#SEA URCHIN BLASTOPORE SERIES#

The inhibitory activity is quantitatively estimated as the value of k A, the slope here, k A = 1.26 × 10 10 M −1. The intercept, Y 0 (here Y 0 = 0.78 of total probe in the reaction) represents the probe occupancy in the absence of antibody ( A = 0).

#SEA URCHIN BLASTOPORE SERIES#

Relative occupancy of oligonucleotide probe ( Y) is plotted against an antibody dilution series ( A). MATERIALS AND METHODS DNA-Binding Inhibition Assays.įigure 1 Measurement of inhibitory activity of mAb with respect to specific P3A2–DNA complex formation. Our objectives in this work were threefold: first, to develop a TKO vector that would effectively sequester endogenous P3A2 transcription factor ( αP3A2⋅ TKO), the functionality of which could be assayed by determining its effects on spatial expression of CyIIIa⋅ CAT second, to determine whether we could affect the expression of the endogenous CyIIIa gene during embryonic development and third, to look for any other phenotypes in αP3A2⋅ TKO embryos that may indicate additional embryonic functions of the P3A2 transcription factor. Similarly, if the endogenous P3A2 factor is titrated away from the CyIIIa⋅ CAT expression construct by cointroduction of excess target-site, ectopic oral ectoderm expression is also observed ( 15). CyIIIa⋅ CAT expression constructs reproduce the aboral expression of the parent gene, but if either of the P3A2 sites in the wild-type construct is destroyed, expression spreads dramatically to the oral ectoderm ( 14). This gene is normally expressed only in aboral ectoderm lineages beginning early in development. P3A2 was cloned and characterized ( 9, 13) after identification of two of its target sites in the cis-regulatory element of the CyIIIa cytoskeletal actin gene, which were found to be required for correct spatial expression of this gene.

The TKO vector described herein was designed to attack the SpP3A2 transcription factor ( 9), the initial member of a small family of transcription factors that now includes Drosophila erect wing ( 10), chicken IBR/F ( 11), and human NRF-1 ( 12). More generally, it would provide a means of determining downstream functions of the targeted factor. This would afford the opportunity to compare the effects of canceling transcription factor activity with the effects of mutations of the relevant target sites in an expression vector ( 8). Antisense oligonucleotides can be used to destroy maternal mRNAs encoding transcription factors however ( 7), there has been no general or direct way to examine the function of specific transcription factors in sea urchin embryos by blocking their activity in vivo.

Many relevant sea urchin transcription factors have been purified by using affinity chromatography after identification of their cis-regulatory target sites and subsequently have been cloned ( 6). Quantitative temporal and spatial patterns of reporter-gene expression can be conveniently assessed (e.g., refs. Sea urchin eggs and embryos have thus emerged as a major experimental system for analysis of developmental cis-regulatory functions ( 1– 3). Sea urchins have a relatively simple process of embryogenesis, and an efficient and straightforward method of gene transfer has been developed, affording the opportunity to introduce expression constructs into thousands of eggs per day. In this communication we describe a means of blocking a specific gene regulatory interaction in living sea urchin embryos.

In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer. Widespread expression of αP3A2⋅ TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. αP3A2⋅ TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Furthermore, the αP3A2⋅ TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an α P3A2⋅ TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. “TKO” is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control.

Sea urchin blastopore