Developmental Biology Research Research Articles

Standardizing CRISPR-Cas13 knockdown technique to investigate the role of cdh2 gene in pituitary development through growth hormone expression and transcription factors.

Congenital hypopituitarism (CH) is characterized by the deficiency of pituitary hormones. Among CH patients, 85% lack a molecular diagnosis. Whole Exome Sequencing (WES) identified a homozygous variant (c.865G>A, p.Val289Ile) in the CDH2 gene, responsible for N-Cadherin production, crucial for cell-cell adhesion. Predicted to be likely pathogenic, the variant was found in a patient deficient in GH, TSH, ACTH, and LH/FSH. Its impact on cell adhesion was confirmed in L1 fibroblast cell lines. Create a cdh2 knockdown in zebrafish for investigating its role in pituitary development through growth hormone and transcription factors expression. Utilized pET28B-RfxCas13d-His plasmid for Cas13 mRNA production via in vitro transcription, guiding Cas13 to cdh2 with three RNAs. Injected the complex into single-cell embryos for analysis up to 96 hpf. Assessed gene expression of cdh2, prop1, pit1, and gh1 using RT-qPCR. Evaluated cdh2 protein expression through the western blot technique. Knockdown animals displayed developmental delay. The cdh2 expression decreased by 75% within 24 hours, rebounded by 48 hours, and reached wild-type levels by 96 hpf. gh1 expression decreased at 48h but increased by 96 hpf, aligning with WT. No significant differences in prop1 and pit1 expression were observed. Our findings underscore cdh2's role in pituitary development and hormonal regulation, offering insights for developmental biology research.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers invitro and invivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Immunohistochemical characterisation of the adult Nothobranchius furzeri intestine.

Nothobranchius furzeri is emerging as an exciting vertebrate organism in the field of biomedicine, developmental biology and ecotoxicology research. Its short generation time, compressed lifespan and accelerated ageing make it a versatile model for longitudinal studies with high traceability. Although in recent years the use of this model has increased enormously, there is still little information on the anatomy, morphology and histology of its main organs. In this paper, we present a description of the digestive system of N. furzeri, with emphasis on the intestine. We note that the general architecture of the intestinal tissue is shared with other vertebrates, and includes a folding mucosa, an outer muscle layer and a myenteric plexus. By immunohistochemical analysis, we reveal that the mucosa harbours the same type of epithelial cells observed in mammals, including enterocytes, goblet cells and enteroendocrine cells, and that the myenteric neurons express neurotransmitters common to other species, such as serotonin, substance P and tyrosine hydroxylase. In addition, we detect the presence of a proliferative compartment at the base of the intestinal folds. The description of the normal intestinal morphology provided here constitutes a baseline information to contrast with tissue alterations in future lines of research assessing pathologies, ageing-related diseases or damage caused by toxic agents.

Prediction and Analysis of Transcription Factor Binding Sites: Practical Examples and Case Studies Using R Programming.

Transcription factors (TFs) bind to specific regions of DNA known as transcription factor binding sites (TFBSs) and modulate gene expression by interacting with the transcriptional machinery. TFBSs are typically located upstream of target genes, within a few thousand base pairs of the transcription start site. The binding of TFs to TFBSs influences the recruitment of the transcriptional machinery, thereby regulating gene transcription in a precise and specific manner. This chapter provides practical examples and case studies demonstrating the extraction of upstream gene regions from the genome, identification of TFBSs using PWMEnrich R/Bioconductor package, interpretation of results, and preparation of publication-ready figures and tables. The EOMES promoter is used as a case study for single DNA sequence analysis, revealing potential regulation by the LHX9-FOXP1 complex during embryonic development. Additionally, an example is presented on how to investigate TFBSs in the upstream regions of a group of genes, using a case study of differentially expressed genes in response to human parainfluenza virus type 1 (HPIV1) infection and interferon-beta. Key regulators identified in this context include the STAT1:STAT2 heterodimer and interferon regulatory factor family proteins. The presented protocol is designed to be accessible to individuals with basic computer literacy. Understanding the interactions between TFs and TFBSs provides insights into the complex transcriptional regulatory networks that govern gene expression, with broad implications for several fields such as developmental biology, immunology, and disease research.

An interview with Aimee Jaramillo-Lambert.

