Development and application of novel cloning strategies for analysis of genes controlling embryo development.

Abstract

Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes.