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Results for “developmental biology”

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bioRxiv2020

DELocal: Chromosomal neighbourhoods having genes of diverse functions allow improved identification of differentially expressed genes

Although most genes share their chromosomal neighbourhood with other genes, distribution of genes has not been explored in the context of individual organ development; the common focus of developmental biology studies. Because developmental processes are often associated with initially subtle changes in gene expression, here we explored whether neighbouring genes are informative in the identification of differentially expressed genes. First, we quantified the chromosomal neighbourhood patterns of genes having related functional roles in the mammalian genome. Although the majority of protein coding genes have at least five neighbours within 1 Mb window around each gene, very few of these neighbours regulate development of the same organ. Analyses of transcriptomes of developing mouse molar teeth revealed that whereas expression of genes regulating tooth development changes, their neighbouring genes show no marked changes, irrespective of their level of expression. Finally, we test whether inclusion of gene neighbourhood in the analyses of differential expression could provide additional benefits. For the analyses, we developed an algorithm, called DELocal that identifies differentially expressed genes by comparing their expression changes to changes in adjacent genes in their chromosomal regions. Our results show that DELocal removes detection bias towards large changes in expression, thereby allowing identification of even subtle changes in development. Future studies, including the detection of differential expression, may benefit from, and further characterize the significance of gene-gene neighbour relationships. Author SummaryDevelopment of organs is typically associated with small and hard to detect changes in gene expression. Here we examined how often genes regulating specific organs are neighbours to each other in the genome, and whether this gene neighbourhood helps in the detection of changes in gene expression. We found that genes regulating individual organ development are very rarely close to each other in the mouse and human genomes. We built an algorithm, called DELocal, to detect changes in gene expression that incorporates information about neighbouring genes. Using transcriptomes of developing mouse molar teeth containing gene expression profiles of thousands of genes, we show how genes regulating tooth development are ranked high by DELocal even if their expression level changes are subtle. We propose that developmental biology studies can benefit from gene neighbourhood analyses in the detection of differential expression and identification of organ specific genes.

bioinformatics↗

bioRxiv2018

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.\n\nSummary statementStructural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms.

cell biology↗

bioRxiv2019

Two distinct bacterial biofilm components trigger metamorphosis in the colonial hydrozoan Hydractinia echinata

In the marine environment bacterial-induced metamorphosis of larvae is a widespread cross-kingdom communication phenomenon and critical for the persistence of many marine invertebrates. However, the identities of most inducing bacterial signals and the underlying cellular mechanisms remain enigmatic. Larvae of Hydractinia echinata provide an excellent model for investigating bacteria-stimulated settlement as they transform upon detection of the signal into the colonial adult stage within 24 h. Although H. echinata served as cell biological model system for decades, the influence of bacterial signals on the morphogenic transition remained largely unexplored. Using a bioassay-guided analysis, we first identified that specific bacterial (lyso)phospholipids, naturally present in bacterial biofilms, elicit metamorphosis in Hydractinia larvae in a dose-response matter. In particular, lysophospholipids as single compounds or in combinations at 50 {micro}M concentrations induced metamorphosis in up to 50% of all larvae phospholipid within 48 h. By using fluorescence-labeled bacterial phospholipids, we demonstrated their incorporation into the larval membranes, where interactions with internal signaling cascades could occur. In addition, two structurally distinct exopolysaccharides, the newly identified Rha-Man polysaccharide from Pseudoalteromonas sp. P1-9 and curdlan from Alcaligenes faecalis caused up to 75% of all larvae to transform within 24 h. We also found that combinations of (lyso)phospholipids and curdlan induced the transformation in almost all larvae within 24 h, thereby exceeding the morphogenic activity observed for single compounds and axenic bacterial biofilms. Our results demonstrate that multiple and structurally distinct bacterial-derived metabolites converge to induce high transformation rates of Hydractinia larvae, which might ensure optimal habitat selection despite the general widespread occurrence of both compound classes. Significance StatementBacterial biofilms profoundly influence the recruitment and settlement of marine invertebrates, critical steps for diverse marine processes such as coral reef formation, marine fisheries and the fouling of submerged surfaces. Yet, the complex composition of biofilms often makes it challenging to characterize the individual signals and regulatory mechanisms. Developing tractable model systems to characterize these co-evolved interactions is the key to understand fundamental processes in evolutionary biology. Here, we characterized for the first time two types of bacterial signaling molecules that induce the morphogenic transition and analyzed their abundance and combinatorial activity. This study highlights the crucial role of the converging activity of multiple bacterial signals in development-related cross-kingdom signaling. AreasMajor: Chemical Biology, Microbiology, Developmental Biology

ecology↗

bioRxiv2023

BERLIN: Basic Explorer for single-cell RNAseq analysis and cell Lineage Determination.

