Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution

Abstract

The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established in ovo electroporation and an ex ovo culture system to address neural stem cell dynamics, neuronal differentiation and migration. Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex.

Pattern of Neurogenesis and Identification of Neuronal Progenitor Subtypes during Pallial Development in Xenopus laevis

“…The analysis of the pallium throughout development in the anuran amphibian Xenopus laevis (anamniote tetrapod) serves to shed light on the anamniote-amniote transition, since particular attention has been given only to reptiles and birds in recent years (Nomura et al, 2013a,b, 2015, 2016; Suzuki and Hirata, 2014; Martínez-Cerdeño et al, 2016; Montiel et al, 2016). Although general patterns of cell proliferation in the anuran brain have been studied (Wullimann et al, 2005; Chapman et al, 2006; Raucci et al, 2006; Simmons et al, 2006; Coen et al, 2007; Denver et al, 2009; Tao et al, 2015; Thuret et al, 2015), no detailed analysis of the pallium has been performed.…”

Efficient harvesting methods for early‐stage snake and turtle embryos

“…Recently, an ex ovo culture system and method of gene transfer using in ovo electroporation in turtle eggs have been reported (Nomura et al . ). Owing to these resources, P. sinensis embryos can now be used as a powerful model system for studying development in the Testudines.…”

“…; Nomura et al . ). In order to better understand the detailed morphogenetic processes in reptiles and utilize this newly available information and technology, we need to establish improved methods for manipulating reptile embryos.…”

Embryonic development of a parthenogenetic vertebrate, the mourning gecko (Lepidodactylus lugubris)

“…Interest in how developmental processes have influenced morphological diversity has led to the recent investigation of squamate (lizard, snake, and amphisbaenian) development. These studies have demonstrated the immense utility of squamates as models for the development of amniote bauplans . Ideally, model clades or species in evo‐devo will meet several criteria (reviewed by Jenner and Wills).…”

“…Despite these factors, few parthenogenetic vertebrates are routinely maintained in laboratory settings . Several additional characteristics make L. lugubris a desirable model organism, including reproductive output (they are highly fecund, laying eggs year round), there are published protocols for husbandry and embryo collection, they can easily be targeted for field collection (including from populations in the Hawaiian Islands) or commercial purchase, and, unlike the majority of squamates, have hard‐shelled eggs which make embryological dissection and manipulation substantially easier …”