Fig. 1. Anteroposterior patterning in the early neural tube. Morphological analysis and whole-mount double in situ hybridization of wild-type embryos with indicated markers and stages. (A-C) The transition between forebrain and midbrain is marked by altered morphology, the posterior pax6.1 gene expression boundary and location of the posterior commissure (PC). (A-C) Morphology of the diencephalic-mesencephalic boundary (DMB): confocal microscopy of a living Bodipy-FL-ceramide C5 stained embryo at 26 hpf (B); (A) scanning plane. The white arrow indicates the position of the biotinylated rhodamine-dextran labeled cell and white circles indicate the position of the PC as a morphological landmark of the DMB. (C) The same embryo after in situ hybridization with a pax6.1-probe and antibody staining against biotin to visualize the position of the labeled cell (red arrow). (D-R) Analysis of expression patterns at the territory of the DMB: expression pattern of pax6.1 and pax2.1. pax6.1 is first detectable at 90% of epiboly in a `salt and pepper' distribution (D; black arrows). A horseshoe-like pattern is visible at 100% (E). (G) Cross-section of the embryo in F visualizes the gap between pax6.1 and pax2.1 at tailbud stage (arrowheads). (H,I) Pattern of gap at 6 ss and 24 hpf. (J-N) Expression pattern of pax6.1 and eng2. From the onset (J) until midsomitogenesis (M) there is no gap between the expression domains of pax6.1 and eng2. At 24 hpf (N), the expression of eng2 is much broader on the MHB compared with pax2.1 (I) and fades out into the midbrain. (O-R) Expression pattern of pax6.1 and fgf8. fgf8 is first expressed in a wide band spreading from the MHB primordium over to the anterior hindbrain (O). At tailbud stage the expression is confined to the MHB territory and the presumptive rhombomere 4 (P). In the early phase of neural patterning, 10 cell wide gap is shown between the expression domains (O,P). At 6 ss the gap increases in size to 20 cells (Q). At 24hpf, the expression of fgf8 is refined to the posterior part of the MHB (R). Arrowheads demarcate the gap between the expression domains of pax6.1 and the indicated marker (D-R). Asterisks mark the pax6.1 free territory (E,J,O). dd, dorsal diencephalon; eye, eye anlage; fb, forebrain; fec, facial ectoderm; DMB, diencephalic-mesencephalic boundary; mb, midbrain; MHB, midbrain-hindbrain boundary; ost, optic stalk; PC, posterior commissures, somites per stage; tb, tailbud stage; tec, tectum opticum. Percentage specifications are percent of epiboly indicated in the figures.