Inter-Species Rescue of Mutant Phenotype—The Standard for Genetic Analysis of Human Genetic Disorders in Drosophila melanogaster Model

Abstract

Drosophila melanogaster (the fruit fly) is arguably a superstar of genetics, an astonishing versatile experimental model which fueled no less than six Nobel prizes in medicine. Nowadays, an evolving research endeavor is to simulate and investigate human genetic diseases in the powerful D. melanogaster platform. Such a translational experimental strategy is expected to allow scientists not only to understand the molecular mechanisms of the respective disorders but also to alleviate or even cure them. In this regard, functional gene orthology should be initially confirmed in vivo by transferring human or vertebrate orthologous transgenes in specific mutant backgrounds of D. melanogaster. If such a transgene rescues, at least partially, the mutant phenotype, then it qualifies as a strong candidate for modeling the respective genetic disorder in the fruit fly. Herein, we review various examples of inter-species rescue of relevant mutant phenotypes of the fruit fly and discuss how these results recommend several human genes as candidates to study and validate genetic variants associated with human diseases. We also consider that a wider implementation of this evolutionist exploratory approach as a standard for the medicine of genetic disorders would allow this particular field of human health to advance at a faster pace.

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Citation: Ecovoiu, A.A.; Ratiu, A.C.;
Micheu, M.M.; Chifiriuc, M.C.
Inter-Species Rescue of Mutant
Phenotype—The Standard for
Genetic Analysis of Human Genetic
Disorders in Drosophila melanogaster
Model. Int. J. Mol. Sci. 2022,23, 2613.
https://doi.org/10.3390/
ijms23052613
Academic Editors: Athanassios D.
Velentzas and Dimitrios J.
Stravopodis
Received: 2 February 2022
Accepted: 24 February 2022
Published: 27 February 2022
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International Journal of
Molecular Sciences
Review
Inter-Species Rescue of Mutant Phenotype—The Standard
for Genetic Analysis of Human Genetic Disorders in
Drosophila melanogaster Model
Alexandru Al. Ecovoiu 1, Attila Cristian Ratiu 1, *, Miruna Mihaela Micheu 2and Mariana Carmen Chifiriuc 3
1Department of Genetics, Faculty of Biology, University of Bucharest, 060101 Bucharest, Romania;
alexandru.ecovoiu@bio.unibuc.ro
2Department of Cardiology, Clinical Emergency Hospital of Bucharest, 014461 Bucharest, Romania;
miruna.micheu@yahoo.com
3The Research Institute of the University of Bucharest, Faculty of Biology, University of Bucharest,
050095 Bucharest, Romania; carmen.chifiriuc@bio.unibuc.ro
*Correspondence: attila.ratiu@bio.unibuc.ro; Tel.: +40-722250366
Abstract:
Drosophila melanogaster (the fruit fly) is arguably a superstar of genetics, an astonishing
versatile experimental model which fueled no less than six Nobel prizes in medicine. Nowadays, an
evolving research endeavor is to simulate and investigate human genetic diseases in the powerful
D. melanogaster platform. Such a translational experimental strategy is expected to allow scientists
not only to understand the molecular mechanisms of the respective disorders but also to alleviate
or even cure them. In this regard, functional gene orthology should be initially confirmed
in vivo
by transferring human or vertebrate orthologous transgenes in specific mutant backgrounds of
D. melanogaster. If such a transgene rescues, at least partially, the mutant phenotype, then it qualifies
as a strong candidate for modeling the respective genetic disorder in the fruit fly. Herein, we review
various examples of inter-species rescue of relevant mutant phenotypes of the fruit fly and discuss
how these results recommend several human genes as candidates to study and validate genetic
variants associated with human diseases. We also consider that a wider implementation of this
evolutionist exploratory approach as a standard for the medicine of genetic disorders would allow
this particular field of human health to advance at a faster pace.
Keywords:
human genetic disorder; Drosophila melanogaster model; heterologous rescue; functional
complementation; genetic analysis
1. Introduction
Advances in animal model-based research markedly increased our understanding of
molecular mechanisms that regulate physiological and pathological processes. Perhaps
one of the greatest achievements in this field is the development of genetically engineered
animal models which offer a valuable platform for disease modelling and testing of po-
tential therapeutic strategies. Accordingly, choosing reliable animal models represents a
critical step to speed up the successful integration of precision medicine into daily clinical
practice [
1
]. With its short generation time, low cost, large brood size and ease of genetic
manipulation, Drosophila melanogaster (the fruit fly) has emerged as a key organism to
explore disease-related genetic mechanisms [2].
Homo sapiens and D. melanogaster share strong similarities regarding many biological
functions such as reproduction, embryo development, locomotion, respiration, circulatory
system and neurodevelopment [
3
–
5
]. This relies on a high degree of evolutionary con-
servation of important genomic features such as genes, core regulatory mechanisms and
genetic pathways.
These analogies endorse phenotypic rescue experiments conceived to reveal inter-
species functional gene orthology. A crucial rescue assay for modelling a human genetic
Int. J. Mol. Sci. 2022,23, 2613. https://doi.org/10.3390/ijms23052613 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022,23, 2613 2 of 37
disorder (hGD) in D. melanogaster is the functional complementation (heterologous rescue)
of an appropriate mutant fruit fly strain by the orthologous human or mammalian transgene
associated with the respective hGD. If the two genes or proteins with similar nucleotide or
amino acid sequences are also functionally related, namely, if the molecular functions are
evolutionary conserved, the human wild-type allele (hWT) of the gene of interest (hGOI)
is expected to rescue, at least partially, the fruit fly mutant phenotype. In other words, to
rescue or save a phenotype means to restore it to wild type with a transgenic copy of the
orthologous gene from other species.
In the technical jargon, various synonyms are used for the inter-species rescue of
the mutant phenotype. For example, scientists working with a humanized yeast model
(
Saccharomyces cerevisiae
) coined the terms cross-species functional complementation, stand-
ing for heterologous rescue, and heterologous expression, meaning the ectopic expression
of a human gene in yeast strains, regardless of whether the yeast is wild type or mutant [
6
,
7
].
Herein, we conversely utilize the terms heterologous rescue and functional complementa-
tion used by FlyBase [
8
], but the more intuitive phrase phenotype rescue (such as lethality
rescue) is also used whenever appropriate. The rescue term is also used for saving a fruit
fly mutant phenotype with a dWT (Drosophila wild-type) transgene to confirm that the
phenotype is indeed determined by the presumed gene and not by a hidden mutation
present in the genetic background. We will further refer the phenotype rescue with dWT as
intra-specific rescue, to differentiate from inter-specific, heterologous rescue or functional
complementation equivalent terms. A different type of rescue is the chemical rescue, which
is not a genetic one but instead is part of an endeavour to find chemicals able to alleviate or
save mutant phenotypes, allowing the screening for potential new drugs.
D. melanogaster is suitable for different modelling approaches of human genetic dis-
eases. One strategy involves targeted mutagenesis of Drosophila gene of interest (dGOI)
in conserved sequences shared with hGOI, or RNAi inactivation of dGOI, to reproduce
phenotypes resembling pathologic aspects of the hGD in D. melanogaster. An alternative is
the replacement of dGOI with a disease-specific allele of hGOI to mirror clinical phenotypes
in D. melanogaster. Last but not least, a different avenue is to introduce into fruit flies
either a wild-type or a mutant copy of a hGOI having no evident structural ortholog in
the D. melanogaster genome, but which may be useful to reproduce
in vivo
some molecular
interactions important for understanding of the hGD. Whichever experimental alterna-
tives are to be considered in practice, either individually or overlapping, a key step is to
perform preliminary inter-specific phenotype rescue experiments, namely, to check if the
wild-type copy of hGOI is able to functionally compensate a mutant allele of the ortholo-
gous dGOI. A positive result shows evolutionary functional conservation between the two
species and reinforces D. melanogaster as a suitable experimental platform for modelling
that particular hGD. On the other hand, a failure of the inter-specific phenotype rescue
attempt, either an intrinsic or a false negative one, may induce the geneticists to decide on
a mammalian model.
Since the experimental strategies used to model hGDs on D. melanogaster are already
detailed in a few excellent papers [
9
,
10
], we choose to focus on various examples of inter-
species phenotype rescues relevant for the medical research.
High-quality sequenced and assembled genomes have become increasingly available
and allow experts to identify genes and regulatory sequences relevant for human medical
research. This achievement relies on a deep comparative scanning of the two genomes
with state-of-the-art bioinformatics tools. If structural orthologous gene pairs of interest are
identified, targeted mutagenesis may be induced in D. melanogaster by an array of highly
effective methods developed for this experimental model. The mutant alleles are then
subjected to genetic analysis methods to check for functional orthology to human genes
responsible for the aberrant phenotypes underlying the respective hGD.
Briefly, the modus operandi of this experimental approach is the identification of
a human gene associated with the hGD of interest, bioinformatics comparative analysis
to scan D. melanogaster genome for a candidate structural ortholog of the human gene,

Int. J. Mol. Sci. 2022,23, 2613 3 of 37
generation and analysis of relevant mutant alleles in D. melanogaster, delivery of the or-
thologous human cDNA into the appropriate fruit fly mutant background by means of
effective molecular constructs and checking for partial or complete rescue phenotype of
the transgenic fruit flies. Commonly, the heterologous rescue experiments target mutant
phenotypes determined by loss-of-function (LOF) alleles, which are either hypomorphic
alleles with reduced activity or null alleles with no residual activity [
11
]. The experimental
steps of heterologous rescue, which rely on the modular and versatile UAS-GAL4 system,
are outlined in Figure 1[12,13].
Int. J. Mol. Sci. 2022, 23, 2613. https://doi.org/10.3390/ijms23052613 www.mdpi.com/journal/ijms
Figure 1.
General outline of the heterologous rescue of D. melanogaster mutant phenotypes determined
by lethal LOF alleles of dGOIs with transgenic cDNAs corresponding to the orthologous hGOIs
associated with a hGD. (1) Bioinformatics analysis is deployed to search in the fruit fly genome for an
orthologous gene for a human gene associated with a genetic disorder. (2) A cDNA copy of the wild-type
allele of hGOI is cloned into an insertional vector under the control of a UAS enhancer sensitive to GAL4

