Materials and methods

Unless otherwise stated, flies were raised at 25ºC under standard procedures (Roberts, 1998). Embryos were collected on apple juice plates with fresh yeast for further analysis.

2.2. 2R maternal screen

In order to identify new genes required for blastoderm cellularization we took advantage of a collection of maternal mutants previously isolated in the laboratory of Dr. Ruth Lehmann (Barbosa et al., 2007). Due to methodological constraints related to the induction of germ-line clones all mutants mapped to the right arm of the second chromosome (2R).

A primary screen isolated 137 independent mutant lines with abnormal blastoderm cellularization and/or germ-band extension defects, but normal formation of PGCs. Mutant lines with egg-laying defects were discarded to avoid mutations in housekeeping genes. A secondary screen isolated 47 lines from the previous 137. These mutant lines showed absence or “scraps” of embryonic cuticle. This phenotype is frequently associated with loss of embryonic ectoderm integrity.

The FLP/FRT ovoD _{system (Chou and Perrimon, 1992) allows}

the generation of germ-line clones and the isolation of maternal mutant embryos. Mutagenized P[FRT 42B] / CyO virgin females were crossed with P[hs-FLP22_{]/Y; P[FRT42B] P[w}+ _ovoD_{]/ CyO hs-hid males. F1}

progeny was heat shocked for 1h at 37°C during the second and third larval instar stages. Since induction of hs-hid expression in larvae is

lethal, and since ovoD _{is a dominant mutation that inhibits oogenesis,}

only the germ-line stem cells that induced FRT-mediated mitotic recombination after induction of the flipase (hs-FLP) will produce mature eggs. Mature eggs were necessarily homozygous for isolated maternal mutations.

2.3. Mapping and isolation of complementation groups

Different alleles of the same gene (complementation group) were identified by crossing the mutant lines with each other. We assumed the isolated mutations were recessive and intragenic complementation was unlikely. Lethal and female sterile complementation groups were directly mapped using the Bloomington 2R deficiency kit (Bloomington Drosophila Stock Center). All together these deficiencies (deletions) are known to cover ~80% of the 2R chromosome-arm. To avoid secondary site lethal mutations not related with the isolated phenotypes, deficiency mapping was always performed using at least two different alleles of each complementation group. Any non-complementation result was confirmed with all alleles of each complementation group and with the reciprocal crosses. Once a complementation group was mapped to a given cytological (genetic) interval, a candidate gene approach was taken for all available lethal and sterile mutations known to map to that relevant cytological interval.

2.4. DNA sequencing of candidate genes

To molecularly characterize the isolated mutations on a given complementation group, genomic polymerase chain reaction (PCR) was carried out from mutant males of each isolated allele. An allele from a different complementation group but also isolated on the 2R screen (similar genetic background) was used as a control in order to

identify DNA polymorphisms when compared to the published Drosophila genome sequence (Flybase). Two independent genomic PCR fragments from each allele were sequenced and compared with each other and with the control.

2.5. Molecular biology and generation of transgenic lines 2.5.1. UAS-san generation

To generate UAS-san, san open reading frame (ORF) was amplified by PCR with a forward primer containing an XbaI restriction site and a reverse primer containing a KpnI restriction site. The Amplified PCR product was subcloned into pGEM-T Easy vector (Promega). After verification by digestion and DNA sequencing, san cDNA was subcloned into a P[UAST] vector (Brand and Perrimon, 1993). Transgenic san-UAS lines were generated by microinjecting the P[UAST-san] construct into w1118 embryos. UAS-san transgenic lines were created at BestGene Inc. (Chino Hills, CA, USA).

Fly lines with the genotypes w; B16-79/CyO; Actin5C- Gal4/TM6B and w; B50-26/CyO; san-UAST/TM6B were generated to perform rescue experiment. Rescue of complementation group 2 mutation alleles was performed by crossing w; B50-26/CyO; san- UAST/TM6B virgin females with w; B16-79/CyO; Actin5C-Gal4/TM6B males. B50-26 and B16-79 are two mutant alleles of san isolated in the maternal screen.

2.5.2. Genomic fragments for complementation group 3

The bacterial artificial chromosome BACR25I01 (Berkeley Drosophila Genome Project) containing the entire interval to which the complementation group 3 was mapped, was partially digested with the

restriction endonuclease XbaI. The larger DNA fragments (between approximately 8 and 15Kb) were subcloned in bulk into the XbaI site of the pCasper 2 vector. Size of the subcloned fragments were identified by digestion and identification was performed by DNA sequencing

using the flanking primers pCasperFw 5’-

CGCAAAGCTTGGGCTGCAGGTCGA-3’ and pCasperRev 5’-

TACTAGAATTCGTTAACAGATCT-3’. Six non-overlapping clones were used to generate transgenic flies (table 1, fig. 6). Given the difficulty of cloning the gene affected in complementation group 3 and to facilitate our work, these clones were given names of deserts.

Clone Clone_name Clonesize (bp)

Cloned region of Drosophila genome Beginning sequence End of sequence

1 Kalahari 17 494 CTAGAAGCGCCACCGGGC… …ATTAGCAAATATGTAGCT

2 Sonora 15 400 CTAGAGAACGAAAGCGAG… …AATTAAACATAAATATAT

3 Libya 15 555 CTAGATGGAAATTGAAAT… …TCGCCATTTTCTGCTCAT

4 Sahara 12 922 CTAGAGCGGACCAAGTAA… …CACGCTTATCGGAGCTGT

5 Atacama 6 671 CTAGATTTATATGTTGAT… …TCTATAGGAAATTCAACT

6 Mojave 6 528 CTAGAAAAGGTAGTATGT… …ATCGCGGCATTCTCTGGT

Table 1: Genomic DNA fragments used to map complementation group 3.

2.6. DNA staining of complementation group 3 embryos

For primary phenotypic analysis of complementation group 3 mutants, embryos at 4-5 hours of age were collected and fixed (after dechorionation in 50% bleach for 5 min) by gentle shaking for 1 hour in 4 mL heptane, 0.125 mL 37% formaldehyde and 0.875 mL PBS. Fixation was followed of devitellinization by addition of 4 mL methanol and shaking vigorously during 1 minute. Following rehydration, embryos were three times 5-min washed with phosphate buffered saline (PBS, pH 7.4) containing 0.1% Tween-20. DNA was stained with OliGreen at 1:5000 (Invitrogen) with the addition of 5 μg/mL RNAse A.

Embryos were mounted in Fluorescent Mounting Medium (DakoCytomation, Inc) and immunostainings were visualized using a Leica SP5 confocal microscope. All images are confocal sections.