Can clues from evolution unlock the molecular development of the cerebellum?

The cerebellum sits at the rostral end of the vertebrate hindbrain and is responsible for sensory and motor integration. Owing to its relatively simple architecture, it is one of the most powerful model systems for studying brain evolution and development. Over the last decade, the combination of molecular fate mapping techniques in the mouse and experimental studies, both in vitro and in vivo, in mouse and chick have significantly advanced our understanding of cerebellar neurogenesis in space and time. In amniotes, the most numerous cell type in the cerebellum, and indeed the brain, is the cerebellar granule neurons, and these are born from a transient secondary proliferative zone, the external granule layer (EGL), where proliferation is driven by sonic hedgehog signalling and causes cerebellar foliation. Recent studies in zebrafish and sharks have shown that while the molecular mechanisms of neurogenesis appear conserved across vertebrates, the EGL as a site of shh-driven transit amplification is not, and is therefore implicated as a key amniote innovation that facilitated the evolution of the elaborate foliated cerebella found in birds and mammals. Ellucidating the molecular mechanisms underlying the origin of the EGL in evolution could have significant impacts on our understanding of the molecular details of cerebellar development.

Absence of an external germinal layer in zebrafish and shark reveals a distinct, anamniote ground plan of cerebellum development.

The granule cell layer of the cerebellum comprises the largest population of neurons in the vertebrate CNS. In amniotes, its precursors undergo a unique phase of transit amplification, regulated by Sonic hedgehog. They do so within a prominent but transient secondary proliferative epithelium, the external germinal layer, which is formed by tangential migration of precursor cells from the rhombic lip. This behavior is a hallmark of bird and mammal cerebellum development. Despite its significance for both development and disease, it is unclear whether an external germinal layer is a requirement for granule cell production or an expedient of transit amplification. Evidence for its existence in more basal vertebrates is contradictory. We therefore examined cerebellum development in the zebrafish, specifically in relation to the expression of the basic helix-loop-helix gene Atonal 1, which definitively characterizes granule cell precursors. The expression of Atoh1a-Atoh1c, in combination with patterns of proliferation and fate maps, define precursor pools at the rhombic lip and cerebellar midline but demonstrate that an external germinal layer is absent. Sonic hedgehog signaling is correspondingly absent in the zebrafish cerebellum. Sustained roof-plate-derived signals suggest that, in the absence of transit amplification, primary granule cell precursor pools are maintained throughout development. To determine whether this pattern is specific to zebrafish or reflects a more general anamniote organization, we examined the expression of similar genes in the dogfish, Scylliorhinus canicula. We show that these anamniotes share a common ground plan of granule cell production that does not include an external germinal layer.

Reiterative pattern of sonic hedgehog expression in the catshark dentition reveals a phylogenetic template for jawed vertebrates.

For a dentition representing the most basal extant gnathostomes, that of the shark can provide us with key insights into the evolution of vertebrate dentitions. To detail the pattern of odontogenesis, we have profiled the expression of sonic hedgehog, a key regulator of tooth induction. We find in the catshark (Scyliorhinus canicula) that intense shh expression first occurs in a bilaterally symmetrical pattern restricted to broad regions in each half of the dentition in the embryo jaw. As in the mouse, there follows a changing temporal pattern of shh spatial restriction corresponding to epithelial bands of left and right dental fields, but also a subfield for symphyseal teeth. Then, intense shh expression is restricted to loci coincident with a temporal series of teeth in iterative jaw positions. The developmental expression of shh reveals previously undetected timing within epithelial stages of tooth formation. Each locus at alternate, even then odd, jaw positions establishes precise sequential timing for successive replacement within each tooth family. Shh appears first in the central cusp, iteratively along the jaw, then reiteratively within each tooth for secondary cusps. This progressive, sequential restriction of shh is shared by toothed gnathostomes and conserved through 500 million years of evolution.

Early Palaeozoic dentine and patterned scales in the embryonic catshark tail.

Regular scale patterning, restricted to the caudalmost tail and organized into two opposing rows on each side of the tail, is observed in few chondrichthyans. These evenly spaced scales, in dorsal and ventral rows, develop in an iterative sequence from the caudal tip, either side of the notochord. They are subsequently lost as a scattered pattern of placoid scales develops on the body and fins. An identical organized pattern is observed in tail scales of Scyliorhinus canicula (catshark), where the expression of sonic hedgehog signal is restricted to the epithelium of developing scales and remains localized to the scale pocket. Regulation of iterative scale position by sonic hedgehog is deeply conserved in vertebrate phylogeny. These scales also reveal an archaic histological structure of a dentine type found in the oldest known shark scales from the Ordovician and Silurian. This combination of regulated pattern and ancient dentine occurs only in the tail, representing the primary scalation. Scattered body scales in elasmobranchs such as S. canicula originate secondarily from differently regulated development, one with typical orthodentine around a central pulp cavity. These observations emphasize the modular nature of chondrichthyan scale development and illustrate previously undetected variation as an atavism in extant chondrichthyan dentine.