Int. J. Mol. Sci. 2022,23, 2613 4 of 37
activator. The UAS–cDNA construct is delivered by micro-injection into mutant embryos heterozy-
gous for dGOI
LOF
allele (obtained by targeted mutagenesis), and the resulting transgenic adults
are subsequently crossed with a heterozygous dGOI
LOF
fruit fly strain able to produce the GAL4
activator. (3) Transgenic analysis of descendant dGOI
LOF
/dGOI
LOF
flies that contain a functional
hGOI activated by GAL4 may reveal two distinct situations. YES (complete or partial heterologous
rescue). The functional hGOI1 encodes a human protein of interest (hPOI1) which is able to properly
interplay with an interacting protein (dIP
α
) in D. melanogaster molecular background; since dIP
α
is the proximal interactor of the normal protein encoded by wild-type copy of dGOI1, the correct
interaction between hPOI1 and dIP
α
rescues lethality of LOF/LOF transgenics. NO (heterologous
rescue fails). On the other hand, the human protein hPOI2, encoded by a different hGOI2 transgene,
does not interact accurately with dIP
β
and the heterologous rescue fails. Created with BioRender.com
(accessed on 23 February 2022).
As an example, if the LOF allele is a recessive null lethal one, very young heterozygous
mutant embryos are microinjected with an insertional vector containing the orthologous
hGOI cloned under an UAS enhancer control. Transgenic adults containing both the LOF
allele and UAS–hGOI construct are crossed with a strain containing both the LOF allele and
a GAL4-driver with either specific or generic pattern of expression. If, in the F1 generation
of this cross, the LOF/LOF homozygous individuals are viable, the heterologous rescue
was successful due to activation of UAS-hGOI by GAL4. Therefore, the structural ortholog’s
genes are also functionally orthologous, indicating that at least some of their functions
were conserved during evolution.
A successful heterologous rescue result is a very strong indicator for functional or-
thology between the members of human–fruit fly gene pairs. It is important to mention
that any functional improvement of the mutant phenotypes of LOF flies such as rescue of
lethality, proceeding through a later developmental stage, increased lifespan, increased
fertility, improved behavior, etc., deserves attention and qualifies the functionally rescued
dGOI as an attractive candidate for modeling hGDs in D. melanogaster [
14
]. Even when
heterologous rescue of mutant fruit flies was not performed with a hWT but with a mam-
malian orthologous gene [
15
,
16
], this functional conservation is a strong genetic logic to
start a research project on that gene model [14,17].
FlyBase reports the rescue experiments as “heterologous rescue” in the Overview
tab of the report of a human genetic disorder modelled in D. melanogaster. The link to
the respective fruit fly orthologous gene opens a Gene Report webpage which contains a
Functional Complementation Data Table. If functional data are available, links under the
Ortholog tabs showing functional complementation and Supporting References are present.
In practice, many studies reported in FlyBase describe rescuing of abnormal phenotypes
induced by RNAi suppression of GOI, but care should be taken when interpreting such
data. A recent report dealing with the difficulties arising from the RNAi method reveals
that residual functional activity of some genes in D. melanogaster still exists even when this
technology is improved [18].
We reviewed data obtained from heterologous rescue experiments supporting human–
fruit fly functional gene equivalence and their value for genetic analysis of hGDs. To this
end, we present relevant examples of neurodegenerative and neuromuscular disorders,
cardiac pathophysiology, cancer and infectious diseases. To our best knowledge, the
present paper is the first attempt to scrutinize up-to-date scientific literature and FlyBase
(FB2021_06) for the vast majority of the heterologous rescue experiments performed in
D. melanogaster. We argue that preliminary experiments of mutant phenotype rescue should
be the paradigm for any relevant genetic analysis of hGDs on the D. melanogaster model.
2. Neurodegenerative and Neuromuscular Disorders
For more than 20 years, D. melanogaster has been employed to tackle neurodegenerative
and neuromuscular human afflictions [
19
,
20
]. Due to its relatively complex brain, which har-
bors around 300,000 neurons organized into specialized areas with discrete functions [
21
],
D. melanogaster displays complex behaviors such as learning, memory, depression, anxiety,

Int. J. Mol. Sci. 2022,23, 2613 5 of 37
competitiveness, aggressiveness and alcoholism. Consequently, D. melanogaster represents
a valuable system for the study of neuronal dysfunction and related disorders particular to
several neurodegenerative diseases such as Alzheimer’s disease (AD), amyotrophic lateral
sclerosis (ALS), Angelman’s syndrome (AS), autism spectrum disorder (ASD), Charcot–
Marie–Tooth (CMT) disease, Friedrich’s ataxia (FA) and Parkinson’s disease (PD), to name
a few [22].
2.1. Parkinson’s Disease
PD is one of the most common neurodegenerative diseases, accompanied by specific
tremors and slow movement caused by degradation of dopaminergic (DA) neurons in the
midbrain. To decipher the poorly understood mechanisms of selective degeneration of DA
neurons, interactions between the products of human
α
-Synuclein (
α
-Syn), parkin RBR E3
ubiquitin protein ligase (PRKN) and PAELR genes were modelled in D. melanogaster brain
neurons [
23
]. Co-expressing PRKN and
α
-Syn in transgenic flies rescued the loss of DA
neurons and reduced the aggregation of
α
-Syn, a mutant phenotype, which endogenous
parkin (park) from D. melanogaster was not able to rescue in
α
-Syn transgenic flies. This
example can be viewed as a type of phenotypic rescue reflecting a putative incapacity of
Drosophila’s park gene product to interact with human α-Syn.
On the same topic, Burchell et al. [
24
] showed that overexpression of human Fbxo7
gene, associated with a severe form of autosomal recessive early-onset PD [
25
], significantly
rescued several mutant phenotypes such as locomotor defects, DA neuron loss and muscle
degeneration determined by LOF of parkin. Pathogenic mutant Fbxo7 alleles were not able
to rescue the loss of park, a result reinforcing the notion that the corresponding proteins
share a common role in mitochondrial maintenance and mitophagy.
Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) human gene
negatively impact the oxidative phosphorylation processes in mammalian cells [
26
] and
are associated with an autosomal dominant form of late-onset PD. In D. melanogaster, LOF
and hypomorphic Chchd2 alleles affect the maintenance of mitochondrial crista structure
and lead to neuronal phenotypes associated with PD, such as sensitivity to oxidative stress,
motor dysfunction, short lifespan and loss of DA neurons with age [
27
]. Expression of either
transgenic dWT Chchd2 or hWT CHCHD2, but not missense alleles of the latter, successfully
rescued the mitochondrial morphology and DA neurons loss induced by hypomorphic
Chchd2H43 in D. melanogaster.
Another study concerning PD [
28
] focuses on exploring a specific subset of human
iPLA2-VIA/PLA2G6 mutations that direct
α
-Syn aggregation and DA neurodegeneration
specific for the PARK14-linked PD with α-synucleinopathy. The iPLA2-VIA/PLA2G6 gene
codifies for an enzyme that is fundamental to phospholipids synthesis by the remodeling
pathway or Lands’ cycle [
28
]. IPLA2-VIA-null allele impacts the early developmental stages
of Drosophila mutants and leads to alterations of neurotransmission and midbrain DA
neurons’ degeneration, causing gradual locomotor defects and sleep disruption [
28
]. When
transgenic hWT iPLA2-VIA was expressed in the neurons of mutant flies, the motor and
paralytic phenotypes were rescued, pointing to functional conservation between the two
orthologous genes.
In addition to the previous examples, complex molecular interactions characterizing
PD, such as the imbalance in trace metal levels characteristic for some forms of PD and
AD, may be also addressed. The metal-responsive transcription factor 1 (MTF-1) gene is
evolutionary conserved between D. melanogaster and mammals [
29
] and counteracts the
effects of heavy metal loads. In mammals, MTF-1 was found to induce transcription of
specific target genes in response to oxidative stress and infection [
30
]. A study focusing
on the interactions between metal homeostasis and park function established that mutants
expressing both park and MTF-1 LOF alleles in homozygous condition define a genetic
assembly termed synthetic lethality [
31
]. The introduction of a transgene of MTF-1 in the
double homozygous park and MTF-1 mutants rescued the lethality and has significantly
increased the lifespan of park homozygous mutants. Alternatively, human MTF-1 has

Int. J. Mol. Sci. 2022,23, 2613 6 of 37
been able to rescue the short lifespan phenotype of park mutants [
31
], and largely, but not
completely, rescued the metal sensitivity characterizing the LOF MTF-1 flies [32].
Loss of function pink1 mutant flies experience PINK1 deficiency and display motor
disturbances as well as corrupted function of Complex I of the mitochondrial respiratory
chain, and thus increased sensitivity to apoptotic stress. In humans, mutations in PTEN
(phosphatase and tensin homologue)-induced kinase1 (PINK1) are strongly correlated with
recessive forms of PD. HWT allele, but not mutant PINK1, rescued the phenotype exhibited
by LOF Pink1 mutant flies [33].
2.2. Amyotrophic Lateral Sclerosis
ALS is arguably the most prevalent motoneuron disorder that leads to fatal adult-onset
neurodegenerative progression [
34
,
35
]. The genetic basis of ALS overlaps at least partially
with that of frontotemporal dementia, and often, the ALS patients concurrently develop
cognitive and behavioral alterations [36,37].
Among over 30 genes that could harbor ALS causing mutations, some of the most no-
ticeable genes are superoxide dismutase 1 (SOD1), chromosome 9 open reading frame 72 (C9orf72),
fused in sarcoma RNA binding protein (FUS) and TAR DNA-binding protein (TARDBP) [38].
Various studies focused on validating the effects of human SOD1, as well as of other
ALS associated genes, were performed on the fly phenotype in order to establish an ALS
experimental model, as reviewed elsewhere [
38
–
40
]. In D. melanogaster it has been shown
that null alleles of resident Sod1 determine impaired locomotor activity and lethality. These
severe phenotypes are fully rescued by SOD1
WT
but not by its clinically relevant mutant
alleles such as SOD1
A4V
,SOD1
G37R
or SOD1
I113T
[
41
], which confirm the impaired functions
of these alleles in human patients. In addition, it was revealed that even a localized adult
motor neuron expression of SOD1WT restored the lifespan of null Sod1-null flies to 60% of
the normal controls [42].
In humans, both TARDBP and FUS code for DNA- and RNA-binding proteins involved
in RNA processing of thousands of targets and share some common functionality under-
lined by similar pathogenic outcomes stemming from mutations [
43
–
45
]. D. melanogaster
harbors TAR DNA-binding protein-43 homolog (TBPH) and cabeza (caz) as orthologs of
TARDBP and FUS, respectively. LOF alleles of TBPH cause the disruption of mitochondrial
trafficking accompanied with severe motor dysfunctions revealed by low rates of eclosion,
altered larval crawling and adult climbing capacity [
46
]. Except the adult climbing mutant
phenotype, all of the others are fully rescued by expressing either TBPH
WT
or TARDBP
WT
transgenes. Intriguingly, the mitochondrial transport defects were also rescued by express-
ing TARDBP
M337V
, an ALS-linked allele. This particular example of phenotypic rescue
reveals that the pathogenic variant can display normal function in D. melanogaster, indicat-
ing that this allele is not involved in ASL. The same study found that caz
1
-null mutants [
47
]
presented a significant decrease in mitochondria and vesicle transport. These phenotypes
were rescued by expressing FUS
WT
in mutant flies, but the caz
WT
transgene was able to fully
rescue both phenotypes only at 29
◦
C, when it is overexpressed, and only partially at 25
◦
C.
Surprisingly, the FUS
P525L
pathogenic allele as well as its equivalent caz
P938L
successfully
rescued the mitochondrial transport defects but not the vesicle transport. Regardless of
expressing WT or pathogenic FUS alleles, other phenotypes particular to caz
1
mutants
such as eclosion, larval crawling and climbing defects were fully rescued. To cement the
functional overlap between TBPH and caz, the reduced viability, lifespan, eclosion and
climbing ability of TBPH mutants were fully rescued by neuronal overexpression of caz, but
not vice versa. Overexpression of cazWT did not rescue the mitochondrial transport defects
or larval crawling impairment. Consistent with these findings, it was previously shown
that TARDBP
WT
transgene is also able to rescue the drastically reduced locomotor speed of
mutant flies lacking TBPH, just as overexpressing cazWT [47].

