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bioRxiv2024

Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes towards a Purkinje Fiber Fate

Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as SCN5A, HCN4 and ID2, and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing in silico models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings in vitro, demonstrating that our new cell-specific in vitro model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.

developmental biology↗

bioRxiv2026

Fetal sex influences molecular regulation of human placental vascular development across gestation

Males experience elevated mortality and morbidity during the perinatal period and infancy; however, the biological origins of this vulnerability remain unclear. Emerging evidence suggests that sex-dependent placental development may contribute to differences in fetal growth and outcomes. Vascular networks are central to placental function, established early in the first trimester to support growth and mitigate harmful exposures, and maturing by term to sustain late fetal development. Although these processes are tightly regulated by gene-expression programmes, the role of sex-specific vascular signalling in placental development remains poorly understood. To address this, we analysed raw, publicly available RNA-sequencing transcriptomics data from first trimester and term placental samples. We conducted parallel differential gene expression and functional enrichment analyses across the datasets to ensure direct comparability and to disentangle the specific contribution of sex chromosomes. We identified multiple differentially expressed genes and enriched pathways between male and female placentas. In the first trimester, male placentas exhibited significant enrichment of vascular signalling pathways, particularly Notch signalling, cell adhesion, and migration, consistent with heightened proliferative and remodelling activity. At the end of gestation, sex differences remained detectable; however differentially expressed genes was clearly sex-chromosome driven, with autosomal single-gene sex differences and autosomal-sex-specific pathways markedly reduced. This reflected a mature placental vascular system with minimal sex-based differences outside of the sex chromosome genes. These results reveal gestational stage-dependent sex differences in placental vascular development, with early male fetuses exhibiting increased angiogenic activity and immature vascular signalling, potentially contributing to accelerated growth and increased vulnerability in male neonates.

developmental biology↗

bioRxiv2024

Spatiotemporal dynamics of primary and motile cilia throughout lung development

Cilia are specialized structures found on a variety of mammalian cells, with variable roles in the transduction of mechanical and biological signals (by primary cilia, PC), as well as the generation of fluid flow (by motile cilia). Their critical role in the establishment of a left-right axis in early development is well described, as is the innate immune function of multiciliated upper airway epithelium. By contrast, the dynamics of ciliary status during organogenesis and postnatal development is largely unknown. In this study, we define the progression of ciliary status within the endothelium, epithelium, and mesenchyme of the lung. Remarkably, we find that endothelial cells (ECs) lack PC at all stages of development, except in low numbers in the most proximal portions of the pulmonary arteries. In the lung epithelium, a proximodistal ciliary gradient is established over time, as the uniformly mono-ciliated epithelium transitions into proximal, multiciliated cells, and the distal alveolar epithelium loses its cilia. Mesenchymal cells, interestingly, are uniformly ciliated in early development, but with restriction to PDGFR+ fibroblasts in the adult alveoli. This dynamic process in multiple cellular populations both challenges prior assertions that PC are found on all cells, and highlights a need to understand their spatiotemporal functions. HighlightsO_LIPrimary cilia are found broadly throughout early embryonic tissues. C_LIO_LIPrimary cilia are observed in both epithelial and mesenchymal cells in the early lung. C_LIO_LIPulmonary endothelial cells largely do not possess primary cilia during embryonic development. C_LIO_LIDifferential multiciliation and loss of epithelial cilia in a proximal-distal axis. C_LIO_LIMaintenance of cilia in adult pulmonary PDGFR+ fibroblasts. C_LI

developmental biology↗

bioRxiv2020

Impact of gestational low-protein intake on embryonic kidney microRNA expression and in the nephron progenitor cells of the male offspring fetus

