Biology subjects

Shylo, N. A.

Publications and source records attributed to Shylo, N. A..

7 recordsLinked to original sources

bioRxiv2026

A versatile cryopreservation method for peri-gastrulation squamate embryos optimised using the veiled chameleon (C. calyptratus)

Stem cell technologies have become a vital component of conservation efforts around the globe. Biobanks and pluripotent stem cell lines help to ensure species and their genetic diversity are preserved. These efforts have however, focussed mostly on mammals and birds, and the cryopreservation protocols for embryos and cells were developed decades ago laying the basis for artificial reproductive techniques for species conservation. With over 20% of non-avian reptile species facing extinction, it is imperative to establish protocols for reptiles to ensure species preservation and also to facilitate the establishment of new reptile model organisms to match the standard of mammals. Here, we have generated a cryopreservation method for preserving early gastrulating veiled chameleon embryos as a representative squamate species. To this end, we first developed a tissue culture method for maintaining cells extracted from peri-gastrulation chameleon embryos and then tested different cryopreservation methods altering the concentration of the penetrating cryoprotectant DMSO and assessing the effect of the addition of non-penetrating cryoprotectants Trehalose and Sucrose. We then optimised a protocol for whole embryo vitrification in 20% DMSO with added Trehalose or Sucrose that can easily be adapted for fieldwork. Taken together, our method not only provides a protocol for conservation efforts but also lays the basis for mechanistic studies of early squamate embryo development by enabling cryopreservation of whole embryos in a fieldwork setting, which facilitates their live transport back to a laboratory for functional experiments or molecular analyses.

developmental biology↗

bioRxiv2025

Oogenesis and germinal bed morphology of the brown anole (A. sagrei)

BackgroundThe brown anole is a model species of the genus Anolis, a squamate (encompassing lizards and snakes) group widely studied in evolutionary, behavioral, and developmental biology. Full genome annotation, the establishment of gene editing techniques, and comprehensive description of reproductive tract morphology and embryogenesis in this species, has laid the foundation for functional studies. However, analysis of brown anole oogenesis is still required and vital to optimize genome modification, mutant line establishment, and analyses of the evolution of reproductive developmental mechanisms. ResultsHere, we characterize ovary morphology and gametogenesis in the female brown anole, A. sagrei using brightfield imaging, microCT, histology staining, electron microscopy, and confocal imaging. We define 10 stages of oocyte maturation which commences inside the oogonial nest within the germinal bed and concludes with the mature follicle ready to ovulate based on follicle size, yolk-acquisition, and follicular, cellular, and basement membrane architecture. ConclusionsWe describe the complete oogenesis of the brown anole in 10 stages and report that oogenesis is highly conserved within iguanids, a suborder of lizards. With our staging framework, we lay the foundation for functional studies of oogenesis and optimized gene-editing.

developmental biology↗

bioRxiv2025

Biophysical mechanisms of morphogenesis in lizard lungs

The lungs of squamate reptiles (lizards and snakes) are highly diverse, exhibiting single chambers, multiple chambers, transitional forms with two to three chambers, along with a suite of other anatomical features, including finger-like epithelial projections into the body cavity known as diverticulae. During embryonic development of the simple, sac-like lungs of anoles, the epithelium is pushed through the openings of a pulmonary smooth muscle mesh by the forces of luminal fluid pressure. This process of stress ball morphogenesis generates the faveolar epithelium typical of squamate lungs. Here, we compared embryonic lung development in brown anoles, leopard geckos, and veiled chameleons to determine if stress ball morphogenesis is conserved across squamates and to understand the physical processes that generate transitional-chambered lungs with diverticulae. We found that epithelial protrusion through the holes in a pulmonary smooth muscle mesh is conserved across squamates. Surprisingly, however, we found that luminal inflation is not conserved. Instead, leopard geckos and veiled chameleons appear to generate their faveolae via epithelial folding downstream of epithelial proliferation. We also found experimental and computational evidence suggesting that the transitional chambers and diverticulae of veiled chameleon lungs develop via apical constriction, a process known to be crucial for airway branching in the bird lung. Thus, distinct morphogenetic mechanisms generate epithelial diversity in squamate lungs, which may underpin their species-specific physiological and ecological adaptations.