Int. J. Mol. Sci. 2022,23, 2613 7 of 37
2.3. Autism Spectrum Disorder
ASD comprises of complex developmental conditions and it is mainly characterized by
behavioral symptoms such as impaired communication skills, defective social interaction,
repetitive behavior, limited capacity to live independently, etc. but also by a high prevalence
of gastrointestinal problems such as diarrhea, constipation, vomiting, and abdominal pain,
just to mention a few [48–52].
SFARI Gene (https://gene.sfari.org/, accessed on 20 December 2021) is a database
indexing the genes associated with ASD and categorizes these genes according to evidence
of their involvement in ASD [
53
]. Within Category 1 of high confidence for implication in
ASD, there are currently 207 genes, which are also present in other similar gene lists or were
previously identified in an extensive exome sequencing study [
54
]. Out of these, 203 have
orthologs in D. melanogaster, with 141 genes having a Drosophila RNAi Screening Center
integrative ortholog prediction tool (DIOPT) score of at least of 0.6 [
55
]. DIOPT scores are
provided by the DIOPT integrative tool [56], which is currently at version 8.5 and enables
the search of orthologs in different species among the data provided by 18 large-scale
ortholog prediction tools. The aforementioned list of orthologs with conclusive DIOPT
scores includes the D. melanogaster genes Fmr1,Pten or ubiquitin protein ligase E3A (Ube3a),
with DIOPT scores of 0.73, 0.87 and 0.93, respectively [55].
In D. melanogaster,Fmr1 gene is a structural ortholog of FMRP translational regulator 1
(FMR1) human gene. Mutations in FMR1 gene are causing human fragile X syndrome
(FXS), which is probably the most common heritable foundation of autism disorders and
mental retardation [
57
]. The two paralogs of FMR1,FMR1 Autosomal Homolog 1 (FXR1)
and FXR2, also share a strong sequence similarity with Fmr1, thus making it difficult
to choose a certain gene for testing the functional orthology. The Fmr1
50M
-null allele
determines a wide range of mutant phenotypes, the most striking in neurons and germ
cells of adult flies. All three paralog human genes were tested in order to assess whether
mutant phenotypes can be rescued in Fmr1-null mutants. Targeted neuronal expression
of both hWT FMR1 and dWT Fmr1 transgenes rescued the characteristic FXS phenotypes
such as higher brain protein levels, abnormal circadian rhythm patterns determined by
small ventrolateral neurons’ synaptic arbor overgrowth defect and increased synaptic
branching of neuromuscular junction. Expression of FXR1 and FXR2 failed to rescue the
neuronal mutant phenotypes, however, all three human paralogs were equally competent
to overcome non-neuronal symptoms in Fmr1 mutants such as severely reduced fertility
caused by immotile sperm exhibiting defects in sperm tail microtubule organization [
58
].
These results highlight that care should be taken when testing structurally similar functional
orthology candidates, especially when working with genes that manifest both evolutionary
conserved and shared roles.
The PTEN gene has tumor suppressor activity, and even a partial loss of PTEN activity
leads to cancers [
59
] or PTEN hamartoma syndrome, consisting of a variety of disorders
such as macrocephaly, epilepsy, mental retardation and ASD [
60
,
61
]. In mammals as well
as in D. melanogaster, PTEN, which is a dual lipid and protein phosphatase, is a critical
repressor of phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/AKT) pathway [
62
].
In D. melanogaster,Pten
100
/Pten
117
represents a strong hypomorphic heteroallelic combi-
nation that leads to increased larval growth and, consequently, increased pupal volume
and adult weights. Expression of the PTEN
WT
allele in embryos and larvae successfully
rescued the hypomorphic mutants [
63
]. Within the same study, the authors devised a
scalable experimental platform to functionally test about 100 human PTEN alleles with
potential clinical relevance. To do this, they overexpressed an activated Phosphatidylinositol
3-kinase 92E (Pi3K92E) allele (PI3K92E–CAAX–PI3K
act
) in the wing imaginal disc, resulting
in adults harboring enlarged wings. This phenotype was rescued by PTEN
WT
but not by
PTEN
C124S
, which lacks both protein and lipid phosphatase activity, or by the PTEN
G129E
lipid phosphatase dead alleles. PTEN
Y138L
, which is deficient for protein phosphatase
activity, is able to partially alleviate the wing size phenotype. These either failed or partially
successful heterologous rescue experiments demonstrated that PTEN-dependent suppres-

Int. J. Mol. Sci. 2022,23, 2613 8 of 37
sion of PI3K/AKT tissue growth in Drosophila is dependent upon both lipid and protein
phosphatase activities. Using this wing size-based system, they successfully tested the
functionality retained by the PTEN alleles by scoring their ability to partially or completely
rescue the oversized fly wing.
Mutations affecting UBE3A gene are the main cause for AS, a relatively common
human disorder involving aberrant central nervous system development and characterized
by mental retardation and locomotor impairment [
64
]. In our laboratory, we obtained a new
Ube3a allele, symbolized as As
m1.5-R
, consecutive to transposon-mediated mutagenesis [
65
].
This allele is semilethal for mutant homozygous males, which, when surviving to adult-
hood, elicit decreased locomotor performances. We successfully rescued this abnormal
phenotype in flies raised for more than a year on culture medium supplemented with
omega-3 polyunsaturated fatty acids, i.e., eicosapentaenoic and docosahexaenoic acids [
66
].
A true heterologous rescue was demonstrated for the learning abilities of both larvae and
adult Ube3a-null mutants [
67
]. Individuals from both mutant categories exhibited impaired
learning abilities as scored by using the aversive phototaxis suppression assay, which tests
the ability of fruit flies to link an unpleasant taste stimulus with light. Expressing UBE3A
WT
transgene by the pan-neuronal elav-GAL4 driver in the mutant background rescued the
mutant learning defects.
Altogether, the previous rescue and disease modelling examples reinforce the power
of the D. melanogaster experimental model. In addition, many other mammalian genes
associated with neurodegeneration-related diseases were studied in the fly model, as
presented in Table 1.
Table 1.
Successful examples of heterologous rescue experiments related to neurodegeneration.
Within the vertebrate gene column, (h) indicates a human gene while (m) stands for a mouse gene.
Unless otherwise indicated, WT alleles are implicitly considered. HR is the acronym for heterologous
rescue and indicates that the references designate HR studies.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
motor neuron
diseases (h)VAPB Vap33 loss of Vapp33 determines larval
lethality, with few adult escapers
expression of (h)VAPB alleviates the
lethal phenotype determined by
loss of Vap33
[68]
Huntington’s disease
(h)UCP2 UCP5
expression of mutant Huntingtin
protein in glia determines altered
locomotor performances and
uncommon vulnerability to
mechanical stress
co-expression of (h)UCP2 [69]
(h)HTT htt
htt-null flies have severe thorax
muscle loss and accelerated
deterioration in mobility
and lifespan
(h)HTT rescues the htt loss
associated phenotypes [70]
PD
(h)MIC60 MIC60
MIC60mut-null allele determines
pupal lethality in homozygous
individuals; MIC60mut/+ flies
are normal
expression of (h)MIC60 in
MIC60
mut
/+ flies provides a normal
phenotype, while expression of
mutant (h)MIC60A4V, T11A or C17F
leads to severe adult lethality and
reduced larval crawling
[71]
(h)PRKN park
park-null flies exhibit reduced
lifespan, locomotor and fly
defects, infertility, lower cell size
and number, progressive
degeneration of certain
DA neurons
co-expression of (h)PRKN rescues
the neurotoxicity; muscle-specific
expression of (h)PRKN rescues the
flight ability
[72,73]
(h)LRRK2 Lrrk
Lrrk-null mutants elicit autophagy
defects and DA degeneration
overexpression of (h)LRRK2 rescues
the mutant phenotype [74,75]
(h)VPS35 Vps35
downregulation of Vps35 in brain
determines supernumerous
neuroblast phenotype
expression of (h)VPS35 fully rescues
the brain tumor phenotype
exhibited by Vps35 mutants
[76]

Int. J. Mol. Sci. 2022,23, 2613 9 of 37
Table 1. Cont.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
PD; frontotemporal
dementia (h)MAPT tau
loss of tau determines lethality;
deletion of tau in neurons
determines neurodegeneration
expression of (h)MAPT partially
rescues the
neurodegenerative phenotype
[77]
ALS (h)PFN1 chic
RNAi-mediated downregulation
of chic in motor neurons
determines pupal lethality
the chic mutant phenotype is
rescued by expressing (h)PFN1 in
motor neurons
[78]
ALS and other
neurodegenerative
diseases
(h)VCP TER94 TER94 mutations determine
tubular lysosome dysfunction
expression of (h)VCP rescues the
phenotype determined by
mutant TER94
[79]
late-onset AD (h)TM2D3 amx strong neurogenic phenotype
when amx is maternally mutated
(h)TM2D3 is able to partially rescue
the neurogenic phenotype and
embryonal lethality
[80]
Parkinsonism with
spasticity, X-linked;
intellectual
developmental
disorder, X-linked,
syndromic,
Hedera type
(h)ATP6AP2
ATP6AP2
ATP6AP2 depletion is lethal;
RNAi knockdown of ATP6AP2 in
wing pouch leads to abnormal
wing development and
growth defects
expression of (h)ATP6AP2 in
ATP6AP2 RNA1 background
rescues the specific
mutant phenotype
[81]
pediatric-onset
neurodegenerative
disorder
(h)ADPRHL2 Parg
Parg LOF determines decreased
survival in response to
oxidative challenge
lethality is rescued by expressing
(h)ADPRHL2 [82]
neurodegeneration (h)TARDBP TBPH
TBPH-null mutants experience
loss of the ventral nerve cord
neurons (bursicon neurons)
expression of (h)TARDBP rescues
the bursicon neurons [83]
neurodegeneration;
cancer; metabolic
disorder
(h)TOMM70 Tom70
Tom70-null mutation conducted to
pupal lethality
the lethality is rescued by the
expression of (h)TOMM70 [84]
neurodegeneration;
Boucher–Neuhäuser,
Gordon Holmes,
Laurence–Moon and
Oliver McFarlane
syndromes
(h)PNPLA6 sws
the sws1-null mutation causes
locomotion deficits and
neurodegeneration
(h)PNPLA6 rescues the mutant
sws phenotype [85]
the sws1mutants showed
characteristic vacuoles in central
brain and optic lobes
(h)PNPLA6 partially rescues the
vacuolization of mutant sws [86]
pantothenate
kinase-associated
neurodegeneration
(h)PanK2 fbl
a hypomorphic mutation in
fumble results in flies that have
brain lesions, defective
neurological functions and severe
motor impairment
the paralysis and impaired climbing
activity are rescued by expressing
(h)PanK2
[87]
in mice, neonatal
lethality, slow
progressive
neurodegeneration,
enhanced
limb-clasping
reflexes, impaired
motor activity,
cognitive deficits and
hypomyelination [
88
]
(h)NRD1 Nrd1 LOF allele causes
neurodegeneration
expression of (h)NRD1 rescues the
pupal lethality and
electroretinogram defects
[89]
chorea-
acanthocytosis,
neurodegeneration,
progressive loss of
cognitive and
locomotor functions
(h)VPS13A Vps13
mutant flies have age-linked
neurodegeneration and
reduced lifespan
overexpression of (h)VPS13A in
mutant flies rescues the
characteristic phenotype
[90,91]
Alkuraya-Kucinskas
and Oliver
Mcfarlane syndromes
(h)DENND4A Crab
flies lacking Crab activity
experience age-dependent decline
in photoreceptor function and
structural integrity
expression of (h)DENND4A rescues
the eye defects exhibited by the
mutant flies
[92]