Authors have demonstrated that gestational low-protein (LP) intake offspring showed a lower birth weight, reduced nephrons numbers and renal salt excretion, chronic renal failure, and arterial hypertension development when compared to normal (NP) protein intake group in adult life. The current study was aimed to evaluate the miRNAs and predicted gene expression patterns in the fetal kidney at 17 days of gestational (17-DG) protein-restricted male offspring (LP) in an attempt to elucidate the possible molecular pathways and disorders involved in renal cell proliferation and differentiation during kidney development. By NGS and RT-qPCR were identified, 44 differentially expressed miRNAs, which 19 miRNA were up- and 25 downregulated in LP compared to NP offspring metanephros. Among the top 10 deregulated miRNAs, the study selected 7 miRNAs which its biological targets were related to proliferation, differentiation, and cellular apoptosis processes. As could be seen below, both miRNA-Seq and TaqMan data analysis have shown a consistent change of miRNA expression in LP animals relative to control NP age-matched animals. By immunofluorescence, the LP fetus showed a significant 28% reduction of Six-2 labeled cells when compared to NP offspring associated with a reduced percentage of cell number and c-Myc metanephogenic cap and ureter bud immunostained cells in LP relative to NP offspring. Additionally, the Ki-67 labeled area in CM was 48% lesser in LP compared to NP age-matched fetus. On the other hand, in LP, the CM and UB {beta}-catenin marked area were 154 and 85% raised, respectively. Mtor immunoreactivity was also significantly higher in LP CM (139%) and UB (104%) compared to the NP fetus. In the LP offspring, the TGF{beta}-1 in UBs cells staining increased (about 30%). In contrast, Zeb1 metanephros-stained, located in the CM nuclei cells, enhanced 30% in LP and Zeb2 immunofluorescence, although present in whole metanephros structures, was similar in both experimental groups. In conclusion, the present study demonstrates that the miRNAs, mRNAs, and proteins are modified in LP animals with 17 DGs leading to the reduction of the reciprocal induction between CM and UB, and hence the number of nephrons. These findings will facilitate new functional approaches and further studies to elucidate the regulatory mechanisms involved in the processes of proliferation, differentiation, and apoptosis that occurs during renal development.

developmental biology↗

bioRxiv2024

PFAS Compounds PFOA and Gen X are Teratogenic to Sea Urchin Embryos

Per-and polyfluorinated substances (PFAS) are synthetic chemicals that are used to make fluoropolymer coatings found in many products, such as non-stick pans, clothing, cosmetics, and food packaging. These highly persistent molecules are known as "forever chemicals" since they neither degrade environmentally nor break down enzymatically within biological systems. PFAS compounds readily contaminate water sources, and as a result, certain PFAS molecules have bioaccumulated in exposed species including humans. The purpose of this study was to define the effect of two PFAS molecules, the ostensibly more toxic perfluorooctanoic acid (PFOA) and the more recent, reportedly safer chemical hexafluoropropylene oxide dimer acid (Gen X), on the development of Lytechinus variegatus sea urchin embryos. We examined the effects of PFOA and Gen X on development and patterning using morphological analysis, immunostaining, HCR-FISH, and Particle Image Velocimetry (PIV). The results show that both PFAS compounds are teratogenic to sea urchin embryos. PFOA and Gen X each function at different intervals during development and provoke distinct phenotypic and gene expression outcomes. Despite beliefs that Gen X would be a safer alternative, our findings indicate that Gen X has earlier and more severe effects on endomesoderm and dorsal-ventral axis specification, neural development and function, and pattern formation compared to PFOA. These results illustrate the dangerous teratogenic potential of environmentally accumulating PFAS like Gen X, underscoring the negative ecological implications that accompany continuing commercial and industrial use of PFAS in the absence of remediation strategies. HIGHLIGHTSO_LIGen X is more teratogenic in sea urchins than PFOA C_LIO_LIGen X acts earlier than PFOA and perturbs axial and germ layer specification C_LIO_LIEach PFAS reduces neural numbers and perturbs ciliary behavior and swimming C_LI

developmental biology↗

bioRxiv2020

Cell-to-cell heterogeneity in Sox2 and Brachyury expression guides progenitor destiny by controllingtheir movements.

Although cell-to-cell heterogeneity in gene and protein expression within cell populations has been widely documented, we know little about its potential biological functions. We addressed this issue by studying progenitors that populate the posterior region of the vertebrate embryos, a cell population known for its capacity to self-renew or to contribute to the formation of the neural tube and paraxial mesoderm tissues. Posterior progenitors are characterized by the co-expression of Sox2 and Brachyury (Bra), two transcription factors related to neural and mesodermal lineages, respectively. In this study, we show that the respective levels of Sox2 and Bra proteins display a high degree of variability among posterior progenitors of the quail embryo. By developing forced expression and downregulation approaches, we further provide evidence that the value of the Sox2-to-Bra ratio in a given progenitor directs its choice of staying in place or exit the progenitor zone to generate neural or mesodermal cells. Time-lapse imaging together with mathematical modeling then reveal that variations of the Sox2-to-Bra ratio confer these cells heterogeneous motile behaviors. While high Bra levels display high motile properties that push cells to join the mesoderm, high levels of Sox2 tend to inhibit cell movement making cells get integrated into the neural tube. Our work thus provides evidence that the spatial heterogeneity of posterior progenitors, with regards to their relative contents of Sox2 and Bra and thus to their motile properties, is fundamental to maintain a pool of resident progenitors while others segregate to contribute to tissue formation. More broadly, our work reveals that heterogeneity among a population of progenitor cells is critical to ensure robust multi-tissue morphogenesis.