Aimee Jaramillo-Lambert is currently an Assistant Professor at the University of Delaware. Aimee was awarded the Society for Developmental Biology 2023 Elizabeth D. Hay New Investigator Award in recognition of her outstanding research in developmental biology during the early stages of her independent career. We caught up with Aimee over a video call to talk about her research into sexual reproduction in Caenorhabditis elegans, the importance of mentorship, and what drives her work in diversity, equity and inclusion.

Striped Expression of Leucine-Rich Repeat Proteins Coordinates Cell Intercalation and Compartment Boundary Formation in the Early Drosophila Embryo.

Planar polarity is a commonly observed phenomenon in which proteins display a consistent asymmetry in their subcellular localization or activity across the plane of a tissue. During animal development, planar polarity is a fundamental mechanism for coordinating the behaviors of groups of cells to achieve anisotropic tissue remodeling, growth, and organization. Therefore, a primary focus of developmental biology research has been to understand the molecular mechanisms underlying planar polarity in a variety of systems to identify conserved principles of tissue organization. In the early Drosophila embryo, the germband neuroectoderm epithelium rapidly doubles in length along the anterior-posterior axis through a process known as convergent extension (CE); it also becomes subdivided into tandem tissue compartments through the formation of compartment boundaries (CBs). Both processes are dependent on the planar polarity of proteins involved in cellular tension and adhesion. The enrichment of actomyosin-based tension and adherens junction-based adhesion at specific cell-cell contacts is required for coordinated cell intercalation, which drives CE, and the creation of highly stable cell-cell contacts at CBs. Recent studies have revealed a system for rapid cellular polarization triggered by the expression of leucine-rich-repeat (LRR) cell-surface proteins in striped patterns. In particular, the non-uniform expression of Toll-2, Toll-6, Toll-8, and Tartan generates local cellular asymmetries that allow cells to distinguish between cell-cell contacts oriented parallel or perpendicular to the anterior-posterior axis. In this review, we discuss (1) the biomechanical underpinnings of CE and CB formation, (2) how the initial symmetry-breaking events of anterior-posterior patterning culminate in planar polarity, and (3) recent advances in understanding the molecular mechanisms downstream of LRR receptors that lead to planar polarized tension and junctional adhesion.

An interview with Hernan Garcia.

Hernan Garcia is a Principal Investigator and Associate Professor of Genetics, Genomics and Development and Physics at the University of California, Berkley (USA). His research aims to understand, predict and control developmental programs. In 2022, Hernan was awarded the Elizabeth D. Hay New Investigator award by the Society for Developmental Biology (SDB) recognizing his outstanding research in developmental biology. We spoke to Hernan to learn more about his education, career path and approach to running a lab.

The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells

AbstractIn many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF‐β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder‐free substrates. The results showed that TGF‐β signaling agonists (IDE1 and Activin‐A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF‐β, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency‐associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.

Lifting the cloche: Jeroen Bakkers interviews Didier Stainier.

Didier Stainier is Director of the Department of Developmental Genetics at the Max Planck Institute for Heart and Lung Research in Bad Nauheim, Germany. He became acquainted with the zebrafish model as a PhD student in Walter Gilbert's lab at Harvard, which motivated him to champion the use of this powerful model organism to study heart development as a postdoctoral fellow with Mark Fishman at Massachusetts General Hospital. Although his scientific focus has expanded significantly since then, zebrafish modelling and heart development and regeneration remain key topics in his research. The developmental biology and zebrafish modelling communities have embraced him as an inspiring mentor and advocate for basic research. Jeroen Bakkers is a group leader at the Hubrecht Institute for Developmental Biology and Stem Cell Research and Professor of Molecular Cardiogenetics at the University Medical Center Utrecht, The Netherlands. Jeroen did hid PhD with Herman Spaink at Leiden University, The Netherlands. A short visit to Massachusetts Institute of Technology during his doctoral training introduced him to the zebrafish model, which he applied to his PhD project. Zebrafish development remained the focus of his career, including during his postdoctoral training in the lab of Matthias Hammerschmidt at the Max Planck Institute of Immunology and Epigenetics in Freiburg and in his own lab at the Hubrecht Institute, where his group uses this powerful model organism to investigate cardiac development, disease and regeneration. Jeroen and Didier met up at a recent conference to talk about their shared interest in cardiac regeneration, a zebrafish mutant with a curious name and Didier's commitment to mentorship.

Practical pursuit in stem cell biology: Innovation, translation, and incomplete theorization

This paper aims to contribute to the study of practical pursuit-worthiness in science by engaging with a case in therapeutic stem cell biology. Induced pluripotent stem cell (iPSC) research emerged from research in developmental biology and the molecular biology of cell fate conversion. It took on practical significance when proposed as an alternative to therapeutic stem cell research that used human embryonic stem cells. The supposed ability of iPSC research to tackle ethical and regulatory constraints on research at the beginning of the twentieth century was a central part of the heuristic assessment of iPSC. However, the development and transfer of knowledge from experimental and theoretical biology to preclinical pursuit conflicted with the framing of biomedical innovation in public policy. The framing of innovation operated as part of the heuristic assessment of the pursuit-worthiness of iPSCs in the United States and was characterized by attempts to underdetermine conflicting ethical and socio-economic values – to seek innovations that are “incompletely theorized” in the sense that they purportedly allow stakeholders to refrain from engagement with the divisive values that created impediments to research in stem cell biology. When conflict arose with the epistemic standards in preclinical pursuit required to ensure the safety and efficacy of biomedical innovations, it resulted in the critical appraisal of the values used to rationalize policies for the distribution of federal resources for biomedical research. The case demonstrates how non-epistemic values impinge on standards of assessment in translational science, how background assumptions about innovation can drive practical pursuit, and how conflicting values and goals in research creates an important context for the appraisal of emerging science, technology and policy.

Facile methods for reusing laboratory plastic in developmental biology experiments

Plastic pollution negatively affects ecosystems and human health globally, with single-use plastic representing the majority of marine litter in some areas. Life science laboratories prefer pristine conditions for experimental reliability and therefore make use of factory standardized single-use plastic products. This contributes to overall plastic waste in the United States and globally. Here, we investigate the potential of reusing plastic culture dishes and subsequently propose methods to mitigate single-use plastic waste in developmental biology research laboratories. We tested the efficacy of bleach and ethyl alcohol in sterilizing used dishes. We then tested the feasibility of washing and reusing plastic to culture Xenopus laevis embryos subjected to various manipulations. Cleaning and reusing laboratory plastic did not affect the development or survival of X. laevis, indicating that these cleaning methods do not adversely affect experimental outcome and can be used to sterilize plastic before reuse or recycling. Lastly, we performed a survey of various life science laboratories to estimate both waste reduction and savings associated with recycling single-use plastics. Standardization of these procedures would allow research laboratories to benefit economically while practicing environmentally conscious consumption.

Fritz Müller: 200 years of a pioneer evolutionist

Abstract Fritz Müller is known for explaining the evolutionary consequences of what is now called Müllerian mimicry, but his scientific contributions encompass a wide range of organisms and both ecological and evolutionary processes. His originality is seen in aspects as different as the discovery of the mutualistic nature of ant–fungus symbiosis, pioneering research in evolutionary developmental biology and the first mathematical treatment in a Darwinian framework. From his intimate knowledge of the natural history of Brazil and original observations of morphology and development, Müller provided evidence supporting natural selection. From the 20th century onwards, however, his scientific legacy has been re-examined only for single topics. To celebrate the bicentenary of his birth in 2022, this article briefly discusses his importance for current ecological and evolutionary knowledge.

Past, Present, and Future of Direct Cell Reprogramming.

Budding off from the broader developmental biology and stem cell research fields, cellular reprogramming is now established as a prominent discipline in its own right. Direct cell reprogramming is defined as the cell fate conversion of a somatic cell toward another identity without a pluripotent intermediate state. In addition to the opportunity for mechanistic dissection of lineage commitment in human cells, the field offer the promise of diverse applications such as for disease modeling, cell replacement therapy, regenerative medicine, and immunotherapy that have recently spurred innovation and out of the box thinking to unleash the potential of cellular plasticity.

Effect of Embryonic Alcohol Exposure on Craniofacial and Skin Melanocyte Development: Insights from Zebrafish (Danio rerio).

Alcohol is a common addictive substance and prenatal alcohol exposure could cause fetal alcohol spectrum disorder (FASD) and can lead to various birth defects. The small teleost zebrafish (Danio rerio) has been identified as a fine animal model in developmental biology and toxicological research. Zebrafish models are widely used to study the harmful effects of alcohol and limited studies are available on the craniofacial and skin malformations associated with FASD. The present study attempts to investigate the effect of alcohol on early zebrafish embryonic development. The effects of prenatal alcohol exposure on neural crest cell-derived organ formation, including pharyngeal dentition, palatal bones and skin melanocytes were analysed. Whole-mount cartilage and bone staining and imaging techniques were applied to determine the effects of alcohol on the above-mentioned structures. The tooth size and shape were affected by alcohol exposure, but the number of teeth in the pharyngeal dentition was not affected. Only first-generation teeth showed size differences. The alcohol-exposed ethmoid bone, which is homologous to the human hard palate, was smaller and less dense in cell arrangement compared with the control medial ethmoid bone. The skin pigmentation defects included reduced melanocyte density, melanin contraction, smaller melanocyte surface area and aberrations in melanosome dispersion, revealing that alcohol significantly influenced and downregulated each and every step of the melanocyte developmental process. This descriptive study summarises the effects of alcohol on the development of neural crest cell-derived structures and highlights the importance of zebrafish in studying the phenotypic characteristics of fetal alcohol spectrum disorder.

Upregulation of miRNA-26a Enhances the Apoptosis of Cerebral Neurons by Targeting EphA2 and Inhibiting the MAPK Pathway

Neural tube defects (NTDs) constitute the second most common congenital malformation of the central nervous system. The pathogenesis of NTDs is not entirely clear. In recent years, microRNAs (miRNAs) have become a hot spot in genetic and developmental biology research. The present study aimed to explore the potential role of miRNA-26a in NTDs and the underlying pathogenesis thereof. First, we found significantly increased miRNA-26a expression in fetuses with NTDs (p < 0.0001), which significantly downregulated EphA2 and ERK1 mRNA and protein expression levels in fetuses with NTDs compared to normal controls (p < 0.01). In addition, a dual-luciferase reporter assay showed that miR-26a negatively regulated EphA2 by directly binding with the 3′-untranslated region of EphA2. Second, the upregulation of miRNA-26a expression increased caspase 3 and 9 protein expression levels (p < 0.01) and decreased EphA2 mRNA and protein expression levels (p < 0.01), as well as ERK1 and SRF protein expression levels (p < 0.01) in mouse neural stem cells (NE-4C) and human astroblastoma cells (U87MG). Furthermore, the upregulation of miRNA-26a inhibited cell proliferation and enhanced apoptosis of NE-4C and U87MG cells (p < 0.05). Similar results were observed with the MAPK inhibitor PD98059 (p < 0.01). These results suggest that miR-26a targets EphA2, modulates phosphorylation of the MAPK/ERK (MEK) pathway, regulates SRF, and participates in regulating nervous cell proliferation and apoptosis. Dysregulation of the aforementioned mechanism may be involved in the pathogenesis of NTDs.

Establishment of a Mouse Submandibular Salivary Gland Organ Culture.

The salivary glands produce saliva and are important in maintaining oral health. Saliva keeps the mouth moist, cleanses the oral cavity, aids digestion, and has antibacterial properties. Saliva also helps in swallowing and speech. Investigating the development of the salivary glands is thus relevant in the context of both health and disease. Various cell culture methods have been used to study salivary gland development, including culturing cells in two dimensions (2D). Under physiological conditions, cells constantly interact with other cells and the extracellular matrix, which controls complex biological functions such as cell migration and apoptosis, and can modulate gene expression. Since many of these functions are not accurately represented or reproduced in 2D culture, the results of in vitro experiments using such culture methods are often not reflected in vivo. The use of 3D cultures, such as organ cultures, has helped address this issue and has emerged as a model that better reflects the in vivo physiological environment. Here, we describe a protocol for establishing submandibular salivary gland organ culture that is more concise and simpler than previous methods and includes the separation and dissection of the salivary glands. We also describe the use of environmental stress (hypoxic stimulation) and inhibitors (U0126, LY294002, and rapamycin) to elucidate signaling pathways involved in salivary gland development. This protocol can provide researchers with a simpler and more robust method of salivary gland organ culture, enabling analysis of organ-based signaling pathways to advance developmental biology research. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Submandibular salivary gland organ culture Basic Protocol 2: Analysis of salivary gland development in the presence of hypoxia and signaling pathway inhibitors Basic Protocol 3: Western blotting using submandibular salivary gland organ culture.

A vasculogenesis model based on flow-induced stresses on endothelial cells

Vascular network formation and sustenance in both normal and pathological froms of angiogenesis has been a focus of research in developmental biology. The assembly and remodeling of vascular structures play major roles in numerous pathologies, including the angiogenesis of tumors. Endothelial morphogenesis is dependent on a number of chemical and mechanical stimuli and cell–cell signaling. To understand the nature of angiogenesis and vasculogenesis, many models have been developed to simulate these phenomena based on the defined responses of endothelial cells to these stimuli. Among the mechanical signals affecting these cells, flow-related stresses, including shear stress, play a major role in migration, elongation, attachment to the matrix and neighboring cells, and eventually the morphogenesis of vascular networks. Here, we proposed a model to describe the cellular responses to shear and tensile stress induced by fluid flow, which can describe some of the morphological behaviors observed in in vitro and in vivo studies. The lattice Boltzmann method was utilized to model the flow, and the cellular Potts model was used to simulate the cellular responses to the flow. This model is based on the hypothesis that endothelial cell binding energy to the matrix is regulated by shear stress and tensile stress acting on the attachment site and is increased by shear stress and decreased by tensile stress. It was demonstrated that these rules can predict the development of vascular networks and the sustenance of lumens and regression in the low flow regions. The results of this study can be further improved to investigate endothelial dysfunctions, such as atherosclerosis, as well as tumor angiogenesis and vascular permeability, which are directly related to the flow rate and endothelial responses to shear stresses.

A reflection of life - my tale of microdiversity, equity and inclusion in cell biology (and beyond).

A reflection of life - my tale of microdiversity, equity and inclusion in cell biology (and beyond).

Structural Modeling of Drosophila melanogaster Gut Cytochrome P450s and Docking Comparison of Fruit Fly Gut and Human Cytochrome P450s.

Drosophila melanogaster is a prominent organism in developmental biology research and in studies related to pathophysiological conditions like cancer and Alzheimer's disease. The fruit fly gut contains several cytochrome P450s (CYP450s), which have central roles in Drosophila development and in the normal physiology of the gut. Since the crystal structures of these proteins have not been deciphered yet, we modeled the structure of 29 different D. melanogaster gut CYP450s using Prime (Schrödinger). The sequences of chosen D. melanogaster gut CYP450s were compared with that of their human counterparts. The common gut (and liver) microsomal CYP450s in humans were chosen for structural comparison to find the homology and identity % of D. melanogaster CYPs with that of their human counterparts. The modeled structures were validated using PROCHECK and the best fit models were used for docking several known human pharmacological agents/drugs to the modeled D. melanogaster gut CYP450s. Based on the binding affinities (ΔG values) of the selected drug molecules with the modeled fly gut CYPs, the plausible differences in metabolism of the prominent drugs in humans and flies were projected. The gut is involved in the absorption of oral drugs/pharmacological agents, and hence, upregulation of intestinal CYP450 and their reactions with endobiotics and xenobiotics is envisaged. The insights gleaned from this work can validate D. melanogaster as a model organism for studying intestinal drug metabolism, particularly in the context of a) toxicology of pharmacological agents to the gut cells and b) how gut P450 metabolites/products can influence gut homeostasis. This work can help establish a platform for further in vitro investigations on how intestinal CYP450 metabolism can influence gut health. The data from this work can be used for further in silico studies and this work can serve as a platform for future in vitro investigations on intestinal CYP450-mediated metabolism of endo- and xeno-biotics in D. melanogaster.

The GRN concept as a guide for evolutionary developmental biology.

Organismal phenotypes result largely from inherited developmental programs, usually executed during embryonic and juvenile life stages. These programs are not blank slates onto which natural selection can draw arbitrary forms. Rather, the mechanisms of development play an integral role in shaping phenotypic diversity and help determine the evolutionary trajectories of species. Modern evolutionary biology must, therefore, account for these mechanisms in both theory and in practice. The gene regulatory network (GRN) concept represents a potent tool for achieving this goal whose utility has grown in tandem with advances in "omic" technologies and experimental techniques. However, while the GRN concept is widely utilized, it is often less clear what practical implications it has for conducting research in evolutionary developmental biology. In this Perspective, we attempt to provide clarity by discussing how experiments and projects can be designed in light of the GRN concept. We first map familiar biological notions onto the more abstract components of GRN models. We then review how diverse functional genomic approaches can be directed toward the goal of constructing such models and discuss current methods for functionally testing evolutionary hypotheses that arise from them. Finally, we show how the major steps of GRN model construction and experimental validation suggest generalizable workflows that can serve as a scaffold for project design. Taken together, the practical implications that we draw from the GRN concept provide a set of guideposts for studies aiming at unraveling the molecular basis of phenotypic diversity.