Int. J. Mol. Sci. 2022,23, 2613 10 of 37
Table 1. Cont.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
neurodegenerative
encephalopathy (h)TBCD TBCD
projection neurons expressing
TBCD1mutant allele have
affected axonal branches
overexpression of (h)TBCD
extensively suppresses the axonal
mutant phenotype
[93]
neuronal K+–Cl−
cotransporter;
epilepsy
(h)SLC12A5 kcc
kccDH1 hypomorphic allele acts as
a seizure-enhancer mutation and
exacerbates the bang-sensitive
paralytic behavior
(h)KCC2 rescues the mutant
phenotype induced by kccDH1 [94]
progressive
myoclonus epilepsy (h)GOSR2 membrin homozygosity for membrin-null
allele causes larval lethality
expressing (h)GOSR2 fully rescues
the larval lethality, but the adults,
although normal looking, display
severe motor impairments
[95]
early infantile
epileptic
encephalopathy
(EIEE)
(h)ACTL6B
(BAF53B)Bap55
mutations in Bap55 affect the
synaptic connections in
olfactory neurons
(h)ACTL6B rescues the mutant
phenotype of Bap55-null individuals
[96]
photosensitive
epilepsy (PSE) (h)SGMS1 -
cpes-null mutants show
compromised ceramide
phosphoethanolamine synthase
and fail to complete neuronal cell
body encapsulation in the
neuronal cortex
expression of (h)SGMS1 rescues the
PSE and cortex glial aberrations [97]
ASD
(h)TaoK2 Tao
loss of Tao determines overgrowth
of dendritic branching and
behavioral defects
(h)TaoK2 restores the aberrant
dendritic branches to control levels [98,99]
(h)DAT
(SLC6A3)DAT DAT KO flies are hyperactive DAT KO flies expressing (h)DAT
have reduced locomotion [100,101]
(h)SCAMP1,
(h)SCAMP5 Scamp
Scamp-null flies exhibit shortened
lifespan, compromised climbing,
heat-induced seizures and
compromised learning and
long-term memory
both (h)SCAMP1 and (h)SCAMP5
rescue the climbing mutant
phenotype; (h)SCAMP1
significantly improves the learning
index of Scamp-null flies
[102]
autism; multiple
myeloma (m)Nbea rg
rg-null mutants exhibit aberrant
associative odor learning,
modification of gross brain
morphology and of
synaptic architecture
the transgene (m)Nbea is able to
rescue only aversive odor learning
and synaptic architecture
[103]
affected development
of distinct cell types
in the central nervous
system and in
sensory systems
(m)Math1 ato
mutant ato embryos lack
precursor cell selection and
chordotonal organ specification
expression of (m)Math1 under the
control of the ato
embryonic enhancer [104]
in mouse, (m)Math1-null animals
do not succeed to initiate
respiration and die soon
after birth
replacing (m)Math1 coding region
with ato allowed the animals to
survive to adulthood
CMT type 2A, axon
degeneration [105]
(h)MFN1,
(h)MFN2 Marf
mutant flies have affected
mitochondria and, as a
consequence, their nerves cannot
send out signals to muscles; in
addition, Marf is lost in the ring
gland affecting the production of
a hormone required for larva
transition to adult, the mutants
dying in their larval stage
expression of both (h)MFN1 and
(h)MFN2 is necessary for hormone
production and the rescue of
all phenotypes
[106]
CMT neuropathy (h)GDAP1 Gdap1 knockdown mutants experience
retina and muscle degeneration
the mutant phenotype is rescued by
(h)GDAP1 [107]
dominant-
intermediate CMT
neuropathy
(h)YARS TyrRS RNAi-silenced TyrRS determines
specific bristle phenotypes
expressing (h)YARS rescues the
abnormal bristle phenotype [108]

Int. J. Mol. Sci. 2022,23, 2613 11 of 37
Table 1. Cont.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
CMT neuropathy
type 2D (h)GARS GlyRS
GlyRS-null flies lack dendritic and
axonal terminal arborization
(h)GARS rescues the arborization
defects in GlyRS-null flies [109]
autosomal recessive
cerebellar ataxia (h)UBA5 Uba5
Uba5-null mutants have reduced
lifespan and locomotor activity as
well as neuromuscular junction
(NMJ) defects
(h)UBA5 expression significantly
rescues the NMJ mutant phenotype [110]
FA (h)FXN fh
fh mutants have altered
mitochondrial functions and
exhibit age-dependent
neurodegeneration
expression of (h)FXN rescues the
neurodegeneration [111]
ataxia determined by
defects of autophagy (h)ATG5 Atg5 flies lacking Atg5 activity are
unable to walk and fly properly
(h)ATG5 restores the mutant flies’
normal movements; (h)ATG5E122D
slightly improves the
defective mobility
[112]
X-linked
Snyder–Robinson
syndrome
(h)SMS Sms
Sms mutants have critically
lowered transcript levels that
reduce viability
(h)SMS rescues the viability
of mutant flies [113]
Delpire–Mcneill
syndrome
(h)SCL12A2
(NKCC1)Ncc69
Ncc69 mutants reach adulthood
but their abdominal nerves are
swelled and form bulges
this neuropathy is rescued by
(h)SCL12A2 [114,115]
microcephaly; Zika
virus target (h)ANKLE2 Ankle2
mutations in Ankle2 can lead to
loss of peripheral nervous system
organs in adults and severely
reduced brain size in hemizygous
third instar larvae
expression of (h)ANKLE2 rescues
the mutant phenotype [116,117]
neural network
formation; tumor
progression
(m)Bsg Bsg
mutations in Bsg alter the cell
architecture and can lead to high
embryo or larval lethality
Bsg LOF in adults’ eyes determines
mislocalization of photoreceptor
nuclei, a phenotype rescued by
expressing (m)Bsg
[118]
global developmental
disorders, intellectual
disability
(h)CAPZA2 cpa cpa-null allele determines first
instar lethality
(h)CAPZA2 rescues the lethal
phenotype of cpa-null individuals [119]
autosomal recessive,
nonsyndromic
intellectual disability
(h)ZC3H14 Nab2
Nab2-null flies experience
developmental and
locomotor defects
(h)ZC3H14 expressed in neurons
rescues the Nab2-null phenotype [120]
Troyer syndrome (h)SPG20 spartin
loss of spartin is associated with
motor dysfunctions and brain
neurodegeneration
synaptic overgrowth in spartin-null
flies is rescued by presynaptic
expression of Myc-tagged
(h)ZC3H14
[121]
intellectual disability (h)OPHN1 Graf loss of Graf affects the mushroom
body (MB) development
expression of (h)OPHN1
significantly ameliorates the MB
mutant phenotype
[122]
intellectual disability,
X-linked (h)CASK CASK
affected expression of CASK
negatively impacts middle-term
and long-term memory
overexpression of (h)CASK in
neurons of CASK mutants fully
rescues the memory
[123]
intellectual disability,
X-linked (h)ACSL4 Acsl
Acsl mutants exhibit
neuromuscular
junction overgrowth
expression of (h)ACSL4 rescues the
mutant phenotype particular to
Acsl mutants
[124]
intellectual disability (h)SMARCA5 Iswi
Iswi LOF is related to decreased
body size and movement in larvae
and decreased brain size and
locomotor dysfunctions in adults
(h)SMARCA5 expression rescues
the Iswi specific mutations [125]
nervous system
developmental
defects
(h)EBF3 kn homozygous kn-null genotype is
embryo lethal (h)EBF3 rescues the lethality [126]

Int. J. Mol. Sci. 2022,23, 2613 12 of 37
Table 1. Cont.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
autosomal recessive
neurologic disorder (h)TMTC3 Tmtc3
neuron-specific knockdown of
Tmtc3 rises the incidence of
mechanically induced seizures
neuron-specific expression of
(h)TMTC3 [127]
intellectual
developmental
disorders
(h)IQSEC1 siz
loss of siz affects the growth cones
and causes embryonal lethality
overexpression of (h)IQSEC1 in WT
fly background is toxic; lowered
expression of (h)IQSEC1 in siz-null
mutants partially rescues the
embryonal lethality
[128]
developmental delay,
movement disorders
and metabolic
decompensation
(h)OGDH Ogdh
LOF allele is associated with early
developmental lethality
the expression of (h)OGDH rescues
the mutant phenotype [129]
infantile
encephalopathy
(lethal)
(h)DNM1L Drp1
Drp1 mutants have altered
mitochondrial trafficking and die
as larvae
ubiquitous expression of (h)DNM1L
rescues the lethality [130]
schizophrenia (h)DTNBP1 Dysb
Dysb mutants have compromised
memory, elevated climbing
activity, abnormal male-male
courtship behavior,
hypoglutamatergic and
hyperdopaminergic activities
pan-neuronal or glial expression of
(h)DTNBP1 rescues various Dysb
mutant phenotypes
[131]
Pitt–Hopkins
syndrome
(h)TCF4-A,
(h)TCF4-B da
da-null allele severely impacts the
embryonic nervous
system development
both (h)TCF4-A and (h)TCF4-B
rescue the mutant
embryo phenotype
[132]
neurofibromatosis,
type 2 (h)NF2 Mer Mer-null mutations
determine lethality
isoform 1 of (h)NF2 is able to rescue
the lethality of Mer-null mutants [133]
3. Cardiac Disorders
Several key characteristics related to cellular processes, signalling pathways and gene
conservation endorse D.melanogaster as a model of choice for studying human cardiac devel-
opment, function and diseases. First, although seemingly simplistic, the fruit fly circulatory
system, consisting of a tube-like heart that pumps the haemolymph, shows developmen-
tal and functional similarities to the vertebrate heart [
134
–
137
]. Second, both organisms
(D. melanogaster and H. sapiens) share some regulatory cardiogenic networks encompass-
ing critical cardiac transcription factors such as tin/Nkx2.5,Mef2/Mef2C,pannier/GATA
family and Hand/HAND1 and HAND2, which are required for cardiac progenitor specifica-
tion [
137
–
143
]. Third, there is a strong gene conservation with human genes, particularly
with disease-related genes [
144
]. By performing a systematic BLAST analysis of 929 human
disease gene entries associated with at least one variant in the Online Mendelian Inheritance
in Man (https://www.omim.org/, accessed on 18 December 2021) database against the
reference sequence of D. melanogaster, Reiter and colleagues revealed that 77% of disease
genes queried had Drosophila counterparts [
144
]. Of note, 26 of them were associated with
various cardiovascular diseases such as cardiomyopathies, hypertension and conduction
defects [3].
3.1. Congenital Heart Defects
Congenital heart defects (CHDs) are the most common birth disorders, affecting 0.8%
to 1.2% of live infants [
145
]. Although it is largely acknowledged that genetic factors are
strongly involved in CHD pathogenesis, the great majority of responsible genes remain
elusive [
146
]. Drosophila-based research enabled the recognition of new CHD-related genes.
Zhu et al. [
147
] developed a Drosophila-based functional system to rapidly and efficiently
screen large numbers of candidate genes detected in patients with severe CHDs. By using
heart-specific RNAi silencing, they tested 134 genes, of which more than 70, including a sub-
group encoding histone modifying proteins, were found to be essential for the development,

Int. J. Mol. Sci. 2022,23, 2613 13 of 37
structure and function of the fruit fly heart. The silencing of genes responsible for H3K4 and
H3K27 methylation (i.e., kis/CHD7,wds/WDR5,Trx/MLL2) caused developmental lethality
(up to 84%) and severe structural heart anomalies and reduced adult longevity. Moreover,
a gene substitution strategy comprising concurrent heart-specific silencing of the fly gene
homolog and expression of either a wild-type variant or a pathogenic one was applied to
validate the role of these genes in CHDs. As a proof-of-concept, the authors explored the
potential of the WDR5
WT
human allele to rescue the pathogenic phenotype generated by
silencing of the endogenous wds Drosophila homolog. WDR5
WT
overexpression significantly
reduced developmental lethality and restored abnormal heart morphology, as opposed to
the CHD patient-derived WDR5
K7Q
mutant allele, which resulted in similar pathogenic
cardiac manifestations.
3.2. Cardiomyopathy Phenotypes
Cardiomyopathies are a heterogeneous group of myocardial diseases in which the
cardiac muscle is structurally and functionally abnormal, often due to a genetic cause [
148
].
The injury can be limited to the heart or part of a generalized systemic disorder; either way,
the genetic architecture is very diverse [149,150].
Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy,
affecting at least 1 in 500 individuals in the general population [
151
,
152
]. The underlying
genetic etiology is complex, mainly involving variation in sarcomeric or sarcomeric-related
genes, but mutation in other genes can cause similar phenotypes comprising left ventricular
hypertrophy (LVH). Of the 57 candidate genes included in diagnostic HCM gene panels,
only 8 have been nominated as having definitive evidence, including myosin light chain 2
(MYL2) [153,154].
Recently, Manivannan and colleagues identified a novel recessive frameshift variant
in MYL2 (p.Pro144Argfs*57) resulting in early-onset HCM and death in infancy [
155
]. A
fly model was used to demonstrate that p.Pro144Argfs*57 variant was in fact a LOF allele.
The expression of the Drosophila ortholog Mlc2 was knocked down using transgenic RNAi
lines, which led to multiphasic lethality, with progenies dying before the pupal stage, and
impaired systolic function. Both the developmental lethality and cardiac dysfunction were
partially rescued by MYL2
WT
but not by the frameshift variant. The incomplete restoration
of fly phenotype was most likely due to sequence differences between the two organisms,
given that the Mlc2 N-terminal region has additional sequences that are required for its
function [156].
An HCM-like phenotype can be encountered in other conditions involving LVH,
such as FA, which is a neurodegenerative disease caused by a GAA trinucleotide repeat
expansion in frataxin gene (FXN) [
157
,
158
]. Over time, the cardiomyopathy potentially
progresses to a dilated form. It has been shown in D. melanogaster that RNAi-mediated
frataxin (fh) depletion prompted enlargement of cardiac diameters and reduction in systolic
function, which were fully rescued by complementation with FXNWT human allele [159].
Gonçalves et al. [
160
] reported a study concerning three human families A, B and
C affected by mutations in adducin 3 (ADD3) gene encoding for adducin-
γ
, associated
with various disabilities such as intellectual disability, microcephaly, cataracts and skeletal
defects. Patients from family A are also homozygous for a missense mutation of lysine
acetyltransferase 2B (KAT2B) and the expanded pathologic spectrum, including cardiomy-
opathy and renal problems. This allele is symbolized as KAT2B
F307S
and determines the
substitution of highly conserved Phe with Ser at position 307 of the human protein. These
genetic disorders are prone to be modeled on D. melanogaster, since the fruit fly genome
contains the hu li tai shao (hts) ortholog for ADD1 (adducin-
α
), ADD2 (adducin-
β
) and ADD3,
and Gcn5 acetyltransferase (Gcn5) paralog for KAT2A and KAT2B.

Int. J. Mol. Sci. 2022,23, 2613 14 of 37
The hts
null
hemizygous flies die as late larvae and only a few impaired, short living
escapers reach the adult stage. Expression of ADD3
WT
does not rescue viability, but the
ubiquitous co-expression of ADD1
WT
and ADD3
WT
leads to heterologous rescue by increas-
ing the number of viable adults. Alternatively, ADD1
WT
/ADD3
E659Q
co-expression (where
ADD3
E659Q
is a human mutant allele reported for family A) induces only a partial rescue of
hts
null
hemizygous flies, revealing that ADD3
E659Q
behaves as a hypomorphic allele in the
fruit fly mutant background. The hts mutant flies rescued by ADD1
WT
/ADD3
E659Q
did not
show any significant differences in heart period, cardiac output, fractional shortening and
arrhythmia index as compared to those rescued with ADD1WT/ADD3WT.
The genetic analysis of an alleleof Gcn5 gene in D. melanogaster equivalent to KAT2B
F307S
supports the hypothesis that KAT2B is associated with heart and kidney mutant phenotypes
in humans. Specifically, the Gcn5
E333st
allele, also referred as Gcn5
null
,is lethal in hemizy-
gous individuals, which arrest development at the late larval to early pupal stage [
161
].
When KAT2A
WT
and KAT2B
WT
are expressed either individually or synchronously in hem-
izygous Gcn5
null
flies, the rescue of transgenic flies fails, suggesting that these orthologous
genes have functionally diverged during evolution. As expected, transgenic hemizygous
Gcn5
null
flies appear to be completely rescued by the Gcn5
WT
allele, but partially rescued
by the Gcn5
F304S
allele, as lethality still often occurs in pupae or in early adults and the
escapers exhibit morphological impairments. Gcn5
F304S
resembles KAT2B
F307S
and encodes
a protein variant having Ser instead of Phe at position 304. Remarkably, the escapers
expressing Gcn5
F304S
not only have obvious morphological mutant phenotypes, but also
present a prolonged heart period and reduced cardiac output comparative to both a control
strain and Gcn5null hemizygous flies rescued with Gcn5WT.
RNAi silencing of Gcn5 (Gcn5
RNAi
) in D. melanogaster induces functional heart prob-
lems, while silencing of hts (hts
RNAi
) does not. However, silencing of both genes in Gcn5
RNAi
and hts
RNAi
flies aggravates heart period length and the arrhythmia index induced by Gcn5
knockdown alone. These data reveal that Gcn5 is directly involved in heart function in
D. melanogaster, while some hts mutations increase the severity of the Gcn5-null pheno-
type [160], spotting hts as a potential genetic enhancer of Gnc5.
Such complex heterologous rescue experiments of specific D. melanogaster mutants
simulate patients with multilocus genetic diseases, affected by pathogenic mutations lo-
cated in more than one gene. In this case, simultaneous knockdown of hts and Gcn5
concurrently increased the severity of heart phenotype in fruit flies, but the heterologous
rescue succeeded only for hts. Nevertheless, when this partial success is corroborated with
cardiac phenotypes reported for the equivalent KAT2B
F307S
and Gcn5
F304S
alleles and the
RNAi results, the perspectives are encouraging. It is reasonable to conclude that various
interactions between ADD3 and KAT2B variants in patients can be mirrored by interplays
of hts and Gcn5 alleles in D. melanogaster, helping experts to develop novel drugs able to
restore the normal cardiac phenotype.
Intriguingly, a closer inquiry revealed that Gcn5
WT
rescues lethality and the mor-
phology of wings, legs and eyes of hemizygous Gcn5
null
flies, but, similar to Gcn5
F304S
transgenic individuals, these organisms have a smaller diastolic diameter as compared
to control flies. A possible explanation for this phenotype is that Gcn5
WT
transgene is
not in its natural genomic environment where its regulators reside. Nevertheless, when
compared to the Gcn5
WT
rescued flies, the Gcn5
F304S
transgenic ones exhibit supplemental
cardiac impairments as reduced contractility and a more irregular heartbeat. This case is a
very interesting one, as it shows that subtle phenotypes may still be present even when a
complete intraspecific rescue is reported. It seems that sporadic complete phenotype rescue
results may remain partial to some degree, as subtle mutant phenotypes may be difficult to
notice unless specifically searched for, as exemplified in the study of Gonçalves et al. [
160
].

Int. J. Mol. Sci. 2022,23, 2613 15 of 37
Fundamental questions emerge when considering that KAT2A
WT
and KAT2B
WT
coun-
terintuitively fail to rescue hemizygous Gcn5
null
flies, pointing to a functional divergence.
Why are KAT2B
F307S
and Gcn5
F304S
equivalent alleles associated with similar cardiac phe-
notypes in humans and flies, suggesting an inter-specific functional conservation? Should
one always expect that a structural gene orthology is concluded by heterologous rescue
experiments? Why does functional complementation of hemizygous Gcn5
null
flies with
human WT alleles fail? Considering that most heterologous rescue experiments reported
for D. melanogaster were performed using the GAL4-UAS system, it is helpful to consider
the recent work of Casas-Tintóet al. [
162
]. Due to carefully designed experiments, the
authors conclude that the expression of enhancer-Gal4 constructs may be transiently ectopic
and influenced by the genomic insertion site. Added to the fact that not a complete human
gene sequence but a human cDNA, without regulatory sequences, is usually cloned in
a UAS vector, we presume that the unstable activation history of some enhancer-Gal4
constructs may interfere with the heterologous rescue results.
3.3. Other Cardiac Disorders
Other cardiac disorders have been modelled in D. melanogaster, such as channelopa-
thies
[163–167]
and different syndromic [
168
–
170
] or nonsyndromic cardiomyopathies
[171–177].
To our knowledge, currently, none of these diseases benefit from effective human allele-
based functional complementation studies, although some groups successfully tackled the
heterologous rescuing of fly cardiac phenotype, the alleles of choice being of animal origin,
mainly from mice [
173
,
178
,
179
]. For example, Gao’s group reversed the effect of loss of
fly
γ
-sarcoglycan (Scg
δ
) by using a murine counterpart gamma-sarcoglycan (Sgcg) [
179
]. An
engineered form of the Sgcg (termed Mini-Gamma) has been introduced into flies, and
it efficiently rescued the cardiac phenotype of an amorphic allele of Scg
δ
. Mini-Gamma
was generated by removing a portion of extracellular domain of Sgcg that contained a
large frameshift deletion, which led to a premature stop codon. Exon skipping corrected
the reading frame, expression of Mini-Gamma in the heart tube being sufficient to restore
cardiac function to wild-type magnitudes.
A gene involved in cardiac dysfunction independently from canonical Wnt signaling is
pygopus (pygo), which maintains normal heart physiology in aging D. melanogaster [
180
] and
is involved in the differentiation of intra-cardiac valves [
181
] of the fruit flies. Knockdown
pygo mutant allele underpins cardiac arrhythmias and decreased contractility with systolic
dysfunction in fruit flies [
182
]. The cardiac impairments determined by knockdown pygo
allele in D. melanogaster resemble increased incidence of atrial fibrillation in senior humans.
Although Pygo1 and Pygo2 are not essential for heart function and development in mouse,
they may be involved in preventing senescence phenotypes specific for aging hearts in
mammals [180].
An experiment of interest would be the functional rescue of the cardiac phenotype of
pygo knockdown fruit flies with the transgenic Pygo1
WT
allele, a plausible scenario, as the
lethality of pygo
130
-null embryos was rescued by both PYGO1
WT
and hPYGO2
WT
human
alleles [
182
]. Again, the experimental paradigm is that if heterologous rescue of a specific
severe phenotype such as lethality is possible, then rescuing subtle mutant phenotypes
determined by the same orthologs is reasonably plausible.
The use of high-throughput sequencing techniques and wide-ranging cardiac gene pan-
els dramatically increased the detection of variants of uncertain significance (VUS)
[183–186],
whose definite classification requires additional studies including functional ones. The
previously presented data, as well as data from Table 2, demonstrate the structural and
functional homologies between fruit fly and human cardiac genes and advocate the use
of D. melanogaster system as a prime candidate to study and validate genetic variants
associated with cardiac disorders.

Int. J. Mol. Sci. 2022,23, 2613 16 of 37
Table 2.
Successful heterologous rescue experiments related to heart disease. Within the vertebrate
gene column, (h) indicates a human gene, while (m) stands for a mouse gene. Unless otherwise
indicated, WT alleles are implicitly considered. HR is the acronym for heterologous rescue and
indicates that the references designate HR studies.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
cardiac dysfunction
(postulated),
TRiC/CCT complex
(h)CCT4 CCT4
RNAi-silenced CCT4
determines pupal lethality
and growth defects
overexpression of (h)CCT4
rescues the mutant phenotype [187]
lipotoxic
cardiomyopathy,
ceramide/sphingolipid-
related
(h)DEGS1 ifc knockout of ifc results in
larval lethality
(h)DEGS1 rescues the
lethal phenotype of ifc null
individuals
[188]
congenital heart defect
(postulated),
KMT2-related
(h)KMT2A
(MLL)trx
LOF mutations determines
larval to pupal lethality
associated with aberrant
cuticular patterns
expression of (h)MLL partially
rescues the cuticular phenotype [189]
dilated
cardiomyopathy 3B (m)Dmd Dys
loss of Dys function leads to
reduced lifespan,
significantly increased heart
rate, age-dependent
myofibrillar disorganization,
cardiac chamber
enlargement and impaired
systolic function
the mutant phenotype was
partially reversed by expression
of a truncated (m)Dmd
which restores the cardiac
diameters and function
[173,178]
Noonan syndrome (h)PTPN11
(SHP-2)csw csw mutations determine
zygotic lethality
expression of (h)SHP-2 rescues
the zygotic lethality [190]
muscle and aortic
defects, ARIH1-related (h)ARIH1 ari-1
ari-1-null allele is associated
with affected larval muscle,
lethality or reduced lifespan
in adults
(h)ARIH1 rescues
ari-1-related lethality. [191]
4. Cancer
Cancer is a multifactorial and multistep disease characterized by uncontrolled prolifer-
ation of tumor cells that escape the control of physiological growth sentinels, apoptosis de-
fects and metabolic alterations. These signaling pathways are conserved in D. melanogaster,
making the fruit fly an appropriate model organism to study cancer biology [
192
–
195
].
Important processes such as genomic instability, strategies to evade apoptosis, telomerase
reactivation, tumor-promoting inflammation and evasion from the immune system, angio-
genesis, anaerobic glycolysis, competitiveness of cancer stem cells, importance of tumor
microenvironment, invasiveness and metastasis, cancer cachexia, drug screening and resis-
tance have been extensively studied and modeled in D. melanogaster [
196
]. Several examples
are provided in Table 3and the following subchapters.

