Ontogenetic expression of maternal and zygotic genes in Atlantic cod embryos under ambient and thermally stressed conditions Kaja H. Skjærven a, ⁎, Pål A. Olsvik a , Roderick Nigel Finn b,c , Elisabeth Holen a , Kristin Hamre a a National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029 Nordnes, 5817 Bergen, Norway b Institute of Biology, University of Bergen, PO Box 7803, 5020 Bergen, Norway c Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway abstract article info Article history: Received 15 November 2010 Received in revised form 24 February 2011 Accepted 24 February 2011 Available online 3 March 2011 Keywords: Atlantic cod embryos pou2 nanog hsp70 hsp90α Reference genes The embryonic stages of Atlantic cod (Gadus morhua) are especially sensitive to incubation temperature. The purpose of the present study was to follow the ontogenetic expression of selected genes of maternal (pou2 and nanog) and zygotic origin (hsp70, hsp90α and stip1), in Atlantic cod embryos under ambient and thermally stressed conditions. The study also investigated how reference genes can be applied to studies on embryonic development, when maternal genes are degraded and the zygotic transcription stabilizes. Three batches of eggs were reared and gene expression profiles from the reference and target genes were determined. The embryos were reared at ambient 6 °C, and 10 °C for continuous long-term and acute short-term heat exposure. Both pou2 and nanog showed reduced expression whereas the zygotic and reference genes showed increased expression until stabilizing at gastrulation, when a normalized ontogenetic expression profile of target genes could be generated. pou2 and nanog were not affected by thermal stress. In contrast, hsp70 and hsp90α were upregulated after short-term heat exposure at the early blastula (hsp70 only), late blastula, 50% epiboly and 90% epiboly stages (hsp90α only). Long-term heat exposure of Atlantic cod embryos upregulated both hsp70 (90% epiboly) and hsp90α (90% epiboly and 20-somites). The results suggest that a cellular defense mechanism is activated even in the earliest stages of embryonic development, a period critical to developmental temperature. © 2011 Elsevier Inc. All rights reserved. 1. Introduction The timing of the transition from maternal to embryonic gene control varies among different taxa. In mammals this transition occurs as early as the 2-cell stage (Telford et al., 1990), whereas in teleosts, zygotic transcription is delayed until the 1k-cell stage, a period known as the mid blastula transition (MBT) (Kimmel et al., 1995). The MBT in fish marks the beginning of gastrulation, a profound phase that organizes the embryonic axis and subsequently determines the cell fates of the three germ layers. Prior to MBT, during cleavage and the early blastula stage, early morphogenic events are directed by the translation of pre-existing maternal mRNAs accumulated in the oocyte. At cleavage and early blastula stage the embryonic teleost cell size decreases dramatically as the number of blastomeres increases proportionally, and at this time, the cells do not synthesize new cytosol or transcribe their own DNA. No studies have so far investigated the temperature dependent transition from maternal to zygotic gene expression in embryonic stages of Atlantic cod. This early phase of development seems to be especially sensitive to incubation temperature (Brown et al., 2003) and we hypothesized that this sensitivity is connected to transcription of the selected heat shock proteins. The thermal tolerance range for rearing Atlantic cod eggs is 1–12 °C (Galloway et al., 1998), however, the most widely used and recom- mended rearing temperature is 6–8 °C (Puvanendran and Brown, 1999; Baskerville-Bridges and Kling, 2000; Brown et al., 2003; Avery et al., 2009). At the spawning grounds, however, the eggs of the wild coastal cod populations are found at 4 °C in the Barents sea and at 7 °C in the North sea, and the postulated climate mediated increase in temperature is of concern for the Atlantic cod population (van der Meeren and Ivannikov, 2006; Holliday et al., 2008; Kjesbu et al., 2010). It has been shown that increasing incubation temperature to 10 °C, causes accelerated growth and development of Atlantic cod eggs (Hall and Johnston, 2003; Geffen et al., 2006), but a higher rearing temperature is associated with a higher frequency of deformation and reduced survival (Iversen and Danielssen, 1984; van der Meeren and Ivannikov, 2006). The effect of incubation temperature depends on the stage of development, and later stages of development can tolerate a higher temperature than less developed earlier stages (Iversen and Danielssen, 1984; Brown et al., 2003). Recent studies have demonstrated that incubation temperature can have long-term effects on the somatic cell lineages of teleosts so that future growth potential is partially determined in the embryo (Johnston, 2006). Comparative Biochemistry and Physiology, Part A 159 (2011) 196–205 ⁎ Corresponding author. Tel.:+47 41458362; fax: + 47 55905299. E-mail address: ksk@nifes.no (K.H. Skjærven). 1095-6433/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.02.026 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa