Pollution Effects on Sensitive and Resistant Embryos: Integrating Structure and Function with Gene Expression

To explore how gene expression translates to developmental phenotype in both sensitive and resistant Fundulus embryos upon POP exposure, we exposed Fundulus embryos from the Elizabeth River Superfund population and the Magotha Bay, VA clean population to Elizabeth River polluted sediment extracts and measured chemical uptake, gene expression, and altered embryo anatomy, morphology and cardiac physiology during four critical developmental stages: somitogenesis, heart beat initiation, late organogenesis, and pre-hatching. A loop design was used for the microarray hybridizations where each sample is hybridized to 2 arrays using both Cy3 and Cy5 labeled fluorophores (PMID: 11355567; 12933549) modified as per Altman (PMID: 17371610). The loop consisted of Cy3 and Cy5 labeled embryo aRNAs from 4 biological pools for four developmental stages (S: S21, S25, S31, and S35) and four different treatments (MM, ME, EM, and EE where the first letter denotes the family, sensitive (M = Magothas Bay) or resistant (E = Elizabeth River) and the second letter denotes the exposure, clean (M) or polluted (E) sediment extract. In total, 64 biological pools were hybridized to 32 microarrays. Each array had different combinations of biological pools, so that the most direct comparisons (i.e. EE vs MM stage 31 samples) are hybridized to the same array. The loop formed was S21EE -- S21MM --S21EM--S21ME--S25EE--S25MM--S25EM--S25ME-- S31EE-- S31MM--S31EM--S31ME-- S35EE-- S35MM-- S35EM-- S35ME, where each dash represents a separate hybridization (array) with the biological pool at the left of the dash labeled with Cy3 and the biological pool at the right of the dash labeled with Cy5. S21 = somitogenesis; S35 = heart beat initiation; S31 = late organogenesis; S35 = pre=hatching