Paragenetic studies of TgHbox1 during sea urchin embryogenesis

Abstract

The HOM-C homeobox genes are responsible for regional specification along the anterior-posterior axis of the embryo which results in the determination of body structures. TgHbox1, an Antennapedia-class homeobox gene related to homeobox genes in the HOM-C cluster has been previously described in the sea urchin Tripneustes gratilla (Dolecki et aI., 1986). This dissertation is an investigation into the function of the TgHbox1 gene during sea urchin embryogenesis. Due to the lack of the ability to perform classical genetic studies to examine gene function in the sea urchin, a paragenetic approach was designed. This was accomplished by microinjection of batches of sea urchin eggs with one of the following gene-specific reagents: phosphorothioate-modified oligonucleotides with TgHbox1 antisense sequence, TgHbox1-specific polyclonal antibodies, a synthetic lacZ-TgHbox1 fusion mRNA encoding a potential dominant negative protein, or a bacterially expressed truncated TgHbox1 protein to inhibit function of the endogenous protein. The embryos were fertilized and followed visually through embryogenesis. The authenticity of all reagents was confirmed in vitro as well as in vivo. Additionally, the persistence of the reagents or their products were demonstrated to be in excess of the endogenous target molecules and to be present in the embryo during the relevant development stages. Morphological analysis of the embryos injected with the four different TgHbox1-specific reagents yielded no specific mutant embryonic phenotype or perturbation of embryogenesis. The findings suggest are consistent with any of the following: TgHbox1 function was not inhibited sufficiently by any of the gene-specific reagents, redundant homeobox genes or other genes were able to functionally compensate for the loss of TgHbox1 gene function, TgHbox1 has no function in the stages of development studied, or there was an unidentified conceptual or experimental flaw in each of the four approaches.