Following removal operations, embryos were cultured until appropriate stages when brain morphology and pattern could be assayed by in situ hybridisation for various
All wholemounts are viewed form the ventral aspect, anterior to the top. A Normal
hex expression at stage 5+ . B Full restoration of hex expression by stage 5+,
following lower layer removal at stage 4. C Central domain of hex expression in prechordal region is completely absent, several hours after endoderm removal at stage 4+ to 5. D, E Sagittal sections along the midline of a normal stage 5 embryo, anterior to the right and node on the left, showing hex and crescent expression, respectively. Hex expression is seen in the very anterior endoderm, and also in the endoderm and probably mesoderm (see text) of the prechordal region (D). Crescent
expression is seen in all emerged axial mesoderm, and in endoderm stretching right to the periphery of the area pellucida (E). F and G Transverse sections showing
crescent expression in endoderm and head process of a normal embryo (F) and an
embryo following endoderm removal at stage 4+ to 5 (G; level of section shown in K). H Dark field image of embryo in C, showing that prechordal mesoderm structure is intact, even though hex expression is absent in this region. I Normal crescent
expression at stage 6. J Full restoration of crescent expression by stage 6, following lower layer removal at stage 4. K Large area of crecent-expressmg endoderm remains absent several hours after endoderm removal at stage 4+ to 5. Scale bar in A
marker genes or candidate signalling molecules. Analysis at the 12 somite stage was chosen as an informative end-point, since at this stage the forebrain normally shows a distinctive T-shaped morphology due to lateral expansion of the telencephalon and eye cups. Analysis of otx 2 expression at the 12 somite stage, a gene expressed throughout the forebrain and midbrain (Fig3.2), highlights the loss of anterior brain structure following stage 4+ to 5 removals (Fig6.7E-I). However, the anteroposterior extent of
otx 2 expression remained pretty normal following all operations. More specific
forebrain markers were therefore also examined.
GANF, the chick homologue of the mouse homeobox gene hesx 1, is first expressed in
the anterior neural plate at stage 4+. By the 12 somite stage, expression is normally observed in the telencephalon and in ventral head ectoderm (see normal expression chapter; Fig3.3F,G,K). Following lower layer removal at stage 4, this normal pattern of
GANF expression was seen (Fig6.5B). However, GANF expression was not maintained in those embryos showing patterning defects; by the 12 somite stage, neural
GANF expression was completely lost or reduced in 88% of embryos (22/25) following stage 4+ to 5 endoderm removal (Fig6.5E).
The winged helix transcription factor gene BF-1 shows normal early expression restricted to telencephalon and medial eye cup regions (Alvarez et a l, 1998), and is known to be essential for regulating cell proliferation and formation of forebrain architecture (Dou et ah, 1999). Normal BF-1 expression was seen in the majority of embryos following lower layer removal at stage 4 (Fig6.5C; compare with normal expression Fig3.5E,F,I). However, in a manner similar to GANF, BF-1 expression was either completely lost or reduced in 87% of cases (13/15) following removal between stage 4+ and 5 (Fig6.5F).
Dorsal views of wholemount embryos, anterior to the top. A, D 4 somite embryos showing normal GANF expression after endoderm removal at stage 4 (A) and recapitulation of this normal pattern following endoderm removal at stage 4+ to 5 (D). B ,E 12 somite embryos showing normal GANF expression after stage 4 endoderm removal (B) and complete absence of GANF expression following endoderm removal at stage 4+ to 5 (E). C, F 12 somite embryos showing normal BF~ 1 expression after endoderm removal at stage 4 (C) and complete absence of BF-1
expression following endoderm removal at stage 4+ to 5 (F). Scale bar in A = 450pm for A, B and 300pm for C- F
The gene encoding the secreted growth factor FGF 8 also shows a restricted patch of expression in the telencephalon at the 12 somite stage (see Fig3.4E,F). Normal telencephalic expression of FGF 8 was observed in the majority of embryos following endoderm removal at stage 4 (Fig6.6A). Once again, however, this expression was specifically lost or reduced in size in most embryos (75%; 12/16) after stage 4+ to 5 endoderm removals (Fig6.6B). Note that all other aspects of FGF 8 expression, in the midbrain/ hindbrain boundary and neural crest, remained unaffected.
The homeobox gene pax 6 is initially expressed throughout the dorsal aspect of the prosencephalon, but later becomes progressively stronger laterally as the telencephalon and eye cups expand, and is down-regulated at the dorsal midline (see Fig3.6). Interestingly, by the 12 somite stage, pax 6 has an expression pattern complementary to
BF-1 and FGF 8, since it is excluded from the anterior neural folds of the telencephalon but is expressed throughout the rest of the prosencephalon. Following a stage 4 lower layer removal, normal pax 6 expression was observed at the 12 somite stage (Fig6.6C). However, after removing endoderm at stage 44- to 5, pax 6 expression was seen throughout the basic forebrain vesicle and was not down-regulated in the dorso-anterior midline (11/11 embryos; Fig6.6D). Pax 6 expression was not expanded ventrally, however, consistent with the presence of normal inhibitory signals from ventral midline (Fig6.6E,F).