The Drosophila Forkhead/Fox transcription factor Jumeau mediates specific cardiac progenitor cell divisions by regulating expression of the kinesin Nebbish

Forkhead (Fkh/Fox) domain transcription factors (TFs) mediate multiple cardiogenic processes in both mammals and Drosophila. We showed previously that the Drosophila Fox gene jumeau (jumu) controls three categories of cardiac progenitor cell division—asymmetric, symmetric, and cell division at an earlier stage—by regulating Polo kinase activity, and mediates the latter two categories in concert with the TF Myb. Those observations raised the question of whether other jumu-regulated genes also mediate all three categories of cardiac progenitor cell division or a subset thereof. By comparing microarray-based expression profiles of wild-type and jumu loss-of-function mesodermal cells, we identified nebbish (neb), a kinesin-encoding gene activated by jumu. Phenotypic analysis shows that neb is required for only two categories of jumu-regulated cardiac progenitor cell division: symmetric and cell division at an earlier stage. Synergistic genetic interactions between neb, jumu, Myb, and polo and the rescue of jumu mutations by ectopic cardiac mesoderm-specific expression of neb demonstrate that neb is an integral component of a jumu-regulated subnetwork mediating cardiac progenitor cell divisions. Our results emphasize the central role of Fox TFs in cardiogenesis and illustrate how a single TF can utilize different combinations of other regulators and downstream effectors to control distinct developmental processes.


Supplementary Table Legends
Table S1. Quantitative summary and statistical significance of the diverse cell division and positioning defects associated with different genotypes. Table S2. Quantitative summary and statistical significance of the cardial cell number defects associated with different genotypes in RNA interference assays. Table S3. Genes activated by jumu but not by CHES-1-like.

Supplementary Method
Method S1. Assessment of jumu and CHES-1-like mesoderm-targeted RNAi knockdowns of neb transcript expression by reverse transcription quantitative real-time PCR (RT-qPCR).

Supplementary References
Supplementary Figure S1. Cardial cell number defects associated with neb knockdown via RNA interference. (a) A wild-type heart stained to show only Tin-CCs (green) and Svp-CCs (yellow).  Table S1A. Individual null hypotheses that were statistically tested using these data are presented.  Table S2A. Individual null hypotheses that were statistically tested using these data are presented. Table S3. Genes activated by jumu but not by CHES-1-like: genes corresponding to probesets exhibiting log 2 Fold Change < -0.5 and adjusted p-value < 0.05 in purified mesodermal cells homozygous for the jumu null mutation AND exhibiting log 2 Fold Change ≥ 0 in purified mesodermal cells homozygous for the CHES-1-like null mutation.

SUPPLEMENTARY METHOD
Supplementary Method S1. Assessment of jumu and CHES-1-like mesoderm-targeted RNAi knockdowns of neb transcript expression by reverse transcription quantitative real-time PCR (RT-qPCR).

Embryo preparation for total RNA extraction
Stage 11-12 embryos of control and appropriate RNAi knockdown genotypes were collected after being raised at 29°C. Control embryos contained one copy each of the pan-mesodermal twi-GAL4 driver and UAS-Dcr-2, while RNAi knockdown embryos possessed one copy each of the twi-GAL4 driver and UAS-Dcr-2 in addition to one copy of the relevant UAS-RNAi construct for jumu or (Table 1). Embryos were dechorionated by immersion in 50% bleach for 5 m followed by a quick rinse with 0.1% Triton-X and then with water.

Total RNA isolation and quantification
Total RNA was isolated immediately after the dechorionation and rinse step using the Direct Zol TM RNA MicroPrep Kit (Zymo Research) according to the manufacturer's recommendation which included in-column DNase I treatment to remove genomic DNA. Total RNA was eluted in 10 µl of RNase/DNase-Free water. One µl was used to quantify total RNA concentration and quality using a Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Table 1).  Table 1. Genotypes of embryos used as controls and for RNAi knockdowns of specific genes.

Embryo
The concentration and quality of total RNA obtained from these genotypes is listed.

cDNA synthesis for Reverse Transcription quantitative PCR (RT-qPCR)
cDNA was prepared with the SuperScript TM IV VILO TM Master Mix with ezDNase TM Kit (ThermoFisher) using 0.70 µg of total RNA in 20 µl reactions according to the manufacturer's recommendations including the ezDNase TM pre-treatment to remove genomic DNA. The 20 µl cDNA synthesis reactions were diluted 1:5 to a final volume of 100 µl using RNase/DNase-Free water such that they contained the original total RNA at a final concentration of 7.0 ng/µl.

Reverse Transcription quantitative real-time PCR (RT-qPCR)
RT-qPCR was used to quantitate relative transcript expression levels between the different genotypes using the PowerUp TM SYBR TM Green Master Mix (ThermoFisher) in an Applied Biosystems TM QuantStudio TM 3 Real-Time PCR System (ThermoFisher). 10 µl qPCR reactions containing 2 µl (25 ng) of the reverse-transcribed total RNA, 1 pmol each of the relevant forward and reverse primers (Table 2), and 5 µl of the 2X master mix PCR reagent were utilized. qPCR reactions were performed in technical triplicate evaluating C q reproducibility for each condition that was amplified. The standard deviation of the C q varied between 0.022 to 0.184 for the technical replicates for each sample, thereby demonstrating C q reproducibility well below a standard deviation of 0.5 C q .  consisting of a UDG activation step, DNA polymerase activation step, PCR amplification stage, and dissociation curve stage was used for all qPCR reactions (Fig 1). The qPCR experimental parameters utilized the standard curve experimental type setting and determined the C q value using both the Auto-Threshold and Auto-Baseline analysis functions. The qPCR analysis results were exported to Microsoft TM Excel TM for relative gene expression quantification. Relative gene expression was calculated using the 2 -ΔΔC T method [3]. α-Tubulin at 84B (αTub84B) was used as an endogenous reference gene to normalize targets because its primer set demonstrated a low C q variance between all samples of 27.12 ± 0.18 (Mean C q ± Standard Deviation) [4,5].
At least three No Template Control (NTC) qPCR reactions for each primer set were included for each experiment, and they all demonstrated an absence of amplification and thus no template contamination (Fig 2).