Single-cell RNA sequencing has revolutionized the study of immuno-oncology, cancer biology, and developmental biology by enabling the joint characterization of gene expression and cellular heterogeneity in a single platform. As of July 2023, the Gene Expression Omnibus now contains over 4000 published single-cell data sets, providing an invaluable opportunity for reanalysis to identify new cell types or cellular states as well as their defining transcriptional programs. To facilitate the reprocessing of these public datasets, we have devised a single-cell RNA sequencing analysis framework for data retrieval, quality control, expression normalization, dimension reduction, cell clustering, and data integration. Additionally, we have developed a Shiny App visualization platform that enables the exploration of gene expression, cell type annotations, and cell lineages through a user interface. We performed a re-analysis of single-cell RNAseq data generated from acute myeloid leukemia and tumor-reactive lymphocytes and found our pipeline to faithfully recapitulated the cell type assignment as well as expected lineage trajectories. Altogether, we present BERLIN, a single-cell RNAseq analysis pipeline that facilitates the integration and public dissemination of results from the reanalysis.

bioinformatics↗

bioRxiv2024

Flow zoometry of Drosophila

Drosophila serves as a highly valuable model organism across numerous fields including genetics, immunology, neuroscience, cancer biology, and developmental biology. Central to Drosophila-based biological research is the ability to perform comprehensive genetic or chemical screens. However, this research is often limited by its dependence on laborious manual handling and analysis, making it prone to human error and difficult to discern statistically significant or rare events amid the noise of individual variations resulting from genetic and environmental factors. In this article we present flow zoometry, a whole-animal equivalent of flow cytometry for large-scale, individual-level, high-content screening of Drosophila. Our flow zoometer automatically clears the tissues of Drosophila melanogaster, captures three-dimensional (3D) multi-color fluorescence tomograms of single flies with single-cell volumetric resolution at an unprecedented throughput of over 1,000 animals within 48 hours (24 hr for clearing; 24 hr for imaging), and performs AI-enhanced data-driven analysis - a task that would traditionally take months or years with manual techniques. To demonstrate its broad applications, we employed the flow zoometer in various laborious screening assays, including those in toxicology, genotyping, and tumor screening. Flow zoometry represents a pivotal evolution in high-throughput screening technology: previously from molecules to cells, now from cells to whole animals. This advancement serves as a foundational platform for "statistical spatial biology", to improve empirical precision and enable serendipitous discoveries across various fields of biology.

bioengineering↗

bioRxiv2026

Unilateral gene expression via maternally controlled zygotic recombination in Drosophila

Structural and functional asymmetries are ubiquitous in bilaterians, but tools for unilateral genetic access have been lacking, a methodological gap that has limited mechanistic dissections of lateralized cellular functions. We developed the Maternally Controlled Zygotic Recombination (MCZR) system to overcome this limitation in Drosophila, co-opting the regulatory machinery of the maternal-to-zygotic transition to enable somatic recombination during early embryonic development. Targeting recombinase activity to this early window generates large somatic mosaics that frequently split across the anatomical midline. In conjunction with cell type-specific driver lines, this approach provides a reliable, scalable, and user-friendly platform for unilateral transgene expression. In proof-of-concept experiments, we apply our tool to the question of locomotor control, illustrating how MCZR can be used to unilaterally manipulate or monitor a cell type of interest. Overall, the MCZR system unlocks previously inaccessible experimental avenues across neuroscience, cell biology, and developmental biology.

neuroscience↗

bioRxiv2024

CLCNet: a contrastive learning and chromosome-aware network for genomic prediction in plants