developmental biology↗

bioRxiv2020

A preliminary study on the reproductive toxicity of GS-5734 on male mice

BackgroundGS-5734 as a novel and promising medicine for COVID-2019, its biological impact on the mammalian reproductive system has not been systematically studied. The aim of this study was to evaluate the effects of GS-5734 on sperm parameters and spermatogenesis in mice. Materials and MethodsIn this study, GS-5734 was synthesized according to the report. 28 adult male mice were randomly segregated into four groups (n=7 for each group). The group 1 was set as the control group, the group 1, 2, 3 and 4 were administered with GS-5734 at a daily dose of 0, 10, 50, 150 g/mouse respectively, by intraperitoneal injection for 10 days. On the 7th day after the last injection, the testes and cauda epididymides were collected for HE staining and sperm concentration, motility, morphology analysis. ResultsThe results indicated that after treated with GS-5734, the total sperm count and motile sperm rate showed downward trends, the abnormal sperm rate showed an increasing trend. As compared with the control group, GS-5734 at a daily dose of 150 g/mouse caused a significant decrease in sperm concentration and motility, and a significant increased of abnormal sperm rate; the 50 g/mouse drug treatment lead to a significant decrease in sperm motility and an increase in abnormal sperm rate. The HE staining of testicular and epididymal tissues showed that the spermatogenesis of mice was significantly deteriorated with the increasing dosage of GS-5734, especially in the 150 g/mouse group. ConclusionOur findings suggest that a high dosage of GS-5734 may induce testicular toxicity and result in deterioration of sperm parameters in mice. More investigation on the reproductive toxicity of GS-5734 is required.

developmental biology↗

bioRxiv2025

Basal cytoplasmic calcium levels regulate C. elegans germ stem cell proliferation

The C. elegans germline is one of the foremost model systems for the study of germ cell biology. We have created an integrated ratiometric calcium reporter that allows comparative analysis of calcium levels in germ cells. We observe a positive correlation between the rate of cell proliferation and calcium levels. Conditions that decrease proliferation decrease basal cytoplasmic calcium levels, these include starvation and loss of insulin, TGF-{beta}, and folate signaling. Conversely, overproliferating germline tumor cells have increased basal cytoplasmic calcium levels. To determine if basal calcium levels regulate the rate of proliferation, we decreased calcium levels by inactivating the TRPM7-related GON-2 calcium channel. A gon-2 null mutant has significantly decreased basal cytoplasmic calcium levels and germ cell proliferation. Partial reduction of GON-2 activity in a gon-2 mutant heterozygote also reduces calcium levels and proliferation, but to a lower extent. This identifies GON-2 as a calcium channel that is required to maintain basal cytoplasmic calcium levels in germ cells. Conversely, increasing basal cytoplasmic calcium levels in germ cells either by inhibiting the plasma membrane calcium ATPase (which transports cytoplasmic calcium out of cells) or by inhibiting the SERCA channel (which transports cytoplasmic calcium into the endoplasmic reticulum) increased basal calcium levels and germ cell proliferation. This study identifies the germ line as the first tissue in C. elegans whose rate of cell proliferation is regulated by calcium. While transient increases in calcium are associated with cell proliferation in many animal species, our study suggests that the basal level of cytoplasmic calcium acts as a rheostat to modulate the rate of C. elegans germ cell proliferation in response to physiological processes and signals.

developmental biology↗

bioRxiv2025

The differentiation of myeloid progenitors is effected by cascading waves of coordinated gene expression that remodel cellular physiology in a characteristic sequence

