Heterochronic Developmental Shifts Underlying Squamate Cerebellar Diversity Unveil the Key Features of Amniote Cerebellogenesis

Abstract

Despite a remarkable conservation of architecture and function, the cerebellum of vertebrates shows extensive variation in morphology, size, and foliation pattern. These features make this brain subdivision a powerful model to investigate the evolutionary developmental mechanisms underlying neuroanatomical complexity both within and between anamniote and amniote species. Here, we fill a major evolutionary gap by characterizing the developing cerebellum in two non-avian reptile speciesbearded dragon lizard and African house snake-representative of extreme cerebellar morphologies and neuronal arrangement patterns found in squamates. Our data suggest that developmental strategies regarded as exclusive hallmark of birds and mammals, including transit amplification in an external granule layer (EGL) and Sonic hedgehog expression by underlying Purkinje cells (PCs), contribute to squamate cerebellogenesis independently from foliation pattern. Furthermore, direct comparison of our models suggests the key importance of spatiotemporal patterning and dynamic interaction between granule cells and PCs in defining cortical organization. Especially, the observed heterochronic shifts in early cerebellogenesis events, including upper rhombic lip progenitor activity and EGL maintenance, are strongly expected to affect the dynamics of molecular interaction between neuronal cell types in snakes. Altogether, these findings help clarifying some of the morphogenetic and molecular underpinnings of amniote cerebellar corticogenesis, but also suggest new potential molecular mechanisms underlying cerebellar complexity in squamates. Furthermore, squamate models analyzed here are revealed as key animal models to further understand mechanisms of brain organization.

Time in Neurogenesis: Conservation of the Developmental Formation of the Cerebellar Circuitry

“…The existence of a mitotic EGL was recently demonstrated in two reptilian species [Macrì and Di-Poï, 2020]. Our data also evidence a high proliferative capacity of the external surface of the sauropsid cerebellum, leading to a vastly prolonged neurogenic period.…”

“…As an example, the prolonged production of mammalian granule cells [Altman, 1972;Nakashima et al, 2015] could be a mammalian novelty, selected for the generation of highly dense granule cell layers. However, our data and previous research by other authors [Hanaway, 1967;Alvarez Otero et al, 1993;Butts et al, 2011;Hanzel et al, 2019;Macrì and Di-Poï, 2020] show that no significant heterochrony in neurogenesis contributed to the diversification of the cerebellum across amniotes. The vertebrate cerebellum develops following a tight homochrony of neurogenesis in all species tested.…”

“…Briefly, Purkinje cells are the earliest neurons generated in the cerebellar cortex [Hashimoto and Mikoshiba, 2003], and granule cells the last ones [Legué et al, 2016]. In other non-mammalian amniotes, the sequential appearance of immunoreactive markers for each of the populations seems to follow an equivalent pattern [Quesada and Genis-Galvez, 1983;Macrì and Di-Poï, 2020;Hanzel et al, 2019]. However, experiments to directly test the time of development at which each population is generated have not been conducted yet.…”

Transit Amplifying Progenitors in the Cerebellum: Similarities to and Differences from Transit Amplifying Cells in Other Brain Regions and between Species

“…They do not undergo self-renewal, unlike GCPs in the avian and mammal EGL ( Figure 4 ), indicating no transit amplification systems in these animals. The transit amplification system has been reported in lizards and snakes [ 70 ]. In the EGL of lizards and snakes, GCPs undergo self-renewal to increase their progeny, suggesting that reptiles have acquired proliferative potential in the EGL ( Figure 4 ).…”

Incorporation of 5-Bromo-2′-deoxyuridine into DNA and Proliferative Behavior of Cerebellar Neuroblasts: All That Glitters Is Not Gold

“…The cerebellum is an ancient and a prominent part of the vertebrate central nervous system. It presents a great variability in size and complexity from cyclostomes to humans [34][35][36]. In mammalians, the cerebellum presents a central vermis and hemispheres laterally situated.…”