Genomic selection (GS) leverages genome-wide markers and phenotypes to predict breeding values, with its effectiveness largely dependent on the accuracy of genomic prediction (GP) models. However, GP methods often struggle to capture inter-individual variability and are limited by the curse of dimensionality, where the number of SNPs far exceeds the sample size. To address these challenges, we present CLCNet (Contrastive Learning and Chromosome-aware Network), a novel deep learning framework that integrates contrastive learning and chromosome-aware feature modeling. CLCNet comprises two key components: (i) a contrastive learning module that enhances the models ability to capture fine-grained, genotype-dependent phenotypic differences among individuals, and (ii) a chromosome-aware module that captures structured feature selection at both chromosome and genome levels, thereby distilling the most informative SNPs. We evaluated CLCNet across four crop species, covering ten agronomically important traits, and compared it with a diverse set of classical linear, machine learning, and deep learning models. CLCNet achieved superior prediction performance, with statistically significant improvements in Pearson correlation coefficient (PCC), ranging from 0.34% to 12.19% over baseline, together with reduced mean squared error (MSE). Performance gains were more pronounced for traits with moderate linkage disequilibrium (LD; r2 = 0.21-0.36) and high heritability (h2 > 0.66), such as those in maize, rapeseed, and soybean. For cotton traits characterized by high LD (r2 = 0.74) and lower heritability (h2 < 0.50), CLCNet maintained robust performance without degradation. Overall, these results demonstrate that CLCNet is an effective framework for improving genomic prediction accuracy and holds strong potential for practical applications in plant breeding. Short abstractCLCNet is a novel deep learning framework for genomic prediction that integrates contrastive learning with chromosome-aware feature selection. By jointly modeling inter-individual genotype-phenotype variation and chromosomal genomic structure, CLCNet improves prediction accuracy under high-dimensional, low-sample-size conditions. Across four crop species and ten agronomic traits, CLCNet consistently outperformed classical statistical, machine learning, and existing deep learning models. The framework also identified biologically relevant SNPs and candidate genes, demonstrating its potential for practical applications in genomic selection and computational plant breeding. Key pointsO_LIWe propose CLCNet, a multi-task deep learning framework that integrates contrastive learning with chromosome-aware feature selection for genomic prediction, under high-dimensional, low-sample-size conditions. C_LIO_LIThe chromosome-aware module explicitly exploits genomic structural information to select representative and informative SNPs across chromosomes. C_LIO_LIContrastive learning improves model robustness by stabilizing representation learning and reducing the influence of random effects across samples. C_LIO_LIBy complementing GWAS analyses, CLCNet provides additional insights into genotype-phenotype relationships with potential relevance for gene discovery. C_LI Biographical NoteJiangwei Huang is a PhD candidate at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. His research focuses on genomic prediction, deep learning, and computational plant breeding. Zhihan Yang is a PhD candidate at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Her research interests include genomic prediction and bioinformatics. Rongcheng Han is an associate professor at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. His research focuses on bioinformatics and plant phenomics. Yuqiang Jiang is a professor at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. His research interests include plant genomics, plant phenomics and genetic improvement. Organization descriptionThe Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, is a leading research institute focusing on genetics, genomics, molecular breeding, bioinformatics, and systems biology in plants and animals.

genomics↗

bioRxiv2024

The microbiota affects stem cell decision making in Hydra

Research on microbial communities colonizing animals has revealed that the microbiota, despite its typical containment to surfaces, influences virtually all organ systems of the host. In absence of a natural microbiota, the hosts development can be disturbed, but how developmental programs are affected by the microbiota is still poorly understood. Removing the microbiota from Hydra, a classic model animal in developmental biology, causes drastic developmental malformations and leads to polyps that temporarily lack the ability to bud. Recolonizing non-budding germfree Hydra with bacteria reverses this budding inhibition. Single-nucleus ATAC-seq detected a unique chromatin landscape associated with the non-budding phenotype. Single-cell RNA-seq and trajectory-based differential expression analysis showed that epithelial stem cell decision making is disturbed in non-budding polyps, whereby key developmental regulators are not expressed. This process is reversible by adding back bacteria. Transcriptionally silencing of one of the genes that failed to be activated in non-budding animals, GAPR1, led to polyps that have a significantly reduced budding capacity. The results show that maintaining a species-specific microbiota may enable the animal host to maintain its developmental program. Significance StatementAnimal developmental programs work within the context of coevolved associations with microbes. Here, we provide mechanistic evidence of the involvement of the microbiota in maintaining the pattern formation program of Hydra with the asexual formation of buds in the lower part of the body column. We demonstrate that in the absence of bacteria both the epigenetic and transcriptomic landscape is changed and that key regulatory factors are not expressed, causing changes in stem cell trajectories that result in loss of budding capacity. This study provides a new perspective on the role that microbiota plays during animal development and evolution. One Sentence SummaryMicrobiota interfere with Hydras asexual reproduction via modulating its stem cell differentiation programs.

developmental biology↗

bioRxiv2024

Unveiling Vertebrate Development Dynamics in Frog Xenopus laevis using Micro-CT Imaging

