showed significant increase in the number of dividing cells (Figure S6C).
In our study, wefound that newt genes can also modulate cell death inDrosophila developing eyes. When newt
genes were misexpressed in the L
2
mutant background (L
2
;ey>newt gene), the rate of cell death was lower as
compared to the L
2
mutant (L
2
;ey-GAL4) eye (Figures 3H, 3L, S5K–N, and S5R), but minimally higher compared
to the control (ey-GAL4) (Figures 3I, 3L, S5O, and S5R). A minimal rate of apoptosis is observed during the newt
lens regeneration as well (Tsonis et al., 2004). Newt genes can also robustly downregulate cell death caused by
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the overexpression of hid (GMR-hid, GMR-GAL4>vna4)(Figures S6D–S6F). Moreover, it was interesting to note
that newt genes do not upregulate cell proliferation or downregulate cell death in the normal Drosophila eye
(ey>vna4)(Figures 3D, 3J, 3F, and 3L). There is a strong possibility that these newt genes might be hijacking
the same evolutionarily conserved signaling pathway(s) in Drosophila to regulate cell proliferation,and cell death
that might also be involved in promoting regeneration in newts.
Newt genes tightly regulate Wg signaling pathway in Drosophila
We screened for the various signaling pathwaysusing the candidate approach as well as compared them to
our transcriptomic analysis (Mehta et al., 2019). Among the various genetic modifiers of the L
2
mutant
phenotype, we found wg as one of the possible candidates. Wg, a member of the evolutionarily conserved
Wg/Wnt pathway, has been previously reported to promote regeneration, growth, development etc. (Har-
ris et al., 2016;Schubiger et al., 2010;Singh et al., 2012b,2018;Smith-Bolton et al., 2009;Yokoyama et al.,
2007). Misexpression of newt genes in Drosophila wild-typebackgroundmodulatebothpositiveregulators
of Wg/Wnt signaling like frizzled (fz), and microtubule star,(mts) and negative regulators like Casein Kinase
IIb(CKIIb), and sinaH (Figure S10). This modulation in the levels of both inhibitors as well as effectors can be
a homeostatic reaction shown by the Drosophila to normalize expression levels of Wg/Wnt signaling
pathway. As a result, changes in the eye growth after misexpressing newt genes in the wild-type was
non-significant when compared to the wild-type control (ey-GAL4) (Figures 1,3,andS5). We did not
observe any lethality, developmental defects, dysregulation in cell proliferation or cell death etc.
Interestingly, when newt genes were misexpressed in L
2
mutant background, there was significant down-
regulation of Wg/Wnt expression (Figures 4 and S11), which resulted in rescue of the loss-of-ventral eye
phenotype. It is known that overexpressing Wnt, and its receptor Fz promote lens regeneration in newts,
whereas blocking Wnt pathway by expressing Dkk1 inhibits lens induction (Hayashi et al., 2006). Therefore,
our study may have parallels with the regeneration mechanism in newts. Furthermore, we found that when
Wg pathway components are modulated in Drosophila along with the newt genes, it not onl y rescues the L
2
mutant loss-of-ventral eye phenotype but can also rescue the eye-loss caused by the activation of the Wg
signaling pathway in the wild-type background. Hence, our data strongly suggest that the newt genes can
Figure 7. Model of newt genes function during development
These newt genes (vna1-vna5) can negatively regulate evolutionarily conserved Wg/Wnt signaling pathway in the
developing Drosophila eye, promote cell proliferation, and block cell death, which results in the rescue of L
2
mutant’s
loss-of-ventral eye phenotype. This potential to restore missing structures by these newt genes extend from early eye
development to late larval eye and adult eye.
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modulate Wg signaling pathway in loss-of- eye background. Interestingly, these newt genes did not show
any phenotype when they were misexpressed alone suggesting that restoration response is triggered only
to the cues generated because of the tissue damage.
Newt proteins exhibit non-cell-autonomous response
Genetic mosaic experimental approach resulted in the generation of clone(s) of retinal neurons expressing
high levels of newt genes within the L
2
mutant eye disc. These experiments showed that clones expressing
high levels of newt genes can rescue the L
2
mutant phenotype; however, the rescue was not limited to the
clone itself and spreads into the neighboring cells where newt genes were not misexpressed. This non-cell-
autonomous rescue of the missing tissue is possible only if the newt proteins are getting secreted from one
cell to another and/or newt proteins are acting on the signaling molecules. These newt proteins have signal
peptides (Looso et al., 2013), which can facilitate its transport from one cell to another. Similarly, we have
reported that these genes interact with Wg (Figure 4), which is a morphogen that can have a long-distance
effect in the Drosophila eye (Zecca et al., 1996).
It will be interesting to investigate, (a) if newt genes can regulate Wg signaling pathway to promote regeneration
in other tissuesof Drosophila e.g.,wing, leg etc., and in thebackground of othercongenital mutations (Schubiger
et al., 2010;Smith-Boltonet al., 2009), (b) if these proteins can rescue/regenerate missing tissues/organs in other
animals with low regenerative potentials such as mammals because pathways that have roles in regeneration,
growth, development, and cancer are evolutionarily conserved throughout the animal kingdom (Mehta and
Singh, 2019a). For example, the Wg/Wnt signaling pathway, is one such evolutionarily conserved pathway that
apart from promoting regeneration in hydra, planaria, newts, zebrafish, etc. has also been found to promote
cell proliferation during regeneration of mammalian muscles, liver, and bone (Polesskaya et al., 2003;Sodhi
et al., 2005;Zhong et al., 2006). Similarly, its dysregulation has been found to cause cancer in mammals (Nusse
and Varmus,1982;Taipale and Beachy, 2001). Interestingly newts are resistant to carcinogens (Tsonis and Eguchi,
1981), whichcould be because of itspotential to regulateevolutionarilyconserved pathways like Wnt, therefore in
future it will also be interesting toinvestigate (c) potential of these newt genes to regulate overgrowth/cancer in
Drosophila,andinmammals.
Limitations of study
We found that misexpressing these five newt genes (vna1,vna2,vna3,vna4,vna5)intheDrosophila loss-of-
eye mutant background could not completely restore the eye size in the flies as observed in newts. In newts,
there is complete regeneration of the appendage(s). It is possible that there may be many more players in
the regeneration tool kits of newts or in future combining all these five genes in one transgenic fly is war-
ranted to check if we can fully restore the missing eye. There could also be specific domains of these pro-
teins that might be critical for their function, which need to be identified and characterized to investigate
the course of their action in detail. Future studies using the established regeneration system in Drosophila
could help provide more details into the function of these genes. In addition, we have not been able to
determine the regeneration potential of these genes in newt species, because of absence of genetic tools
in the newt model. Furthermore, our data using the dorsal-eye-Gal4 demonstrated that misexpression of
these regeneration tools genes in the dorsal eye can rescue loss-of-eye phenotype non-autonomously
even in the ventral half. However, because of challenges of studying V5 tag expression in the tissue by
immunohistochemistry, we could not validate our observation that these newt genes can direct non-auton-
omous rescue of eye phenotype. Previously it has been suggested that highly regenerative animal models
like newts might be employing unique regeneration tool kit genes, and/or unique regeneration enhancers
that can modulate the evolutionarily conserved signaling pathways to promote restoration (regenerative)
response ( Casco-Robles et al., 2018;Looso et al., 2013;Mehta and Singh, 2019a;Rodriguez and Kang, 2020;
Thompson et al., 2020;Wang and Hu, 2020). In absence of such genes, the low regeneration animal models
like Drosophila, mammals etc. or the animals that lack regeneration potential (Kango-Singh et al., 2001;
Singh et al., 2007) could not exhibit robust response to restore missing structures even though the Wnt/
Wg signaling pathway members are present (Mehta and Singh, 2019a). Thus, introduction of these genes
into flies can coax the genetic machinery to promote restoration response. Such studies will have significant
bearings on our efforts to understand and trigger regenerative response in humans upon injury.
STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:
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dKEY RESOURCES TABLE
dRESOURCE AVAILABILITY
BLead contact
BMaterials availability
BData and code availability
dEXPERIMENT MODEL AND SUBJECT DETAILS
BAnimals
BEthics statement
BDrosophila stocks
dMETHOD DETAILS
BTissue harvest and RNA extraction
BSample collection and storage
BRNA quantitation, and storage
BSample preparation to clone newt genes
BGenerating transgenic flies
BRNA sequencing
BTargeted misexpression studies
BGeneticmosaicanalysis
BImmunohistochemistry
BWestern blotting
BTUNELassayfordetectionofcelldeath
BBright field imaging
dQUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.103166.
ACKNOWLEDGMENTS
We thank Bloomington Drosophila Stock Center (BDSC) for Drosophila strains, and the Developmental
Studies Hybridoma Bank (DSHB) for antibodies. We would like to thank Katia Del Rio-Tsonis, Labib
Rouhana, Madhuri Kango-Singh, Meghana Tare, Neha Gogia, Shilpi Verghese, Pothitos Pitychoutis,
and Deepika K Sodhi for critical comments on the manuscript. We also thank Justin Kumar, Y. Henry
Sun, and Kyung Ok Cho for the gift of fly strains and antibodies. Confocal microscopy was supported
by the core facility at University of Dayton. A.S. is supported by 1R15GM124654-01, 1RO1EY032959-
01 from NIH, Schuellein Chair Endowment Fund and STEM Catalyst Grant from the University of Dayton.
AUTHOR CONTRIBUTIONS
A.S.Mehta,P.A.Tsonis,A.Singhdesignedthestudy.P.Deshpande,A.V.Chimata,A.S.Mehtaperformed
the experiments. A. Singh contributed the resources. A. Singh, A.S. Mehta., A V. Chimata, P. Deshpande
analyzed the data. A. S.Mehta and A. Singh wrote the manuscript with input from all authors. All authors
read and approved the final manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
The author list of this paper includes contributors from the location where the research was conducted who
participated in the data collection, design, analysis, and/or interpretation of the work.
Received:March25,2021
Revised: July 2, 2021
Accepted: September 21, 2021
Published: October 22, 2021
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