Figure 1

Schematic diagram showing the sequential stages in the messenger RNA differential display screen. A. HH st10 chicken embryos were harvested from the eggs and neural tubes dissected as shown in (B). C. Prior to use, total RNA integrity of forebrain, midbrain, rhombomere 1 and hindbrain samples were compared to RNA extracted from an un-manipulated HH st10 chick embryo (lanes 1 [control; C] vs 2–5). D. RNA extracted from F, M, R1 and H samples was subjected to differential display RT-PCR as described in Materials and Methods. E. Differentially expressed cDNA bands were cut from the gel and re-amplified. F. Agarose gel electrophoresis of reamplified band obtained in (E). G. Each re-amplified cDNA was cloned in the TOPO TA vector (Invitrogen, USA). A minimum of six independent clones from each sub-cloning were picked and sequenced H. The differential display fragment was used a probe to isolate a longer clone from a chicken HH st10 cDNA λZAP library by primary (I) and secondary (I') screening. The resulting library cDNA was released from the λ vector using manufacturer's instructions, sequenced on both strands and identity was investigated by comparison to sequence databases (J). K. Expression of the clone was determined by in situ hybridization to HH st10 chick embryos (rostral is at the top of image). F = forebrain, M = midbrain, R1 = rhombomere 1, H = hindbrain (rhombomere 2–7).