NIH Public Access Author Manuscript Dev Biol. Author manuscript; available in PMC 2014 February 19. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Dev Biol. 2008 June 15; 318(2): 360–365. doi:10.1016/j.ydbio.2008.02.056. A new method, using cis-regulatory control, for blocking embryonic gene expression Joel Smith* and Eric H. Davidson Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA Abstract Many genes, and particularly regulatory genes, are utilized multiple times in unrelated phases of development. For studies of gene function during embryogenesis there is often need of a method for interfering with expression only at a specific developmental time or place. Here we show that in sea urchin embryos cis-regulatory control systems which operate only at specific times and places can be used to drive expression of short designed sequences targeting given primary transcripts, thereby effectively taking out the function of the target genes. The active sequences are designed to be complementary to intronic sequences of the primary transcript of the target genes. In this work the target genes were the transcription factors alx1 and ets1, both required for skeletogenesis, and the regulatory drivers were from the sm30 and tbr genes. The sm30 gene is expressed only after skeletogenic cell ingression. When its regulatory apparatus was used as driver, the alx1 and ets1 repression constructs had the effect of preventing postgastrular skeletogenesis, while not interfering with earlier alx1 and ets1 function in promoting skeletogenic mesenchyme ingression. In contrast repression constructs using the tbr driver, which is active in blastula stage, block ingression. This method thus provides the opportunity to study regulatory requirements of skeletogenesis after ingression, and may be similarly useful in many other developmental contexts. Keywords Sea urchin embryo; Skeletogenic mesenchyme; Intron antisense RNA; Gene knockdown Introduction Unraveling the networks of regulatory gene interactions which control development requires technology for specific interference with gene expression. In frogs, fish, ascidians, sea urchins, and sea stars, i.e., in all non-amniote deuterostome research models for embryonic development, introduction of morpholino-substituted antisense oligonucleotides (MASO) into the egg has emerged as the method of choice. An intrinsic problem, due to their very effectiveness, is that MASOs obliterate whatever is the initial function of the transcription factor to which they are targeted, often compromising subsequent development. However, most regulatory genes have multiple successive functions (Howard-Ashby et al., 2006), and in a MASO-treated embryo these later roles can often not be studied. Here we show that in sea urchin embryos, this problem can be solved, and gene expression specifically eliminated *Corresponding author: Fax: +1 626-583-8351, E-mail address: [email protected]. Publisher©s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Smith and Davidson Page 2 at any desired time and place, by transcription of antisense intron RNA under control of a cis-regulatory module which operates at that time and place. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript The approach that we describe is similar in principle to expression of siRNAs from vectors driven by cis-regulatory modules (Sutou et al., 2007), but in sea urchin embryos siRNAs fail to block gene expression. Because of their instability, simple antisense RNAs targeted to cytoplasmic message never remain at high concentration after injection, or if transcribed never accumulate to high concentration. The mRNAs encoding gene regulatory proteins, which are targets of particular interest, turn over at low rates in sea urchin embryos (t1/2 typically > several h), and despite very modest transcription rates, achieve concentrations of tens to hundreds of molecules per cell and sometimes more (Davidson, 1986; Bolouri and Davidson, 2003). The greatly enhanced stability of MASOs provides the solution which enables stoichiometrically favorable ratios of antisense to cytoplasmic regulatory mRNA to be attained, but with the disadvantage that only initial regulatory functions can be assessed. The rationale for attempting to use antisense RNAs targeting intronic sequences is that intranuclear pre-mRNA transcripts are never present at very high concentrations. In sea urchin embryos there are typically only a few molecules of each pre-mRNA species per cell, for the reason that they are processed and turn over rapidly (t~20 min) (Davidson, 1986). Splice-blocking MASOs also prevent gene expression, and in fact prevent the appearance of mature mRNA, in sea urchin as in fish embryos (Imamura and Kishi, 2005; Dickey-Sims et al., 2005). Thus natural antisense RNAs designed similarly to block splicing might be effective as well, and these could be driven off cis-regulatory modules that would only function at given developmental stages and in given cells. The argument is that since the target nuclear pre-mRNAs are at low concentration, the additional stability afforded by the morpholino adduct which is required for cytoplasmic targets would be unnecessary for nuclear targets. We chose the skeletogenic regulatory system of the sea urchin embryo as our test system. As summarized in Fig. 1, the skeletogenic cell lineage arises at the vegetal pole of the egg and its fate is specified early in cleavage by expression of a set of key regulatory genes, viz., alx1, tbr, and ets1, which are together derepressed exclusively in this lineage by operation of a double negative regulatory gate (Oliveri et al., 2002; Revilla-i-Domingo et al., 2007). The gene regulatory network (GRN) which includes this gate is now deeply understood (Revilla- i-Domingo et al., 2007; Oliveri et al, 2008; for always current version, see http:// sugp.caltech.edu/endomes/). This network indicates the genomic code for all subsequent events of skeletogenic regulatory state specification downstream of the double negative gate, as well as for activation of skeletogenic gene batteries. As indicated in Fig. 1, until late blastula stage the 16 skeletogenic cells remain in the epithelial wall of the embryo, whereupon they ingress into the blastocoel (24 h) and commence skeletogenesis (>30 h). Prior to ingression they not only establish and lock down their state of specification, but also activate many skeletogenic differentiation genes, though biomineral deposition begins only after ingression. However, certain genes are activated only following ingression, one of which figures importantly in this work. This is sm30, which encodes a major protein of the biomineral structure (Frudakis and Wilt, 1995). The alx1 and ets1 genes continue to be expressed actively after ingression, during the skeletogenic phase (Ettensohn et al., 2003; Rizzo et al., 2006). MASO directed against these genes and injected into eggs prevents ingression, and thus all subsequent events of skeletogenesis. These MASOs directly or indirectly break multiple GRN linkages, and in consequence the presumptive skeletogenic cells fail to become specified (op. cit.; Oliveri et al., 2008). While these same regulatory genes are very likely to be essential for the later functions of post-ingression skeletogenesis as well, their roles cannot be studied by use of Dev Biol. Author manuscript; available in PMC 2014 February 19. Smith and Davidson Page 3 alx1 or ets1 MASO, because the treated embryos completely lack ingressed skeletogenic cells. This is a specific example of the general problem adduced at the outset: what is needed NIH-PA Author Manuscript NIH-PA Author Manuscriptis a means NIH-PA Author Manuscript for extinguishing alx1 or ets1 expression in skeletogenic cells only after their ingression. Materials and methods Animal husbandry, embryos and microinjection Adult Strongylocentrotus purpuratus were collected along the Southern California coast and maintained in 12°C seawater at Caltech’s Kerckhoff Marine Laboratory. Delivery of nucleic acid vectors was achieved by standard procedures. Gametes were harvested and eggs rinsed for one minute in 1 mM citric acid seawater and placed in seawater containing 300 mg/mL para-aminobenzoic acid. Approximately 1500 molecules of desired DNA construct (450 molecules for large vectors such as BACs) were injected along with a 6-fold molar excess of HindIII-digested carrier sea urchin DNA per egg in a 4pL volume of 0.12 M KCl. The DNA constructs, typically PCR products, were injected into eggs immediately following fertilization. Our injection solutions included both the antisense-expressing construct (either PCR product or linearized BAC vector) along with linearized Tbr BAC-GFP as a marker of incorporations plus excess HindIII-digested sea urchin genomic DNA as a carrier in a 0.12 M KCl solution. We followed injected embryos by observing GFP expression by fluorescent microscopy at various times post-fertilization. Embryos were placed on glass slides for fluorescent imaging