1. An essential cell cycle regulation gene causes hybrid inviability in Drosophila
Authors: Nitin Phadnis, EmilyClare P. Baker, Jacob C. Cooper, Kimberly A. Frizzell, Emily Hsieh, Aida Flor A. de la Cruz, Jay Shendure, Jacob O. Kitzman, Harmit S. Malik
Presenter: Savannah Ulmer

2. Background Speciation Hybrid inviability
D. melangastor and D. simulans
Speciation is the formation of a new biological species due to the evolution of factors like geographic isolation, reduced gene flow and reproductive isolation.
Reproductive isolation can encourage speciation in 3 ways:
1. Mating location, time or rituals have evolutionary changed
2. Incompatible sexual genitalia
3. Hybrid inviability or sterility which is the basis for this paper and seen between sister species, Drosophila melangastor and Drosophila simulans.

3. Introduction
When a genetic cross between a Drosophila melanogaster female and a D. simulans male occurs, they produce only hybrid F1 females because hybrid F1 males are inviable.
Prior research identified two casual elements for this hybrid incompatibility, Hmrmel and Lhrsim , and that there is an additional unknown gene involved.
The purpose of this research is to find the unknown gene(s) involved in inviability.
Prior research using X-ray mutagenesis identified 2 casual elements for the hybrid incompatibility. Hmr which stands for hybrid male rescue on Drosophila melanogaster X chromosome and Lmr which stands for lethal hybrid rescue on D. simulans second chromosome.
Results: When one of these genes are absent it results in viable hybrid males.
But when both incompatible genes are present it results in inviable hybrid males. In contrast, when a transgenic copy of Lhrsim was introduced into the D. melangaster male chromosome it remained viable despite having both alleles. This suggested that there is at least 1 additional unidentified gene necessary to cause the inviability.
Since the absence of 1 of these genes produced a viable progeny, the researchers extended this analogy and sought to find another gene whose ablation would result in viable hybrid males.
Hmr is on X chromosome Lhr is on 2nd and 3rd chromosome

4. Methods for Presumption
55,000 mutated D. simulans males X D. melangaster females
F1 progeny = 32 viable hybrid males (compared to 300,000 females)
26 of them hybrids viable due to non-disjunction
6 of them rescue hybrids viable due to mutations at a locus other than the Lhrsim locus.
These 32 viable hybrids underwent PCR to rule out contamination, PCR and sequencing of the Hmr gene to make sure they carry the D. Melanogaster X chromosome and sequencing of the Lhr gene to make sure there’s no mutation at it’s locus.
In the 6 rescue hybrids, PCR amplified the Lhr gene to confirm they were still intact.
This means they are viable due to a mutation in a different gene, specifically the gene of interest.
Mutated with ethyl methane sulfonate (EMS)

5. Methods/Results for Presumption
Illumina HiSeq 2000 to sequence the DNA of the 6 rescue hybrid males and parental unmutated D. simulans males and D. melangastor females
Only 1 gene was mutated across all 6 viable hybrids.
The gfzf gene on D. Simulans chromosome 3
To map the mutations in the gene of interest, Illumina HiSeq 2000 instrument was used to sequence the genome of the 6 rescue hybrids and the parental strains.
(Genetic crosses couldn’t be done because these progeny are sterile)
This identified all new mutations in the 6 strains.
Most were point mutations but there were also 2 overlapping deletions that were mapped the gene to chromosome 3R
Only 1 D. Simulans gene was disrupted in all the 6 rescue hybrid males, the gfzf gene.

6. Background/Mutations of gfzfsim gene
Involved in DNA damaged induced G2/M cell cycle checkpoint mechanism and regulates cell proliferation
Encodes two alternative transcripts: (1) longer transcript encodes polypeptide with 4 FLYWCH zinc finger domain and a GST domain (2) shorter transcript encodes polypeptide with only a GST domain
Aids in cell cycle arrest by blocking cell proliferation by also essential required for cell proliferation by regulating the RAS/MAPK pathway.
As shown in the table, the longer transcript for the gfzf sim gene was affected by 4 of the 6 mutations that occurred suggesting that it is the longer transcript of the gfzf sim gene that is involved in hybrid incompatibiliy

7. Methods for Confirmation
gfzfsim longer transcript knock down of F1 hybrids using RNA interference pValium20-gfzfsim
Inserted into D. melanogaster X-chromosome
To confirm that mutations of gfzfsim longer transcript are involved in hybrid inviability and to what extent they effect it the researchers knocked down it’s expression in F1 hybrids using RNA interference.
They created 2 RNAi constructs that target the longer transcript at different sites and then inserted it into the D. Melangofaster to make a transgenic fly
These constructs only targets gfzf sim not gfzf Mel

8. Methods/Results for Confirmation
Transgenic D. melanogaster X heterozygous D. melanogaster with CyO balancer on 1 chromosome and GAL4 driver (Actin5C- GAL4) on the other chromosome
F1 progeny X D. simulans male = Hybrid F1 progeny
Knockdown of gfzfsim restored viability
This cross resulted in 2 daughter types
#1 CyO balancer but not Actin5C-GAL4 driver and #2 Actin5C-GAL4 driver + RNIi construct pValium20-gfzfsim
They then crossed both of these females with D. Simulan males and looked at the hybrid progeny and saw only viable hybrid males when the RNA interferences knocked down the gfzf sim expression

9. Methods/Results for Confirmations Continued
They also performed a cross with the same females as before with 3 new strains of D. Simulan which showed consistent data that true rescue viable hybrids are only found with the expression of the RNA construct.
The green are the true rescues that have the D. Mel X chromosome while the ones highlighted in blue are from nondisjuction and have the S. Dimulan X chromosome
On the right is from a RT-PCR that shows the decreased expression of the sim in the rescues.

10. Additional Confirmations
Mutated gfzfmel did not restore viability in the F1 hybrid males
Are these allele differences from positive selection?
Disrupting gfzfmel with mutations does not rescue the viability in the F1 hybrid males.
So the allelic differences of the gene are important just like the alleles of the Hmr and Lhr genes
The 3 viable male progeny were due to nondisjjnction

11. Methods/Results for Testing for Positive Selection
Used a McDonald-Kreitman test to detect adaptive evolution of the gfzf gene along the D. melanogaster and D. simulan lineage
Evolution of the gfzf between the two species
Positive selection along the D. simulan lineage only and localized to the FLYWCH zinc finger domans
Rf= fixed non-synonymous changes
Sf= fixed synonymous changes
Rp= polymorphic synonymous changes
Sp= in gfzf along D. Melangaster and D. Simulan lineages.
Excess of fixed non-synonymous changes occurred which indicates positive selection

12. Background of developmental timing of gfzfsim –associated hybrid lethality
Homozygous gfzfmel null mutants, gfzfmel-pn-Kpn males and gfzf sim mutants
Is hybrid inviability due to cell proliferation defects in hybrid larvae?
Mostly polyploid cells in larvae tissue but diploid cells in nervous system and imaginal discs
During pupation: polyploid tissues are degraded and the imaginal discs proliferate into adult body
Prior research showed that wiithout proper imaginal discs they die during pupation.

13. Methods/Results for developmental timing of gfzfsim –associated hybrid lethality
Gfzfsim knockdown with T80- GAL4 driver, specific to nervous system and imaginal discs in late larval stages
Restores viability in F1 hybrid males
Suggests primary defect of male hybrids is during diploid tissue proliferation
Knocked down the gfzf sim gene but this time they used a T80-GAL4 driver because its specifically expressed in nervous system and imaginal discs during late larval stages.
Rescued viability of hybrid F1 males in adult suggesting melanogaster and simulans cross during diploid tissue proliferation.

14. Confirmation for Defect during Diploid Proliferation
Crossed, dissected and used EdU to track DNA synthesis in proliferating cells
Cell proliferation defects partially rescued in hybrid with knockdown
Found cells were restored in larvae brain tissue

15. Summary and Future Research
Findings suggest that gfzfsim arrests proliferation during late larval stages which is what leads to hybrid inviability. This also suggests that genes that control the cell cycle are involved in evolutionary reproductive isolation.
Since traditional genetic approaches had failed prior at attempting to find missing hybrid incompatibility genes these methods should excel in research other organisms and genes.
More intensive research could be done on the gene in hopes to excel treatment and gene therapy for cancers.
Found the third unknown gene involved in hybrid inviability
Tracked at what point in development the defect caused the hybrid inviability
Suggests other cell cycle regulatory genes may be involved in evolutionary reproductive isolation in other species
Can’t do recombination based methods because even though the F1 hybrids are viable they’re sterile
F1 male hybrid sterility, the genetic disruptions in D. melanogaster and the lack of balancer chromosomes in D. simulans.

16. References
Science  18 Dec 2015: Vol. 350, Issue 6267, pp DOI: /science.aac7504