Early mammillary pouch specification in the course of prechordal ventralization of the forebrain tegmentum.

The mammillary body, a ventral specialization of the caudal hypothalamus, lies close to the transition between epichordal and prechordal parts of the forebrain (Puelles and Rubenstein, 2003). This report examines its presumed causal connection with either prechordal or notochordal mesodermal induction, as well as the timing of its specification, in the context of early ventral forebrain patterning. It was recently found that the ephrin receptor gene EphA7 is selectively expressed in the mammillary pouch from early stages of development (HH14: García-Calero et al., 2006). We used mammillary EphA7 expression as well as ventral hypothalamic expression of the gene markers Nkx2.1 and Shh to analyze experimental effects on mammillary specification and morphogenesis after axial mesoderm ablation at stages HH4+ to HH6. Progressively delayed ablation of the prechordal plate revealed its sequential implication in molecular specification of the entire ventral forebrain, including the mammillary and tuberal regions of the hypothalamus. We observed differential contact requirements for induction by the prechordal plate of all the forebrain regions expressing Shh and Nkx2.1, including distant subpallial ones. In contrast, ablation of the anterior notochordal tip at these stages did not elicit significant patterning changes, particularly no effects on mammillary EphA7 expression or mammillary pouch development.

Correlation of a chicken stage 4 neural plate fate map with early gene expression patterns.

A number of gene markers are currently claimed to allow positive or negative visualization of the early chick neural plate at stages 3d/4, when its fate becomes determined. Some markers labeled by various authors as either "neural" or "non-neural" indeed show ectodermal expression patterns roughly correlative with widespread yet vague ideas on the shape and size of the early neural plate, based on previous fate maps. However, for technical reasons, it is not clear how precisely these expression patterns correlate with any experimentally determined fate boundaries. An eventual mismatch between fate and marker interpretation might bear importantly on ideas about gene functions and causal hypotheses in issues such as the establishment of the neural/non-neural border or the earliest mechanisms of neural regionalization. In this review, we correlated a set of epiblastic and mesendodermal gene expression patterns with the novel neuroectoderm proportions suggested by our recent fate map of the chick neural plate at stages HH 3d/4 [P. Fernández-Garre, L. Rodriguez-Gallardo, V. Gallego-Diaz, I.S. Alvarez, L. Puelles, Fate map of the chicken neural plate at stage 4, Development 129 (2002) 2807-2822.]. This analysis suggests the existence of various nested subregions of the epiblast with boundaries codefined by given sets of gene patterns. No gene expression studied reproduces exactly or even approximately the entire neural plate shape, leading to a combinatorial hypothesis on its specification. This kind of analysis (fate and molecular maps), jointly with competence maps, provides the basis for understanding gene functions and the mechanisms of neural induction, specification and regionalization. Several gene patterns observed are consistent with precocious incipient regionalization of the neural plate along the dorsoventral and anteroposterior axes.

Agreement and disagreement among fate maps of the chick neural plate.

Fate maps are essential to understand embryonic development; they provide a background for deducing maps of differential cellular specification in the context of other experimental data and molecular expression patterns. Due to its accessibility, the chick neural plate has been fate-mapped many times, albeit without complete agreement with respect to its shape, extent and fated subdivisions. In this review, we first comment about avian neural plate fate maps reported since the early period of experimental embryology, referring to the different methods followed. We next review a perfected fate-mapping methodology, which recently allowed us rather precise delimitation of the chick neural plate at stages 3d/4. This leads to a general discussion about the apparent border of the neural plate and the prospective main rostrocaudal and longitudinal divisions of the neural tube.

Fate map of the chicken neural plate at stage 4.

A detailed fate map was obtained for the early chick neural plate (stages 3d/4). Numerous overlapping plug grafts were performed upon New-cultured chick embryos, using fixable carboxyfluorescein diacetate succinimidyl ester to label donor chick tissue. The specimens were harvested 24 hours after grafting and reached in most cases stages 9-11 (early neural tube). The label was detected immunocytochemically in wholemounts, and cross-sections were later obtained. The positions of the graft-derived cells were classified first into sets of purely neural, purely non-neural and mixed grafts. Comparisons between these sets established the neural plate boundary at stages 3d/4. Further analysis categorized graft contributions to anteroposterior and dorsoventral subdivisions of the early neural tube, including data on the floor plate and the eye field. The rostral boundary of the neural plate was contained within the earliest expression domain of the Ganf gene, and the overall shape of the neural plate was contrasted and discussed with regard to the expression patterns of the genes Plato, Sox2, Otx2 and Dlx5 (and others reported in the literature) at stages 3d/4.