developmental biology↗

bioRxiv2025

Epiblast Lumenogenesis is not a mammalian-specific trait

Epiblast lumenogenesis, which leads to amniotic cavity formation, is a hallmark of mammalian embryogenesis, and required for crucial developmental processes such as anterior-posterior patterning and gastrulation. Based on avian model-organisms, the epiblast in reptiles is thought to form a monolayered flat disc that undergoes anterior-posterior patterning and gastrulation. Here, we report that the squamate, veiled chameleon (Chameleo calyptratus), exhibits epiblast lumenogenesis and amniogenesis prior to anterior-posterior patterning. Using SEM, immunofluorescence, and histology techniques, we demonstrate that chameleon epiblast lumenogenesis occurs via a purse-string-like mechanism involving the formation of supracellular actin cables in concentric rings around the epiblast followed by constriction that closes the epiblast lumen. Through expression analyses of Nodal1, Nodal2, Cerberus, Lefty, Brachyury, Wnt3A, and Bmp2, and immunostaining for Brachyury, we uncovered a Wnt3a- and Brachyury-positive ring at the edge of the epiblast concomitant with lumenogenesis, and preceding anterior-posterior patterning and gastrulation. Furthermore, we report anterior-posterior patterning in veiled chameleons occurs independently of Cerberus and Lefty. These processes that mediate epiblast lumenogenesis in chameleons, result in a morphology remarkably similar to human embryos, despite 300 million years of evolutionary separation. Taken together, we show that pre-gastrulation epiblast lumenogenesis is not mammalian-specific, but also occurs in some non-avian reptiles.

developmental biology↗

bioRxiv2024

Pre-oviposition development of the brown anole (Anolis sagrei)

The brown anole, Anolis sagrei, has emerged as a representative squamate species during the past several decades. Novel functional tools have been established to manipulate embryogenesis through genome editing or the introduction of small molecule inhibitors, and their effective use requires a thorough understanding of early anole embryogenesis. To enable precise and reproducible staging of anole embryos, we need knowledge of the progression of anole embryogenesis and morphogenesis. While post-oviposition development has been described, the pre-oviposition period remains to be explored. Here, we provide the first staging series of pre-oviposition development for the brown anole. Analysing the follicles and embryos through brightfield imaging, SEM, STEM, histology, and DAPI staining, we define 26 distinct developmental stages. Our dataset reveals that peri-gastrulation morphogenesis up to the initiation of neurulation diverges significantly from chicken, the common representative model of reptile embryogenesis. Furthermore, we followed heart development, neural crest cell migration and central nervous system development through immunofluorescence analyses and provide new comparative insights into the morphogenesis of each of these organ systems over time. Our work establishes the brown anole as a squamate model organism for cross clade evolutionary studies of early embryogenesis.

developmental biology↗

bioRxiv2022

Nr6a1 controls axially-restricted body elongation, segmentation, patterning and lineage allocation

The vertebrate main-body axis is laid down during embryonic stages in an anterior-to-posterior (head-to-tail) direction, driven and supplied by posteriorly located progenitors. For the vertebral column, the process of axial progenitor cell expansion that drives elongation, and the process of segmentation which allocates progenitor-descendants into repeating pre-vertebral units, occurs seemingly uninterrupted from the first to the last vertebra. Nonetheless, there is clear developmental and evolutionary support for two discrete modules controlling processes within different axial regions: a trunk and a tail module. Here, we identify Nuclear receptor subfamily 6 group A member 1 (Nr6a1) as a master regulator of elongation, segmentation, patterning and lineage allocation specifically within the trunk region of the mouse. Both gain- and loss-of-function in vivo analyses revealed that the precise level of Nr6a1 acts as a rheostat, expanding or contracting vertebral number of the trunk region autonomously from other axial regions. Moreover, Nr6a1 was found to be required for segmentation, but only for trunk-forming somites, with the timely clearance of Nr6a1 critical in supporting tail formation. In parallel with these morphological outcomes, we reveal Nr6a1 as a novel regulator of global Hox signatures within axial progenitors, preventing the precocious expression of multiple posterior Hox genes as the trunk is being laid down and thus reinforcing that patterning and elongation are coordinated. Finally, our data supports a crucial role for Nr6a1 in regulating gene regulatory networks that guide cell lineage choice of axial progenitors between neural and mesodermal fate. Collectively, these data reveal an axially-restricted role for Nr6a1 in all major cellular and tissue-level events required for vertebral column formation, supporting the view that modulation of Nr6a1 expression level or function is likely to underpin evolutionary changes in axial formulae that exclusively alter the trunk region.

developmental biology↗

bioRxiv2018

Presence of midline cilia supersedes the expression of Lefty1 in forming the midline barrier during the establishment of left-right asymmetry

Cilia in the vertebrate left-right organizer are required for the original break in left-right (L-R) symmetry. Subsequently, proper L-R patterning relies on asymmetric expression of Nodal in the lateral plate mesoderm (LPM). Lefty1, expressed in the embryonic midline, has been defined as the midline barrier, restricting the expression of Nodal to the left LPM. Here we use the mouse ciliary transition zone mutant Mks1krc that has left isomerism and bilateral expression of the NODAL target Pitx2, to reveal that the expression of Lefty1 in the midline is insufficient for the establishment of the midline barrier. We further show through a comparison of two Tmem107 mutants that cilia in the midline are required to supplement Lefty1 expression and establish the functional midline barrier. Tmem107null mutants have no cilia in the midline and display left isomerism due to the loss of the midline barrier, whereas Tmem107schlei hypomorphic mutants have numerous cilia in the node and the midline, leading to normal Lefty1 expression and L-R patterning. This study reveals a novel role for cilia in the maintenance of L-R asymmetry.

developmental biology↗