Int. J. Mol. Sci. 2022,23, 2613 17 of 37
Table 3.
Positive heterologous rescue experiments related to cancer. Within the vertebrate gene
column, (h) indicates a human gene. Unless otherwise indicated, WT alleles are implicitly considered.
HR is the acronym for heterologous rescue and indicates that the references designate HR studies.
Clinical Impact Vertebrate
Gene
Fly
Gene Mutant Phenotype (Fly) Heterologous Rescue HR
References
epithelial cancer
(h)LLGL1
and
(h)LLGL2
l(2)gl l(2)gl/l(2)gl genotype
determines lethality
(h)LLGL1 partially rescues the
homozygous l(2)gl lethal
phenotype; imaginal tissues do not
show any neoplastic features, with
Dlg and Scrib exhibiting the correct
localization; animals undergo a
complete metamorphosis and hatch
as viable adults
[197]
(h)HUGL-1 lgl mutations in lgl determine
structural defects in larvae
(h)HUGL-1 expression in the
homozygous lgl mutants leads to a
partial development of rudimental
eyes and larval structures
comparable to wild type
(h)LATS1
and
(h)LATS2
Wts developmental defects,
lethality in flies
(h)LATS1 rescues all developmental
defects including embryonic
lethality in flies
[198]
(h)Scrib scrib
polarity
and neoplastic
overgrowth defects
(h)Scrib rescues the polarity
and neoplastic overgrowth defects
of scrib mutants
[199]
(h)JRK/JH8 ebd1 muscle defects in ebd1 and
ebd1/ebd2 double mutants
(h)JRK/JH8 has rescued the flight
muscle defects in ebd1 as well as in
ebd1/ebd2 double mutants
[200]
(h)CC2D1A
and
(h)CC2D1B
Lgd tissue hyperplasia in Lgd
mutant phenotype
(h)CC2D1A and (h)CC2D1B rescue
the Lgd mutant phenotype [201]
various cancers (h)TP53 p53
p53-null embryos have high
sensitivity to genotoxic
stressors such as irradiation
(h)TP53 partially rescues the
embryo liability to irradiation [202]
acute myeloid
leukemia
(h)MLF1
and
(h)MLF2
Mlf
Mlf LOF phenotypes
include the decrease in
embryonic crystal cell
numbers and adult bristle
and wing phenotypes
expression of (h)MLF1 and (h)MLF2
rescues several Mlf
LOF phenotypes
[203]
(h)CUX1 ct ct deficient flies exhibit
abnormal wing phenotypes
(h)CUX1 rescues the ct mutant
phenotypes [204]
Epithelial cancer is the most studied type of cancer on the D. melanogaster model, as
revealed by the high number of citations recorded in FlyBase. This is at least partially
explained by the fact that the fruit fly larval imaginal discs, which are morphologically
and biochemically comparable to mammalian epithelia, could be used to model different
processes involved in the epithelial cancer onset and progression. The imaginal wing
and eye discs have been successfully used to study tumor growth and invasion, investi-
gate the function of cancer genes, analyze oncogenic cooperation and perform chemical
screenings [205–209].
4.1. Validating Orthologs of Human Tumor Suppressors Using the Drosophila melanogaster Model
In D. melanogaster, three complexes are involved in regulating cell growth and dif-
ferentiation: Crumbs/Stardust/PATJ/Bazooka, Par6/aPKC (atypical protein kinase-C)
and Scrib/Dlg/Lgl (Scribble/Discs large/Lethal giant larvae) complexes [
210
]. The lgl
was the first neoplastic tumor suppressor gene discovered in Drosophila, whose loss leads

Int. J. Mol. Sci. 2022,23, 2613 18 of 37
to an abnormal development (disruption of cell polarity and tissue architecture, uncon-
trolled proliferation and tumor growth) of the imaginal structures and the larval brain.
The apical–basal polarity loss in epithelial cells often occurs in human epithelial cancer,
facilitating invasion and metastasis, and therefore a more aggressive profile of the ma-
lignancy [
211
]. Following their transplantation into wild-type recipients, the lgl mutant
imaginal tumorous tissues could migrate and metastasize in other regions of the fruit fly
body, killing the host, thus resembling the human secondary cancers [
212
]. Other features
shared with human metastasis are represented by the upregulation of type IV collagenase
and NDP kinase in lgl-induced tumors [
213
,
214
]. Mammalian homologues of the lgl gene
(HUGL-1/Llgl1 and HUGL-2) are highly conserved in humans, highlighting their role in
cell growth and initiation of neoplastic lesion. From the two human homologues of the lgl
gene, the HUGL-1 LOF has been reported in different types of human cancer (e.g., breast,
melanomas, prostate, ovarian and lung cancers) and HUGL-1 rescued all the defects of the
fly lgl mutant. For the rescue experiments, the null allele lethal(2)gl4 has been used. The
flies homozygous for the mutant allele are headless pharate, with the eye imaginal disc
structure completely lost in the third instar larval stage [
215
]. The insertion of the HUGL-1
cDNA in the homozygous mutants led to a partial development of rudimental eyes and
larval structures comparable to wild type. The ubiquitous expression of HUGL-1 in lgl-null
fruit flies assured the recovery of viable phenotypes (viable adults or completely developed
pharate). Despite being completely sterile, they did not develop neoplasia during their
lifespan and showed normal imaginal structures, compared to that of the wild-type adults.
These results demonstrate that HUGL-1 can act as a tumor suppressor in D. melanogaster
and thus represents the functional homologue of lgl [197].
4.2. Elucidating the Role of Tumor Microenvironment and Host-Neoplastic Cells Competition in
Gut Adenoma Development
It is largely accepted that the tumor microenvironment plays an important role in the
tumor’s progression, exhibiting either pro-growth or inhibitory effect on the proliferation
and invasiveness of neoplastic cells [
216
]. Cancer cells use cell competition as a form
of interaction within the tumor microenvironment [
217
,
218
]. Cell competition was first
described as a quality control mechanism in Drosophila defined as the ability of wild-type
cells to kill the mutant cells harboring reduced fitness and growth potential [
219
]. However,
normal cells could also be killed by tumor mutant cells called supercompetitor cells, leading
to the development of hyperplasia and adenomas in the adult Drosophila midgut [
220
].
Studies in the Drosophila model have demonstrated that cooperation between the tumor
suppressor adenomatous polyposis coli (APC) gene and wingless (Wnt) is involved in cell
competition. Two APC proteins, APC1 and APC2, with different domains and tissue
distribution are shared by mammals and Drosophila. Fly APC1 and APC2 functions are
partially redundant in regulating Wnt signaling and cytoskeletal reorganization [221].
As also reported for humans, the APC inactivation in Drosophila leads to very high
levels of Wnt target gene expression in different tissues, including the intestine. Akin to the
mammalian intestine, Drosophila adult midgut epithelium cells have a high turnover rate
maintained by the intestinal stem cells (ISCs). Therefore, they have been used as a model
to elucidate the role of different signaling pathways in adenoma formation and the role
of different mutations in tumor development [
222
–
226
]. Loss of function of APC leads to
abnormal proliferation of ISCs, followed by the loss of gut epithelial cell polarity, hyperpla-
sia and epithelial overgrowth [
227
,
228
]. On the other side, the Wnt genes are expressed at
very high levels in colorectal tumors harboring mutations in APC. This proves that upregu-
lation of Wnt expression in human ISCs is associated with adenoma development [
229
].
Importantly, loss of APC1 leads to the activation of Wnt signaling in retinal photoreceptors,
inducing their ectopic apoptosis. Thus, the APC1 mutant eye phenotype could be used to
investigate the roles of Wnt signaling pathways in health and disease [
230
,
231
]. Using this
Drosophila model, it has been shown that the Wnt pathway genes’ expression in Drosophila is
regulated by transcription cofactors such as earthbound1 (ebd1) and erect wing (ewg) [
200
,
232
].

Int. J. Mol. Sci. 2022,23, 2613 19 of 37
From these, ebd1, which harbors Centromere Protein B (CENPB) DNA binding domains, is
essential for the Wnt-dependent control of ISC proliferation. Using the APC1 mutant eye
phenotype, it has been demonstrated that ebd1 heterozygotes induce a partial suppression
of APC1 mutant apoptosis, the rescue being nearly complete in ebd1 homozygotes. The
human homologue of ebd1 (JRK/JH8) is overexpressed in several carcinomas including
colon, breast and ovarian serous cystadenocarcinoma, and has been proven to be associated
with elevated expression levels of Wnt target genes in human colorectal tumors. ebd1 and
its human ortholog share structural and functional similarity and interact directly with
Arm/
β
-catenin associated in a ternary complex with T cell factor (TCF) [
233
]. The human
Jerky has rescued the flight muscle defects in ebd1 as well as in ebd1 and ebd2 double mutants
proving its functional equivalence to Ebd1 and Ebd2 [200].
4.3. Demonstrating the Species-Dependent Pathways of Notch Hyperactivation
Coiled-coil and C2 domain-containing protein (CC2D) 1A and 1B are members of the
Lgd protein family, conserved among metazoans, with many partially redundant functions
such as centrosomal cleavage, molecular signaling, innate immunity response (by modulat-
ing TLR3, TLR4 and RLR pathways) and synapse maturation [
234
,
235
]. The D. melanogaster
ortholog Lgd, a tumor suppressor gene, has been shown to be involved in Notch signal-
ing. Notch is a conserved developmental signaling pathway involved in essential cellular
processes such as differentiation, pattern formation, cell-cycle progression, morphogen-
esis, migration, apoptosis, T cells activation etc., which is dysregulated in many cancer
types [
236
]. The Notch signaling occurs upon the direct contact between a signal-receiving
and a signal-sending cell, mediated by the binding of Delta/Serrate/LAG-2 (DSL) ligand
to the trans-membrane Notch receptor. Upon binding, the extracellular domain cleavage
allows the release of active receptor Notch intracellular domain (NICD), which accumulates
in the nucleus and regulates downstream genes in concert with other proteins [
237
,
238
].
Loss of function of lgd in fruit flies led to constitutive ligand-independent activation of
Notch in epithelial cells and to tissue hyperplasia [
239
–
245
]. However, this effect was not
confirmed in mouse epithelial gut, suggesting that lgd genes are not involved in the Notch
pathway hyperactivation in mammals. In humans, NICD nuclear accumulation was associ-
ated with increased tumor cell growth and cell survival and treatment failure in different
types of malignancies such as breast, lung and pancreatic cancer [
246
–
249
]. The functional
homology between human CC2D1A and CC2D1B and the fly lgd was demonstrated in
rescue experiments. Among the two orthologs, one copy of CC2D1B was sufficient to
completely rescue the mutant [
201
]. Moreover, the expression of CC2D1A and CC2D1B is
under the control of the endogenous promoter of lgd [244].
5. Infectious Diseases
The D. melanogaster model provides a valuable tool for studying the molecular mecha-
nisms of different infectious diseases, the processes involved in anti-infectious immunity
and for pharmacological screenings, as thoroughly detailed in the recent review of Har-
nish et al. [
246
]. This is at least partially because many infectious agents often modulate
highly conserved innate immunity pathways such as the Nuclear Factor kappa B (NF-
κ
B)
and c-Jun N-terminal Kinase (JNK) signaling, phagocytosis and apoptosis, which are also
present in the fruit fly. We will present below some examples illustrating that D. melanogaster
represents an appropriate experimental model to recapitulate Koch–Evans postulates [
247
]
regarding the reproduction of human phenotypes for some infectious diseases.
5.1. Molecular Mechanisms of Neuropathological Effects Caused by Zika Virus NS4A Protein
Zika virus is an emerging mosquito-borne flavivirus closely related to Dengue and
West Nile viruses [
248
]. Zika infection is associated with severe neurological symptoms
and sequelae, such as Guillain–Barre syndrome and congenital microcephaly [
249
,
250
].
These neurological complications are explained by the interaction of the nonstructural 4A
(NS4A) protein of Zika virus with ankyrin repeat and LEM domain containing 2 (ANKLE2),

