1. SpeciesCommon Name (Symbol) HSF1HSF2HSF3HSF4 Danio rerio Homo sapien Mus musculus Canus lupis Equus caballus Xenopus laevis Xenopus tropicalis Gallus gallus Taeniopygia guttata Oncorhynchus mykiss Saccharomyces cerevisiae Drosophila melanogaster Zebrafish (z) Human (h) Mouse (m) Dog (d) Horse (e) Frog (x) Frog (xt) Chicken (c) Zebrafinch (f) Trout (t) Yeast (sc) Fruitfly (dr) NP_571675.1 NP_005517.1 NP_032322.1 XP_532354 XP_001505050.2 NP_001090266.1 P38529.1 NP_001118220.1 NP_011442.1 NP_476575.1 NP_571942.1 NP_004497.1 NP_032323.3 XP_533482.2 XP_001503083.2 NP_001089021.1 NP_001161236.1 XP_002189589.1 NP_001117849.1 BAI50338.1 NP_001039053.1 XP_420166.2 NP_001013335.1 NP_001035757.1 NP_036069.1 NP_001041586.1 XP_001497009.1 NP_001165845.1 XP_002188828 Online Resource 1 List of species and NCBI accession numbers used for heat shock factor protein phylogenic and protein domain analysis

2. Oligo Name Nucleotide Sequence HSE 5’ ctaggGAA ATG GAA GC TTC GG GAA AC TTC GG GTCGG 3’ HSEsn 5’ ctaggGAA ATG GAA GC CTC GG GAA AC TTC GG GTCGG 3’ HSEmod1 5’ ctaggGAA ATG GAG GC GTC GG GAG AC GTC GG GTCGG 3’ HSEmod2 5’ ctaggGAA ATG GCA GC TCC GG GCA AC TCC GG GTCGG 3’ HSEmod3 5’ ctaggGAA ATG GTC GC AGC GG GTC AC AGC GG GTCGG 3’ HSEmod4 5’ ctaggGAA ATG CAA GC TTA GG CAA AC TTA GG GTCGG 3’ CBF 5’ ctaggGAG CCA ATC ACC GAG CTCG 3’ Online Resource 2 Sequence of HSE and CBF oligonucleotides used for EMSA. Nucleotides altered from the perfect HSE sequence are underlined. Over-hanging nucleotides used for end-fill labeling are indicated by lower case letters

3. Online Resource 3 Efficiency of hsf4 gene knockdown using four concentrations of MO. Total numbers of embryos injected with hsf4-MO and hsf4 5bpmm-MO as well as uninjected and danieau injected controls are listed. The percentage of embryo survival 48 hours after injection is also listed. Efficiency of the MO injections in surviving embryos was determined by calculating percentage of embryos that displayed strong flouresceine fluorescence. The number of embryos containing flouresceine and showing non-specific deformities at each MO concentration was tallied to determine the optimal MO concentration for injection ControlsMO Injected Uninjected Danieu Injected 5.00 µg/µL3.46 µg/µL2.595 µg/µL1.73 µg/µL hsf45bpmmhsf45bpmmhsf45bpmmhsf45bpmm Embryos Injected (#) 4612014837266200267247459309 Survival at 48 hours (%) 79634838697470636175 Deformities (%) 002614968857