RIKEN Center for Developmental Biology

2003 Annual Report

Despite their many differences, taxa as diverse as fish, amphibians, reptiles, birds and mammals share a common body plan comprising three regions: the trunk, the hindbrain/pharyngeal region, and the rostral head. Shinichi Aizawa is interested in the molecular bases for and phylogenetic origins of this regionalization, concentrating primarily on the genetic activity and molecular attributes of head development.

All animals develop from the head. The formation of inductive head organizer precedes that of the trunk organizer, as was first demonstrated by Spemann and Mangold in their studies of amphibian embryogenesis. Gene knockout studies in mice, have demonstrated that the rhombomere r1/2 is the ground state in the mammalian body plan. The head organizer induces the rostral head anteriorly to this rhombomere, while the trunk organizer guides trunk development caudally to the same r1/2 landmark. Studying mutations in genes responsible for body patterning in this region, the Aizawa research group hopes to reveal the genetic cascades for the constitution of the anterior head as conserved across vertebrate phyla. The group's research focuses on identifying and studying the functions of factors acting upstream and downstream of Otx2, a master control gene in vertebrate head development, and the molecular bases for the processes of anterior-posterior axis formation, head induction, brain regionalization and cortical development.

Roles of Otx2 in head development

Otx2 plays central roles in each step of head development in vertebrate, but the regulatory mechanisms by which this gene's activity is mediated have remained largely unknown. The development of the head traces back to before gastrulation, and is inseparably linked to the formation of the anterior-posterior axis. In mice, this process begins prior to gastrulation when the cells of the distal ventral endoderm migrate to the region that will become the animal's anterior, forming the anterior visceral endoderm (AVE), which suppresses posteriorizing signals in the adjacent epiblast. In previous work, members of the Aizawa lab demonstrated that Otx2 plays an essential role in triggering this anterior migration. Following gastrulation, organizing centers in the epiblast induce the formation of the anterior neuroectoderm, which subsequently regionalizes into multiple primitive structures destined to form the areas of the brain. In this process, the isthmus and anterior neural ridge act as local organizing centers for mid- and forebrain development.

Otx2 functions as a master gene in each phase of head ontogeny. Aizawa and colleagues have been working to identify and characterize regulatory factors that control the gene's expression in specific sites and stages of development. In the past, the lab found enhancers responsible for promoting Otx2 expression in the visceral endoderm, definitive anterior mesendoderm and the cephalic neural crest cells. These cis-regulatory elements were all located relatively near the transcriptional start site for the gene. More recently, the group has identified and analyzed enhancers that guide Otx2 expression in epiblast, anterior neuroectoderm and fore- and midbrain, which they have named the EP, AN and FM enhancers, respectively. All of these elements are located more remotely from the coding region (more than 80 kb upstream) than are the previously identified Otx2 enhancers.

The activity of the AN enhancer is independent of that of the EP enhancer, and plays an essential role in maintaining the anterior neuroectoderm through Otx2 expression, once that region has been induced. The Aizawa group's studies have also indicated a phylogenetic relationship between these elements, in which FM is the most deeply conserved in the gnathostome (jawed vertebrate) lineage, while the epiblast enhancer appears to have been acquired later, perhaps after the ascent of amphibians, and to include the anterior neuroectoderm enhancer as an essential component. Using these results as a springboard, Aizawa and colleagues next plan to investigate the significance of this phylogenetic specialization of enhancers in terms of the evolution of the vertebrate head, and to continue the search for upstream factors at work in the regulation of Otx2.

Emx genes in early cortical development

In the mouse, the development of the cerebrum is immediately preceded by the closure of the anterior neural plate at around E8.5 in the presumptive forebrain-midbrain junction. In the earliest stages of corticogenesis, the structures of the archipallium, the non-neuronal components of the choroid plexus, the hippocampal complex and the fimbria, are generated. It has been suggested that the last of these structures, the fimbria, which are located at the border of the cerebral cortex and the choroid plexus, function as a local signaling center in cortical development. One model of archipallial patterning involves the expression of ligands, receptors, transcriptional factors and inhibitor molecules to form morphogenetic gradients that direct the differentiation of areas on either side of the cortical hem, but the actions of specific players in this model remain to be worked out. The Aizawa group's studies of Emx1 and Emx2, mouse homologs of the Drosophila head gap gene ems, have shown that these genes cooperate in two phases of cortical development. Previous work demonstrated that Emx1 and Emx2 work together to generate Cajal-Retzius cells and subplate neurons. Recent work now indicates that the two genes also play a combinatory role in establishing the archipallium as distinct from the roof plate, immediately following the closure of the neural tube.