Biology subjects

Sleight, V. A.

Publications and source records attributed to Sleight, V. A..

7 recordsLinked to original sources

bioRxiv2025

Gene editing without a genome: generation and validation of F0 CRISPR mutants in gastropod mollusc Crepidula fornicata

BackgroundCRISPR-Cas9 gene editing is a powerful tool to study gene function but to date is mostly applied in traditional model organisms. Due to specific challenges in spiralian genomics such as genome size, complexity, proportion of repetitive regions and high inter individual genome variation, applications of CRISPR-Cas9 in spiralian models have been limited. Within the Spiralia, molluscs are a strikingly diverse phylum with many unique gene family expansions and novelties, yet there are relatively few applications of CRISPR-Cas9 to unravel gene function. ResultsWe generated pax6 knockout F0 CRISPR-Cas9 mutants in the gastropod mollusc Crepidula fornicata using a de novo transcriptome to design single guide RNAs and genotyping primers. In lieu of an assembled genome, alignments with closely related species were used to determine putative intron-exon boundaries and successfully target gene editing to a specific exon of pax6 containing a homeodomain. F0 pax6 knockout mutants had an eye-loss phenotype. Successful CRISPR-Cas9 gene editing was confirmed genotypically using Sanger sequencing and Interference of CRISPR Edits (ICE) analysis. ConclusionsThe generation of F0 CRISPR-Cas9 knockout mutants with a clear phenotype in a mollusc without an assembled genome enhances the applicability of CRISPR for functional genomics in non-traditional emerging model systems. pax6 is highly conserved and is required for eye development across metazoans, including the snail C. fornicata. The pax6 mutants developed in this study suggests the pax6 homeodomain is specifically required for eye formation in gastropods and sheds light on the evolution of eye development in animals.

developmental biology↗

bioRxiv2022

Wnt signalling, cell fate determination and anteroposterior polarity of the skate gill arch skeleton

The gill skeleton of cartilaginous fishes (sharks, skates, rays and holocephalans) exhibits a striking anterior-posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Shh signalling from a distal gill arch epithelial ridge (GAER) signalling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

developmental biology↗

bioRxiv2022

Evolutionary conservation and divergence of the transcriptional regulation of bivalve shell secretion across life history stages

Adult molluscs produce shells with diverse morphologies and ornamentations, different colour patterns and microstructures. The larval shell however, is a phenotypically more conserved structure. How do developmental and evolutionary processes generate varying diversity at different life history stages? Using live-imaging, histology, scanning electron microscopy and transcriptomic profiling, we have described shell development in a heteroconchian bivalve the Antarctic clam, Laternula elliptica and compared it to adult shell secretion processes in the same species. Adult downstream shell genes, such as those encoding extracellular matrix proteins and biomineralisation enzymes, were largely not expressed during shell development, and instead, a development-specific downstream gene repertoire was expressed. Upstream regulatory genes such as transcription factors and signalling molecules were conserved between developmental and adult shell secretion. Comparing heteroconchian transcriptomic data with recently reported pteriomorphian larval shell proteome data suggests that, despite being phenotypically more conserved, the downstream effectors constituting the larval shell "tool-kit" may be as diverse as that of adults. Overall, our new data suggests that a larval shell formed using development-specific downstream effector genes is a conserved and ancestral feature of the bivalve lineage, and possibly more broadly across the molluscs.

evolutionary biology↗

bioRxiv2022

The little skate genome and the evolutionary emergence of wing-like fin appendages

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins - gene expression, chromatin occupancy and three-dimensional (3D) conformation - we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3 HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

evolutionary biology↗

bioRxiv2021

Conserved and unique transcriptional features of pharyngeal arches in the skate (Leucoraja erinacea) and evolution of the jaw

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers bapx1 and gdf5 and pro-chondrogenic transcription factors barx1 and gsc, and the dorsalising transcription factor pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

developmental biology↗

bioRxiv2020

Embryonic origin and serial homology of gill arches and paired fins in the skate (Leucoraja erinacea)

Paired fins are a defining feature of the jawed vertebrate body plan, but their evolutionary origin remains unresolved. Gegenbaur proposed that paired fins evolved as gill arch serial homologues, but this hypothesis is now widely discounted, owing largely to the presumed distinct embryonic origins of these structures from mesoderm and neural crest, respectively. Here, we use cell lineage tracing to test the embryonic origin of the pharyngeal and paired fin skeleton in the skate (Leucoraja erinacea). We find that while the jaw and hyoid arch skeleton derive from neural crest, and the pectoral fin skeleton from mesoderm, the gill arches are of dual origin, receiving contributions from both germ layers. We propose that gill arches and paired fins are serially homologous as derivatives of a continuous, dual-origin mesenchyme with common skeletogenic competence, and that this serial homology accounts for their parallel anatomical organization and shared responses to axial patterning signals.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

Single-cell sequencing technologies are revolutionizing biology, but are limited by the need to dissociate fresh samples that can only be fixed at later stages. We present ACME (ACetic-MEthanol) dissociation, a cell dissociation approach that fixes cells as they are being dissociated. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, can be sorted by Fluorescence-Activated Cell Sorting (FACS) and are permeable, enabling combinatorial single-cell transcriptomic approaches. As a proof of principle, we have performed SPLiT-seq with ACME cells to obtain around ∼34K single cell transcriptomes from two planarian species and identified all previously described cell types in similar proportions. ACME is based on affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