An artificial promoter construct for heat-inducible misexpression during fish embryogenesis

Abstract

Beside spatial distribution, timing of gene expression is a key parameter controlling gene function during embryonic development. Gain-of-function experiments can therefore have quite opposing results, depending on the time of gene activation. Induction techniques are necessary to control timing in these experiments from outside of the organism. Natural heat shock promoters constitute a simple inducible misexpression system, the main disadvantage is a high background level of expression. We present here a new heat stress-inducible bidirectional promoter consisting of multimerized heat shock elements (HSE). The simplified architecture of this promoter largely improves the properties needed for an efficient induction system: dramatically reduced background activity, improved inducibility, and loss of all tissue specific components. Based on this new artificial promoter, we present a transient induction system for fish embryos. Application of this new induction system for Fgf8 misexpression during embryonic development reveals different windows of competence during eye development. A dramatic early phenotype resulting in loss of the eyes is observed for conventional mRNA injection. Later activation, by using our inducible promoter, uncovers different eye phenotypes like cyclopic eyes. Even after 14 days, an efficient heat stress response could be evoked in the injected embryos. The HSE promoter therefore represents a new artificial heat shock promoter with superior properties, making possible transient experiments with inducible misexpression at various stages of development.

Mutual antagonism of the paired-type homeobox genes, vsx2 and dmbx1, regulates retinal progenitor cell cycle exit upstream of ccnd1 expression

“…To determine the impact of prolonged vsx2 expression on RPC behavior, we used a heat shock inducible full length vsx2 over expression construct (pSGH:vsx2) with a bi-directional promoter driving the expression of vsx2 and GFP (Bajoghli et al, 2004). First, we confirmed that vsx2 RNA and protein levels were up-regulated in the retina at 48 hpf in response to an optimized heat shock protocol (pSGH:vsx2 hs).…”

“…To over-express vsx2 in vivo, the pSGH:vsx2 plasmid (kind gift from Dr. Thomas Czerny) was microinjected (100 pg) into the cytoplasm at the 1-4 cell stage (Bajoghli et al, 2004). This construct is a bi-directional meganuclease transgene vector containing a fragment with eight heat shock elements upstream of a CMV minimal promoter, driving the expression of the GFP gene.…”

Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b

“…Misexpression studies indicate that Fgf signaling is not only necessary but also sufficient for otic induction [11,13,21-25]. However, the region where ectopic otic structures formed was inconsistent in these studies, ranging from limited induction in the region surrounding the endogenous ear [13,21,22] to widespread ectopic otic induction at the expense of other sensory organs [23-25].…”

“…However, the region where ectopic otic structures formed was inconsistent in these studies, ranging from limited induction in the region surrounding the endogenous ear [13,21,22] to widespread ectopic otic induction at the expense of other sensory organs [23-25]. Infection of chick embryonic hindbrain and surface ectoderm with an Fgf3 -expression virus vector at early somite stages results in an enlarged otocyst and ectopic otic vesicles just anterior and posterior to the normal otic vesicle [21].…”

New Ways to Admire Zebrafish: Progress in Functional Genomics Research Methodology

“…This method of induction was improved on when novel artificial synthetic heat-inducible promoter consisting of multimerized heat shock elements (HSEs) used in medaka fish demonstrated a high signal-to-noise ratio (63). …”