Candidate genes etc…

Overview Looking at cell death in inxs embryos via double staining Analysis of inxs candidate genes using Quantitative Real time RTPCR Future Directions Miscellaneous

inxs briefly  Dominant modifier of the irreC-rst phenotype  Phenotypic expression observed in the retina, showing reduction in cell death  Alleles P2, B94, and 3  Referred to in the lab as A, B, and 1237  Question – do inxs embryos have a cell death phenotype?

1237 ♀ TM3 ftz lacZ X 1237 ♂ TM3 ftz lacZ 1237 TM3 ftz lacZ /2 Lac Z expression 1/4 lethal 1/4 No Lac Z expression Proportions expressing ftz- lacZ

Tunel Staining Detection of Apoptosis via the fragmentation of DNA TdT labels the blunt ends of DNA breaks with a (fluorescein)labeled dUTP Can be detected using fluoremetric techniques, or immunohistochemical techniques (ie anti- fluorescein antibodies).

Tunel Staining

Optimization of Double- staining LacZ antibodies from Molecular Probes used at a 1:500 concentration. Anti-mouse Cy3 was used as a secondary antibody. Hot citrix treatment not used Both reactions were fluorometric in order to obtain both signals (that worked the best) Stage 15/16 was chosen because clear midline Lac Z staining could be seen

Double Staining Results 1237/tm3 ftz lacZ 1237/1237

Double Staining Results  Both full counts and rough counts or “eyeballing” were made  OreR embryos showed roughly the same counts as well  Based on this data, inxs may not have a phenotype in embryos.

inxs Candidate Region

Question Can we detect mRNA expression differences between inxs and wild-type tissues using QRTPCR?

Quantitative RTPCR Primers were first tested on embryo RNA Intended to test for differential expression using mutant retina RNA, but stage development was not consistent Used white pre-pupae as a consistent tissue stage for mutant vs OreR RNA Looked for lower expression in mutant vs OreR RNA Best primer sets were used to look for expression in retina RNA

QRTPCR result summary CG32407 and CG32412 were the only genes to show differential expression in the white pp Unfortunately there is no sequence difference between the mutant allele 1237 and OreR in those genes Four new gene predictions in the found in the inxs candidate region from Apollo Genome browser PI38359, PI47229, PI51087, and PI65757 These genes were tested in Retina – only PI65757 worked

QRTPCR result summary CG5592, CG5568, CG32410, CG32408, CG10483, CG10486 primer sets never seemed to work Redesigned CG5568, 10483, 32408, using 1237 sequence 5568 gave a working product in embryo but not in retina – can be eliminated? Caveat – mutation may not affect mRNA levels, negative results are not truly definitive

CG5568 Embryo Retina

CG32412 OreR pp A/1237 pp

Future Directions Further sequencing of mutant DNA in candidate region Further analysis of newly predicted genes using QRTPCR Collection of RNA from A/B retinas for Quantitative Real-time RTPCR Refining the map location of inxs using p- element system and molecular markers

Future Directions Analysis of slit expression in mutant embryos Clonal analysis indicates that inxs may be a signal peptide – analysis of candidates using SignalP and TMHMM CG32412 is the only 100% predicted signal peptide, and CG5592 is predicted to have several transmembrane helices