Transcription profiling during differentiation of the neural tube in HH stage 10 chick embryos

The spinal cord is generated progressively as cells leave the caudal region of the elongating body axis such that the temporal steps of neural differentiation become spatially separated along the head to tail axis. At key stages, it is therefore possible to isolate near-adjacent cell populations from the same embryo in distinct differentiation states. Cells in the caudal lateral epiblast adjacent to the primitive streak (also known as the stem zone, SZ, in the chick) express both early neural and mesodermal genes. Other cells in the stem zone will gastrulate to form the paraxial mesoderm or remain in the epiblast cell sheet and become neural progenitors. These latter cells form a new region called the preneural tube (PNT), which is flanked by unsegmented presomitic mesoderm and represents an early neural progenitor state that can be induced by FGF signalling to revert back to a multi-potent SZ state. Rostral to this, the closed caudal neural tube (CNT) is flanked by somites and is an early site of co-expression of genes characteristic of neural progenitors, and of ventral patterning genes (Diez del Corral et al., 2003). The CNT contains the first few neurons and exposure to FGF cannot revert this tissue to a multi-potent SZ state (Diez del Corral et al., 2002). The transition from the PNT to the CNT thus involves commitment to a neural fate that this is regulated by a switch from FGF to retinoid signalling. More advanced neuroepithelium is then located in more rostral neural tube (RNT), in which neuronal differentiation is ongoing and dorsoventral pattern is refined. This experiment uses the Affymetrix GeneChip chicken genome microarray to compare the transcriptomes of microdissections of these spatially distinct cell populations from the elongating neural axis of HH stage 10 chick embryos. Dissections were carried out in L15 medium at 4°C and explants pooled in TRIzol reagent (Gibco) for RNA extraction. Notochord was removed by controlled trypsin digestion that aimed to keep the neural ventral midline. For the microarrays, at least five tissue samples for each region were pooled to make each of three biological replicates for each (n>15 for each region).

12