BackgroundXenopus laevis, the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus late developmental stages. FindingsIn the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus. ConclusionsOur repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact. Graphical abstract & lay summary3D images of selected developmental stages of X. laevis in a comparison (scale bar = 10 mm). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/598452v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1bc4664org.highwire.dtl.DTLVardef@1b1750dorg.highwire.dtl.DTLVardef@17bddaaorg.highwire.dtl.DTLVardef@115de4e_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryX-ray tomography was used to examine the African clawed frog (Xenopus laevis). An extensive data set of specimens from tadpoles to adult frogs provides novel insights into the changes and developmental dynamics of selected structures, which opens avenues to an improved understanding of this crucial animal model.

developmental biology↗

bioRxiv2024

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin, Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. While the sea urchin is one of the oldest animal models in cell and developmental biology, it has relied on transient manipulations of wild animals, and has lacked a strategy for stable transgenesis. Here we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, to establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2, piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression past metamorphosis. F0 animals were raised to sexual maturity and spawned to determine germline integration, transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrated transgene transmission through the germline, the first example of a germline transgenic sea urchin, and indeed of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility, and efficiency of sea urchin research. Significance StatementTransgenic tools are essential for effective utilization of animal models. Despite being an established model for cell and developmental biology, the sea urchin has not previously benefited from transgenic technology. This study reports the generation of the first germline transgenic sea urchin and opens new avenues for this organism.

developmental biology↗

bioRxiv2022

Microscopic and ultramicroscopic anatomical characteristics of root nodules in Podocarpus macrophyllus during development

To understand the morphological and structural characteristics of root nodules in Podocarpus macrophyllus and their development, this study prepared P. macrophyllus root nodule samples at the young, mature, and senescent stages. Optical microscopy and transmission electron microscopy (SEM) revealed that new nodules can be formed on roots and senescent nodules; new nodules formed on the roots are nearly spherical and have an internal structure similar to finite nodules; new nodules on senescent nodules are formed by extension and differentiation of the vascular cylinder of the original nodules; and these new nodules are nested at the base of the original nodules, which create growth space for new nodules by dissociating the cortical tissue; clusters of nodules are formed after extensive accumulation, and the growth pattern is similar to that of infinite nodules; the symbiotic bacteria of P. macrophyllus root nodules mainly invade from the epidermal intercellular space of the roots and migrate along the intercellular space of the nodule cortex; infected nodule cortex cells have a well-developed inner membrane system and enlarged and loose nuclei; and unique Frankia vesicles, and rhizobia cysts, and bacteriophages can all develop. Compared with common leguminous and nonleguminous plant nodules, P. macrophyllus root nodules are more complex in morphology, structure and composition. From the perspective of plant system evolution, the rhizobium nodules in leguminous angiosperms and Frankia nodules in nonleguminous angiosperms are most likely two branches derived from the nodules in gymnosperms, such as P. macrophyllus. The conclusions of this study can provide a theoretical basis for the developmental biology of P. macrophyllus root nodules and the evolutionary pattern of plant symbionts. HighlightsWe discuss from the perspective of cell developmental biology, the rhizobium nodules in leguminous angiosperms and Frankia nodules in nonleguminous angiosperms are most likely two branches derived from the nodules in gymnosperms, such as Podocarpus macrophyllus.

developmental biology↗

bioRxiv2025

Genomic Resources for the Scuttle Fly Megaselia abdita: A Model Organism for Comparative Developmental Studies in Flies

The order Diptera (true flies) holds promise as a model taxon in evolutionary developmental biology due to the inclusion of the model organism, Drosophila melanogaster, and the ability to cost-effectively rear many species in laboratories. One of them, the scuttle fly Megaselia abdita (Phoridae) has been used in evolutionary developmental biology for 30 years and is an excellent phylogenetic intermediate between fruit flies and mosquitoes but remains underdeveloped in genomic resources. Here, we present a de novo chromosome-level assembly and annotation of M. abdita and transcriptomes of 9 embryonic and 4 postembryonic stages. We also compare 9 stage-matched embryonic transcriptomes between M. abdita and D. melanogaster. Our analysis of these resources reveals extensive chromosomal synteny with D. melanogaster, 28 orphan genes with embryo-specific expression including a novel F-box LRR gene in M. abdita, and conserved and diverged features of gene expression dynamics between M. abdita and D. melanogaster. Collectively, our results provide a new reference for studying the diversification of developmental processes in flies.

developmental biology↗

bioRxiv2018

A mosaic of independent innovations involving eyes shut are critical for the evolutionary transition from closed to open rhabdoms

A fundamental question in evolutionary biology is how developmental processes are modified to produce morphological innovations while abiding by functional constraints. Here we address this question by investigating the cellular mechanism responsible for the transition between fused and open rhabdoms in ommatidia of apposition compound eyes; a critical step required for the development of visual systems based on neural superposition. Utilizing Drosophila and Tribolium as representatives of fused and open rhabdom morphology respectively, we identified three changes required for this innovation to occur. First, the expression pattern of the extracellular matrix protein Eyes Shut (EYS) was co-opted and expanded from mechanosensory neurons to the photoreceptor cells in taxa with open rhabdoms. Second, EYS homologs in these taxa obtained a novel extension of the amino terminus leading to the internalization of a cleaved signal sequence. This amino terminus extension does not interfere with cleavage or function in mechanosensory neurons, but it does permit specific targeting of the EYS protein to the apical photoreceptor membrane. Finally, a specific interaction evolved between EYS and a subset of Prominin homologs that is required for the development of open, but not fused, rhabdoms. Together, our findings portray a case study wherein the evolution of a set of molecular novelties has precipitated the origin of an adaptive photoreceptor cell arrangement.\n\nAuthor SummaryUnderstanding how adaptive morphologies originate is a central question in evolutionary developmental biology. Once confined largely to arguments about the relative frequencies of protein coding vs. regulatory mutations, numerous studies have since revealed more complex interactions involving alterations in gene expression and novel protein-protein interactions as drivers of novel trait evolution. Our study exploits the genetic amenability of Drosophila and utilizes direct comparisons with Tribolium to define a set of cellular mechanisms necessary for the evolutionary transition from a fused (Tribolium) to an open (Drosophila) rhabdom. Our results depict an evolutionary transition involving both non-coding and coding changes that resulted in a novel visual architecture, permitting a subset of diurnal insects to diversify into niches characterized by low light.

evolutionary biology↗

bioRxiv2023

The Mediterranean mussel, Mytilus galloprovincialis, a novel model for developmental studies of mollusks

A model organism in developmental biology is defined by its experimental amenability as well as by resources created for the model system by the scientific community. For the most powerful models, the combination of both has already yielded a thorough understanding of development. However, the number of developmental model systems is still very limited, and their phylogenetic distribution is heavily biased. Members of one of the largest animal phyla, the mollusks, for example, have long been neglected as developmental model organisms. To remedy this shortcoming, we produced a detailed developmental transcriptome for the Mediterranean mussel Mytilus galloprovincialis, a bivalve mollusk, and expanded the list of experimental protocols available for this species. Our high-quality transcriptome allowed us to identify transcriptomic signatures of developmental transitions and to perform a first comparison with the Pacific oyster Crassostrea gigas that can be used in future multi-species analyses. To allow co-labelling studies, we optimized protocols for immunohistochemistry and hybridization chain reaction and combined both techniques to create high-resolution co-expression maps of developmental genes. The resources and protocols we describe here thus represent an enormous boost for the establishment of the Mediterranean mussel as a laboratory model in developmental biology. Summary statementResources and techniques are described for the Mediterranean mussel Mytilus galloprovincialis, which, together, establish a novel model system for studying mollusk development and animal evolution.

developmental biology↗

bioRxiv2025

Growth and patterning in vertebrate limb developmentA timescale perspective on skeletal specification

1The vertebrate limb provides a powerful system to study how growth and molecular signaling interact to shape complex skeletal patterns. However, how these processes are coordinated across space and time is not fully understood. This study introduces a computational tool to examine how growth interacts with positional cues and self-organizing patterning mechanisms to shape skeletal structures in both mice and axolotl limbs. We developed the Growth-Processing-Propagation (GPP) framework, a reaction-diffusion system within a growing domain, in which the relative contribution of growth, processing (reaction) and propagation (diffusion) is modulated through two non-dimensional parameters, informed by experimental morphogen maps. This formulation normalizes the reaction-diffusion equation relative to growth, enabling investigation of how different spatiotemporal regimes of growth, processing and propagation interact to produce whole limb patterning. The GPP framework captures the progressive formation of limb segments and digit patterns by varying the relative contributions of reaction, diffusion, and growth to the pattern. Our models indicate that in the proximal region (humerus, radius/ulna) the contributions of growth, reaction and diffusion are equally important to patterning, but in the distal elements (hands) the reaction and diffusion contributions are much greater than the contribution of growth to formation of the digits. A single framework predicts whole-limb skeletal patterns in both mice and axolotls, despite their morphological differences, highlighting its potential to explore conserved and divergent features of limb development from an evolutionary perspective through a unified mechanism across species. O_TEXTBOXSignificanceUnderstanding limb skeletal patterning is a central question in developmental biology. Numerical models provide a means to explore this process. We introduce the Growth-Processing-Propagation (GPP) framework, which integrates tissue expansion (growth), gene regulation and cellular specification (processing), and signaling spread (propagation). By normalizing patterning dynamics relative to growth, the framework reveals how the relative contributions of growth, processing, and propagation vary across the limb, influencing the timing and positioning of skeletal elements. The model successfully predicts whole-limb skeletal patterning in both mice and axolotls, supporting the idea of a shared underlying mechanism. This cross-species framework offers a versatile computational tool for studying fundamental processes in morphogenesis, with potential applications in developmental biology, and evolutionary studies. C_TEXTBOX

developmental biology↗

bioRxiv2020

Design, construction, and advantages of 100% online laboratories in an upper division undergraduate biology course

For centuries, students have been taught by passively listening to lectures, usually in large groups. In many fields, this type of course delivery is not ideal. In addition, students that have grown up with online access and/or have been forced to take online course during the recent pandemic, are seeking improved online educational experiences. As providers of education, Universities must adapt to this rapidly changing environment. The emergence of exciting, "disruptive" technologies has provided the tools needed for educators to revolutionize the delivery of higher education. The University of Florida, through UF Online, provides students the opportunity to enroll in fully online four-year undergraduate degrees. In this report, we describe the development of fully online laboratories for the upper-division course Evolutionary Developmental Biology, which fulfill the University of Floridas laboratory requirement. This course is taken by Biology, Zoology, and related majors, including majors in the 100% online Biology degree. The experiments in the virtual laboratories would have been impossible to teach in a residential setting due to time constraints, cost, student safety issues, and regulatory/certification requirements. The ability to perform these experiments online provided University of Florida undergraduates located throughout the world access to unique, one-of-a-kind laboratory experiences. The virtual laboratories allowed students to undertake in-depth exploration and application of course content that is not possible in a residential setting. Completion of the Evolutionary Developmental Biology course provided undergraduate students the opportunity to obtain a science degree without ever visiting the universitys residential campus. During the Fall 2020 term, this course was made available to residential students, thus allowing this cohort of students to continue to make progress, 100% online, towards their undergraduate degree during the COVID19 pandemic.

developmental biology↗

bioRxiv2022

Prime editing in chicken fibroblasts and primordial germ cells

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and primordial germ cells (PGCs). By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chick PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

developmental biology↗

bioRxiv2022

Live imaging of echinoderm embryos to illuminate evo-devo

Echinoderm embryos have been model systems for cell and developmental biology for over 150 years, in good part because of their optical clarity. Discoveries that shaped our understanding of fertilization, cell division and cell differentiation were only possible because of the transparency of sea urchin eggs and embryos, which allowed direct observations of intracellular structures. More recently, live imaging of sea urchin embryos, coupled with fluorescence microscopy, has proven pivotal to uncovering mechanisms of epithelial to mesenchymal transition, cell migration and gastrulation. However, live imaging has mainly been performed on sea urchin embryos, while echinoderms include numerous experimentally tractable species that present interesting variation in key aspects of morphogenesis, including differences in embryo compaction and mechanisms of blastula formation. The study of such variation would allow us not only to understand how tissues are formed in echinoderms, but also to identify which changes in cell shape, cell-matrix and cell-cell contact formation are more likely to result in evolution of new embryonic shapes. Here we argue that adapting live imaging techniques to more echinoderm species will be fundamental to exploit such an evolutionary approach to the study of morphogenesis, as it will allow measuring differences in dynamic cellular behaviors - such as changes in cell shape and cell adhesion - between species. We briefly review existing methods for live imaging of echinoderm embryos and describe in detail how we adapted those methods to allow long-term live imaging of several species, namely the sea urchin Lytechinus pictus and the sea stars Patiria miniata and Patiriella regularis. We outline procedures to successfully label, mount and image early embryos for 10-16 hours, from cleavage stages to early blastula. We show that data obtained with these methods allows 3D segmentation and tracking of individual cells over time, the first step to analyze how cell shape and cell contact differ among species. The methods presented here can be easily adopted by most cell and developmental biology laboratories and adapted to successfully image early embryos of additional species, therefore broadening our understanding of the evolution of morphogenesis.

developmental biology↗