Int. J. Mol. Sci. 2022,23, 2613 20 of 37
encoded by a gene associated with autosomal recessive microcephaly in humans [
116
,
251
].
Indeed, the ANKLE2 heterozygous mutations in humans are associated with infants’ severe
microcephaly and later cognitive, neurological, intellectual and developmental deficits [
252
].
Interestingly, Ankle2 is involved in brain development in flies, inhibiting the neuroblast
division in the third instar larval brain; the hypomorphic mutants (Ankle2A) have been
proved to be pupal lethal and exhibited small brain volume phenotype [116,253,254].
The D. melanogaster model was used to demonstrate the physical interaction between
Zika NS4A and human ANKLE2 and its pathological consequences. The ectopic expression
of Zika NS4A in the developing third instar larva brains of Drosophila provoked a reduction
in brain lobe volume, induced apoptosis and reduced neuroblast proliferation. This mutant
phenotype was rescued by wild-type ANKLE2 but not by a microcephaly-associated AN-
KLE2 variant (ANKLE2
Q782X
) [
255
]. The expression of NS4A in fruit flies heterozygous for a
hypomorphic allele of Ankle2 caused a more significant microcephaly in comparison to the
condition induced in wild-type fruit flies [
255
]. Moreover, Ankle2 regulates the function
of genes that control cell polarity during asymmetric division of neuroblasts, including
lethal (2) giant larvae (l(2)gl), atypical protein kinase C (aPKC), bazooka (also known as par-3)
and par-6 and VRK1, for which human orthologs have been described. These genes are
involved, both in flies and humans, in neural stem cell self-renewal and production of
neurons, while being also related to developmental brain disorders [
256
]. From these, the
human VRK1 pathogenic alleles are associated with motor and sensory axonal neuropathy
and microcephaly [
257
]. Mutations in the fly homolog of VRK1,ballchen (ball), induced the
loss of neuroblasts in third star Drosophila larval brain. It has been shown that NS4A, having
the same location as Ankle2 in the endoplasmic reticulum and nuclear envelope, could
interact with ball (VRK1) to regulate brain size in flies. The NS4A expression mimicked the
influence of the Ankle2-Ball (VRK1) pathway on the aPKC and l(2)gl proteins, which are
critical for brain development. The microcephaly induced by NS4A expression has been
rescued by removing a single copy of ball or l(2)gl, demonstrating that NS4A hijacks the
Ankle2-ball (VRK1) pathway, affecting neuroblast division and brain development, leading
to microcephaly [258].
5.2. Elucidating the Molecular Players in the Cytotoxicity of Cholera Toxin
Cholera is an acute diarrheal infection caused by ingestion of food or water con-
taminated with the spiraled bacterium Vibrio cholerae, causing 1.3–4.0 million cases and
21–143 thousand deaths each year [
259
]. The most important virulence factor of V. cholerae
is cholera toxin, a typical AB toxin with ADP-ribosylating action. Cholera toxin stimulates
cAMP production in the gut epithelial cells, generating the hypersecretion of water and
electrolytes responsible of the specific clinical symptoms such as aqueous diarrhea and
rapid, severe dehydration [260].
Drosophila model was used to investigate the molecular mechanisms of cholera toxin
active (A) subunit. For this purpose, the gene for cholera toxin A subunit (CtxA) was
expressed in the developing fly wing, causing a CtxA-dependent weight-loss phenotype.
The mutant phenotype was fully rescued upon co-expression of an active form of Notch
or wild-type Rab11, the most well-represented members of the Ras superfamily GTPases,
involved in intracellular vesicle trafficking and signaling [
261
], and was significantly
worsened when a dominant-negative form of Rab11 or G
α
s was co-expressed. The CtxA
expressed in the midgut epithelial cells affected the intestinal epithelial permeability, as
revealed by the occurrence of gradual wasting and the smurfing phenotype after flies
feeding with food dyed with FD&C blue dye#1 [
262
]. These phenotypes have been also
rescued by co-expression of Rab11, suggesting that cholera toxin A subunit inhibits Rab11-
mediated vesicle trafficking.

Int. J. Mol. Sci. 2022,23, 2613 21 of 37
5.3. Molecular Mechanisms of Apoptosis Induced by Helicobacter pylori Cytotoxin-Associated
Gene A
Helicobacter pylori infection affects about 50% of the human population and around
7% will develop gastroduodenal disease [
263
]. H. pylori is associated with various gastric
pathologies, ranging from peptic ulcer to gastric adenocarcinoma and lymphoma [
264
].
The symptomatic infections are produced by strains harboring the cytotoxin-associated gene
A(cagA), one of the main virulence factors [
265
]. Once delivered in the host cells, CagA
is activated through phosphorylation by Src-family kinases and binds to SHP-2, a protein
phosphatase encoded by the PTPN11 gene in humans. SHP-2 further activates signaling
pathways downstream of receptor tyrosine kinases (RTKs), a class of receptors with a
pivotal role in cancer invasion and metastasis [
266
,
267
]. By interfering with epithelial
cell adhesion, polarity, migration and differentiation, CagA triggers malignant transfor-
mation of gastric epithelial cells, its oncogenic potential being confirmed in transgenic
animals [268,269].
The ectopic expression of CagA in the epithelial cells of the D. melanogaster developing
wing has been shown to induce a dose-dependent apoptotic effect, leading to significantly
lower size wings. This phenotype was similar to that produced by the localized activation
of the JNK pathway within the wing cells. The cagA-induced apoptosis was suppressed
by co-overexpressing a dominant-negative form of Basket (Bsk), a Jun amino-terminal kinase
(JNK) homolog, and enhanced by co-overexpressing of wild-type Bsk [270], suggesting that
CagA is an important mediator of the activation of JNK signaling pathway during H. pylori
infection [271,272].
Since JNK signaling is dependent on the Ras oncogene, it has been further investigated
whether CagA can genetically interact with the constitutively active oncogenic variant of
Ras called p.G12V or Ras
V12
[
272
,
273
]. If the expression of Ras
V12
alone in the fly eye has
been shown to induce the formation of non-invasive tumors, its co-expression with CagA
causes invasive tumors. In addition, CagA has been shown to interact with dlg1 and l(2)gl,
which are considered neoplastic tumor suppressor genes [274].
5.4. Discovering Novel Candidates for Assessing Genetic Susceptibility to Different Infections
The malvolio (mvl) gene of D. melanogaster encodes a protein sharing a high homolog
to natural resistance-associated human macrophage proteins (Nramps), which are inte-
grated in the phagolysosomal membrane and function as cation transporters [
275
–
277
]. In
D. melanogaster,mvl is expressed in macrophages and in differentiated neurons. The loss-of-
function mutations lead to taste behavior defects caused by a reduction in the sensitivity of
the gustatory circuits to stimuli. The ubiquitous expression of human Nramp-1 protein in
mutant fruit flies can fully rescue the taste defect. Moreover, the taste behavioral defects
can be suppressed when the fruit flies are grown on media supplemented with Fe
2+
or
Mn
2+
for a minimum of 2 h before testing, sustaining the role of mvl in the transport of
bivalent cations [278].
The polymorphisms of the Nramp-1 gene have been linked to susceptibility to tubercu-
losis and leprosy in human populations; therefore, the fact that human Nramp-1 can fully
complement the defect in mvl makes D. melanogaster an attractive
in vivo
model system for
Nramp-1 functions in different human infections [279,280].
5.5. Demonstrating the Functional Homology of Human Vasodilator-Stimulated Phosphoprotein
(VASP) and Drosophila enabled
Drosophila enabled (ena), a dominant genetic suppressor of mutations in the Abelson
(Abl) tyrosine kinase, is a member of Ena/human vasodilator-stimulated phosphoprotein
(VASP) protein family, which is associated with actin filaments and focal adhesions. Ena is
a specific substrate for Abl and also interacts with the SH3 domain of Abl [
281
]. Among
its physiological roles, VASP interacts with Listeria monocytogenes Act A protein, which is
required for the internalization of this facultative intracellular pathogen, mediating the
reorganization of actin filaments. The Ena/VASP domain 1 (EVH1) and EVH2 share 58%

Int. J. Mol. Sci. 2022,23, 2613 22 of 37
and 31% homology between Drosophila ena and human VASP, respectively. Moreover,
EVH1 is similar to the WP1 domain found in Wiskott–Aldrich syndrome protein, which is
associated with cytoskeletal defects in T cells and platelets [282].
Using Drosophila model, it has been demonstrated that VASP rescues the lethal phe-
notype associated with ena LOF. The lethal ena mutant alleles which affected EVH1 and
EVH2 and their capability to bind the focal adhesion protein zyxin and the Abelson kinase
were characterized by cytoskeletal defects. A comparison between VASP and three ena
transgenic lines, tested for their ability to rescue the ena null lethal mutants, showed that
VASP partially rescued ena mutant lethality, 25–85% survival rate and normal development
four weeks after eclosion as compared to 79–100% in case of ena transgene. Thus, VASP
represents a functional substitute of ena, the two proteins also sharing the same subcellular
distribution and the same expression pattern in mammalian cells. They are detected at the
level of actin filaments and focal adhesion contact where they bind to zyxin and the SH3
domain of Abl. Finally, lethal Ena mutations have been identified in the most conserved do-
mains of the Ena/VASP family. Further research of this protein family might provide new
insights into the regulation of cytoskeleton changes during physiological and infectious
processes [
283
]. The interaction of Listeria monocytogenes ActA with VASP may explain the
activation of actin reorganization and bacterial cell internalization.
6. Discussion
We emphasize that an a priori validation of the functional orthology of an hGOI/dGOI
pair by heterologous rescue should be the experimental paradigm for
in vivo
modeling
of an HGD. This preliminary approach, even when partially successful, validates the
evolutionary conservation of the matching genes and qualifies the respective HGD as an
appropriate candidate to be modeled on the D. melanogaster platform [13].
Various experimental assays pointing to functional orthology sometimes offer rather
indirect data. Although conceptually overlapped, modeling of a human genetic disease
by expressing human variants and equivalent fruit fly alleles in D. melanogaster does not
equate with heterologous rescue experiments. Sometimes, modelling data are not even
supported by functional complementation data, as in the case of the KAT2B/Gcn5 gene pair,
described above [160].
We consider that whenever a mutant phenotype of D. melanogaster is presumably
determined by a new mutant allele relevant for modeling an hGD, an intra-specific rescue
with the wild-type copy of dGOI is recommended to confirm this link. There is always
a potential risk that the mutant phenotype might have been induced by an unknown
mutation, most probably residing on the same chromosome. In such case, the respective
mutant strain is not appropriate for heterologous rescue experiments.
A positive heterologous rescue reveals that a basic interactome required for normal
functions of the orthologous proteins was conserved between the two species. This aspect
favors modeling of the disorder mainly when a chemical rescue is to be further attempted.
Targeted mutagenesis of an appropriate dGOI often results in impaired fruit flies mimicking
mutant phenotypes specific for the respective hGD. These mutants offer an excellent
perspective for understanding the genetic mechanisms fueling the disorder since they are
ideal to be subjected to chemical rescue assays, namely testing of candidate drugs on them.
Nevertheless, care should be taken when the chemical rescue is efficient in mutant fruit
flies impaired by disease-associated fly or human alleles without a preliminary successful
heterologous rescue, since conclusions may be wrong. The candidate chemical may not
target the mutant protein encoded by dGOI
LOF
but rather a direct or a close interactor of
it. Therefore, it was the adjustment of the stereo-chemical interaction with the defective
protein that led to heterologous rescue instead of a repairing process. If the respective
interactor is different or absent in mammals, the positive effects of the putative drug may
not be reproducible in human patients.

Int. J. Mol. Sci. 2022,23, 2613 23 of 37
Both a partial rescue and the rescue failure may be explained by either a less effective
or an impaired activity of the associated human protein of interest (hPOI) in the context
of fruit fly proteome [
13
]. When heterologous rescue fails, there are a few alternative
explanations, and they should be considered before concluding that the two genes are
indeed not functional orthologs. Most probably, the mammalian proximal interactome
has changed during evolution, therefore that hGOI is per se unable to fit into a fruit fly
genetic pathway. Therefore, the hGOI may be involved in similar biological processes in
mammals but via different molecular avenues. This situation is expected to hinder the
extrapolation of some promising chemical rescue results from fruit fly to a mammalian
organism. Alternatively, hidden mutations in the genetic background of the fruit fly strain
subjected to heterologous rescue may impede the functional complementation results. In
this case, it is worthy to outcross the LOF allele in a new, isogenized background and repeat
the rescue phenotype experiment. Finally, either the molecular construct used for embryo
injection or the GAL4 driver may have inhibitory behaviors, hence alternative approaches
may need to be tested [162].
However, there is also a reverse of the medal, namely when one is performing a
promising heterologous rescue experiment and does not obtain the functional comple-
mentation of the mutant fruit fly. We further discuss various hypothetical scenarios for
successful and failed heterologous experiments, just to emphasize the complexity of data
interpretation when hGDs are modeled on D. melanogaster.
6.1. Issues When Modeling hGDs in D. melanogaster Regardless of Positive Heterologous
Rescue Results
In order to consider a mammalian genetic disorder to be modeled in D. melanogaster, a
preliminary bioinformatics analysis of both nucleotide and amino acid sequences should
be performed. Some scenarios concerning stereo-chemical interactions among an hPOI
WT
and its proximal interacting proteins which are prone to affect the results of heterologous
rescue experiments are summarized in Figure 2.
Let us presume that a structurally orthologous dGOI is identified in the D. melanogaster
genome and the human and fruit fly proteins share a common functional domain D1
(Figure 2A). Particularly, there may be also present an unknown functional domain D2 in
the human protein (Figure 2A), which is not yet characterized and therefore is not described
in the specific databases. In addition, let us assume that research papers refer to D1 as
being affected in many of the reported patients, therefore, a research project is launched to
model the respective disease in D. melanogaster. Nevertheless, in some patients, both D1
and D2 may be distorted, but we expect that there is no medical focus on D2 yet.
To construct the model strains, the fruit flies are subjected to targeted mutagenesis
of dGOI. Further genetic and molecular analysis inquiry may reveal that the mutant
phenotype of adult fruit flies, which mirror medical conditions, are caused by a distorted
D1, and the LOF flies are rescued by hGOI
WT
. Candidate chemicals are then tested on the
impaired flies and one potential drug compensates the steric distortion of D1, resulting
in a partial or complete phenotype rescue of the fruit flies. However, unexpectedly, when
this putative drug is tested in mutant mammals, there is no phenotype rescue, because
hPOI
WT
interacts with the equivalent human interacting protein 1 (hIP1) and hIP2 in a
different manner in mammals, involving also distinct domains of D2 of hPOI
WT
and hIP1
(Figure 2C). The drug tested on D. melanogaster was able to chemically rescue only the
functional domain D1, which is homologous with D1 of hPOI
WT
but not the conformation
of D2. Therefore, a new project is required to understand functions of D2 and then to
identify a different drug capable to rescue its spatial conformation.

Int. J. Mol. Sci. 2022,23, 2613 24 of 37
Int.J.Mol.Sci.2022,23,261325of39


6.1.IssuesWhenModelinghGDsinD.melanogasterRegardlessofPositiveHeterologousRescue
Results
InordertoconsideramammaliangeneticdisordertobemodeledinD.melanogaster,
apreliminarybioinformaticsanalysisofbothnucleotideandaminoacidsequencesshould
beperformed.Somescenariosconcerningstereo‐chemicalinteractionsamonganhPOIWT
anditsproximalinteractingproteinswhicharepronetoaffecttheresultsofheterologous
rescueexperimentsaresummarizedinFigure2.

Figure2.Scenariosforissuesofheterologousrescueresults.(A)D.melanogasterwild‐typeprotein
dPOIWTencodedbyadGOIhasalocalinteractome,representedbytheinteractingproteinsdIP1,
dIP2anddIP3.Whenthesefourproteinsinteractcorrectly,theresultingfunctionalcomplexsup‐
portsanormalphenotype(top).ThefunctionaldomainofdPOIWTisD1,whichisalsopresentin
differenthPOIWTequivalents(bottom);someoftheseequivalentshaveadditionalfunctionaldo‐
mainsD2andD3.(B)hPOIWTallowsheterologousrescue(top)orhasastericconformationleading
toalooseinteractionwithdIP2.Thisconditioneitherpreventsformationofthefunctionalcomplex
(noheterologousrescue)orleadstoanunstablecomplex,whichdeterminesapartialheterologous
rescueresult.(C)Thenormalpathwayinhumansinvolvessupplementalinteractionsamong
hPOIWT,hIP1andhIP2(top);D1ofhPOIWTmayrescuethephenotype(bottom),butifD2isdefect
inpatients,chemicalrepairofD1infruitfliesdrivestoapositiveheterologousresultwhichcannot
beextrapolatedtopatients.(D)Adistinctwaytoensemblethefunctionalcomplexinhumans,when
theinteractomewasnotevolutionaryconserved(upper).hPOIWThassupplementaldomainsD3
andD4;inthiscase,D4domainstericallypreventstheproperinteractionofD1withdIP1,therefore
heterologousrescueintrinsicallyfails(bottom).CreatedwithBioRender.com(accessedon23Feb‐
ruary2022).
Figure 2.
Scenarios for issues of heterologous rescue results. (
A
)D. melanogaster wild-type protein
dPOI
WT
encoded by a dGOI has a local interactome, represented by the interacting proteins dIP1,
dIP2 and dIP3. When these four proteins interact correctly, the resulting functional complex supports
a normal phenotype (
top
). The functional domain of dPOI
WT
is D1, which is also present in different
hPOI
WT
equivalents (
bottom
); some of these equivalents have additional functional domains D2
and D3. (
B
) hPOI
WT
allows heterologous rescue (
top
) or has a steric conformation leading to a
loose interaction with dIP2. This condition either prevents formation of the functional complex (no
heterologous rescue) or leads to an unstable complex, which determines a partial heterologous rescue
result. (
C
) The normal pathway in humans involves supplemental interactions among hPOI
WT
, hIP1
and hIP2 (
top
); D1 of hPOI
WT
may rescue the phenotype (
bottom
), but if D2 is defect in patients,
chemical repair of D1 in fruit flies drives to a positive heterologous result which cannot be extrapolated
to patients. (
D
) A distinct way to ensemble the functional complex in humans, when the interactome
was not evolutionary conserved (
upper
). hPOI
WT
has supplemental domains D3 and D4; in this case,
D4 domain sterically prevents the proper interaction of D1 with dIP1, therefore heterologous rescue
intrinsically fails (bottom). Created with BioRender.com (accessed on 23 February 2022).
A special situation is encountered when IP1, IP2 and IP3 are present in both flies
and humans, but the hPOI
WT
/hIP1/hIP2 complex is organized by different interactions.
The heterologous rescue should not work since, in humans, D1 of hPOI
WT
is required
along with D3 and D4 domains to interact with hIP2 in order to ignite coupling of hIP3
(Figure 2D). Steric constraints caused by D4 prevent formation of hybrid hPOI
WT
/dIP1 and
functional complementation fails, although the targeted D1 is conserved between H. sapiens
and D. melanogaster (Figure 2D).

Int. J. Mol. Sci. 2022,23, 2613 25 of 37
In this case, a preliminary, negative heterologous rescue test suggests that model-
ing of the respective genetic disorder is more appropriately performed in a mammalian
experimental model such as mouse.
6.2. Possible Scenarios Accounting for Heterologous Rescue Failure or Partial Rescue in
D. melanogaster
Let us consider that a protein complex dPOI
WT
(Drosophila POI
WT
)/dIP1 (Drosophila
IP1) interacts with dIP2. The resulting complex dPOI
WT
/dIP1/dIP2 activates dIP3, which
is a key prerequisite for the normal functioning of a biological process (Figure 2B). The
orthologous gene employed for heterologous rescue of specific LOF fruit flies encodes for an
hPOI
WT
, for which bioinformatics reveals the presence of the required domain that allows
a proper interaction with dIP1. The particular stereochemistry of the hybrid hPOI
WT
/dIP1
complex, however, may result in a loose interconnection with dIP2 (Figure 2B). As a
consequence, the hPOI
WT
/dIP1/dIP2 complex is unstable and even unable to activate dIP3.
Therefore, the impaired biological process of LOF fruit flies is either partially rescued or
not rescued at all by the human orthologous gene (Figure 2B).
The partial rescue results are still an incentive for modeling hGDs [
13
], but care should
also be taken to the message send by an incomplete rescue result. Namely, does a partial
rescue result resemble the case depicted in Figure 2B (bottom) or was the complete rescue
phenotype impaired by genetic modifiers already present in the genetic background of a
particular fruit fly strain? The roles of genetic modifiers as enhancers and suppressors in
either precipitation or postponing the onset of symptoms should always be considered, as
they represent the core of personalized medical genetics.
Complete heterologous rescue experiments are more encouraging when starting to
model an hGD on D. melanogaster, but what about the ideal scenario when the orthologous
gene of fruit fly rescues the mammalian mutant phenotypes? The orthologous gene pair
atonal (ato, from fruit flies) and Math1 (from mouse) are both involved in the development
of the nervous system of each species, and null alleles were reported for both genes. In an
impressive experiment, transgenic Math1
WT
allele saved fruit flies with ato null phenotypes.
Nonetheless, a transgenic ato
WT
allele copy also rescued lethality of Math1 null mice,
otherwise unable to initiate breathing movement after birth; the heterologous rescued mice
survived to adulthood [104].
Such examples of functional complementation, when mammalian or even other verte-
brate orthologs save the fruit fly phenotypes, deserve a strong consideration if the respective
gene is associated with an hGD.
7. Conclusions
Interdisciplinary research teaming up experts in genetics, bioinformatics, genomics
and other medical domains strongly relies on D. melanogaster to model both mechanisms
and treatment attempts of several impacting hGDs, such as neurological and cardiac
disorders, cancers and infectious diseases.
Data emerging from functional complementation assays are very valuable for under-
standing the contribution of mutations associated with a genetic disorder in the context of
an individual genetic background. In this context, carefully designed heterologous rescue
experiments are a powerful tool to tackle defiant medical conditions via genetic avenues
kept open by the D. melanogaster experimental model. The functional complementation as-
say is of prime relevance not only for medical use but also for fundamental research. What
are these experiments telling us by revealing that many mammalian genes still function
in the molecular context of the ancient D. melanogaster genome? Perhaps, this spectacular
journey back in time evidences that numerous gene functions are conserved under an
inherent selective pressure force: fundamental biological processes are articulated by using
the same old molecular tools.
Obviously, some other medical challenges, such as aging issues, are prone to be
modeled in D. melanogaster, but heterologous rescue assays’ data are still expected for

Int. J. Mol. Sci. 2022,23, 2613 26 of 37
specific orthologs genes. Although diseases such as Hutchinson–Gilford progeria syndrome
are figured in fruit fly [
284
,
285
], we found no functional complementation experiments
reported so far, neither in FlyBase nor in a recent review [
286
] for genes involved in
aging pathology.
Hence, we conclude that preliminary heterologous rescue assays should serve as the
standard for genetic analysis of hGDs on the D. melanogaster model.
Author Contributions:
Conceptualization, A.A.E.; investigation, A.A.E., A.C.R., M.C.C. and M.M.M.;
data curation, A.A.E. and A.C.R.; writing—original draft preparation, A.A.E., A.C.R., M.C.C. and
M.M.M.; writing—review and editing, A.A.E. and A.C.R.; visualization, A.A.E., A.C.R. and M.C.C.;
supervision, A.A.E.; funding acquisition, M.C.C. All authors have read and agreed to the published
version of the manuscript.
Funding:
This research was funded by the C1.2.PFE-CDI.2021-587 project. This project is financed by
the Ministry of Research, Innovation and Digitalization through Program 1—Development of the
national R&D system, Subprogram 1.2—Institutional performance—Financing projects for excellence
in RDI, Contract no. 41 PFE/30.12.2021. The funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to
publish the results.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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