The differentiation of hematopoietic progenitors into specialized types requires the transmittal of information from a few external or internal regulators to the thousands of genes that produce a cell types characteristic phenotypes. While the main signaling pathways, transcription factors, and the genes eliciting the terminal phenotypes are known, how information flows from a few regulators to thousands of genes to change the state of the cell remains to be fleshed out. To profile this information transfer process, we sampled the differentiation of the PUER myeloid cell line into macrophages and neutrophils at 29 time points over seven days. There is extensive transient regulation; the number of transcripts modulated in time is twice the number differentially expressed between endpoints. Differentiation is marked by two sharp transitions, at [~] 8h and [~] 80h, when transcriptomic state changes suddenly. We utilized non-negative matrix factorization to identify behaviors, characteristic temporal patterns of gene expression, and to classify transcripts by behavior. Only 10 distinct behaviors are sufficient to recapitulate the expression of [~]36,000 transcripts with high fidelity. Gene expression in most of the behaviors occurs in pulses of varying initiation times and durations. This implies that information transfer during differentiation occurs in cascading waves of gene expression culminating in the permanent turning on of certain genes after [~] 80h. Each behavior is enriched in specific biological processes, so that physiological remodeling proceeds in a characteristic order--signal transduction, translation and mRNA processing, metabolism, and, ultimately, myeloid phenotypic processes. The sharp transition at 8h corresponds to the completion of transcriptional and translational remodeling and the initiation of metabolic remodeling; the one at 80h corresponds to the elicitation of myeloid phenotypes. Our analysis shows that differentiation relies upon a series of transient, rapid, and complex gene regulatory events and highlights the importance of profiling it at a high temporal resolution. Author summaryThe maturation of hematopoietic progenitor cells into differentiated cell types occurs over a period of about a week. This process requires the progenitors to respond to external signals by changing the expression of thousands of genes to elicit the required phenotypes. We profiled how information is transferred from a few upstream regulators to thousands of genes by measuring genome-wide gene expression at high temporal resolution during white-blood cell differentiation. We show that the information transfer occurs in cascading waves, some as short as 8 hours and others lasting for 3 days, in which thousands of genes change expression coordinately. The physiological processes remodeled in each successive wave follow a characteristic order, starting with signal transduction pathways, followed by translation and mRNA processing, then metabolism, and culminating in the production of innate immunity phenotypes. Maturation is also punctuated with two sharp transitions, when genome-wide expression changes suddenly, associated with the initiation of metabolic remodeling and the production of terminal phenotypes. Our analysis shows that a complete description of differentiation requires the characterization of transient changes and not just those observable at the endpoints.

developmental biology↗

bioRxiv2014

Comparison of the 3D organization of sperm and fibroblast genomes using the Hi-C approach

The 3D organization of the genome is tightly connected to its biological function. The Hi-C approach was recently introduced as a method that can be used to identify higher-order chromatin interactions genome-wide. The aim of this study was to determine genome-wide chromatin interaction frequencies using the Hi-C approach in mouse sperm cells and embryonic fibroblasts. The obtained results demonstrated that the 3D genome organizations of sperm and fibroblast cells show a high degree of similarity both with each other and with the previously described mouse embryonic stem (ES) cells. Both A- and B-compartments and topologically associated domains (TADs) are present in spermatozoa and fibroblasts. Nevertheless, sperm cells and fibroblasts exhibited statistically significant differences between each other in the contact probabilities of defined loci. Tight packaging of the sperm genome resulted in an enrichment of long-range contacts compared with the fibroblasts. However, only 30% of the differences in the number of contacts are based on differences in the densities of their genome packages; the main source of the differences is the gain or loss of contacts that are specific for defined genome regions. An analysis of interchromosomal contacts in both cell types demonstrated that the large chromosomes showed a tendency to interact with each other more than with the small chromosomes and vice versa. We found that the dependence of the contact probability P(s) on genomic distance for sperm is in a good agreement with the fractal globular folding of chromatin. The similarity of the spatial DNA organization in sperm and somatic cell genomes suggests the stability of the 3D structure of genomes through generations.

Developmental Biology↗

bioRxiv2021

Automatic inference of cell neighborhood in 2D and 3D using nuclear markers

SignificanceEstimating neighboring cells by using only nuclear markers is crucial in many biological applications. Although several strategies have been used for this purpose, most published methods lack a rigorous characterization of their efficiencies. Remarkably, previously described methods are not automatic and depend only on cell-cell distance, neglecting the importance of pair-neighborhood interaction. AimTo develop a robust and automatic method for assessing cell local neighborhood, while analyzing the impact of the physical variables involved in this task. ApproachWe inferred neighbors from images with nuclei labeling by approximating the cell-cell interaction graph by the Delaunay triangulation of nuclei centroids. Each edge of this graph was filtered by thresholding in cell-cell distance and the maximum angle that each pair subtends with shared neighbors (pair-neighborhood interaction). Thresholds were calculated by maximizing a new robust statistic that measures the communicability efficiency of the cell graph. Using a variety of images of diverse tissues with additional membrane labeling to find the ground truth, we characterized the assessment performance. ResultsOn average, our method detected 95% of true neighbors, with only 6% of false discoveries. Even though our methods performance and tissue regularity are correlated, it works with performance metrics over 86% in very different organisms, including Drosophila melanogaster, Tribolium castaneum, Arabidopsis thaliana and C. elegans. ConclusionsWe automatically estimated neighboring relationships between cells in 2D and 3D using only nuclear markers. To achieve this goal, we filtered the Delaunay triangulation of nuclei centroids with a new measure of graph communicability efficiency. In addition, we found that taking pair-neighborhood interactions into account, in contrast to considering only cell-cell distances, leads to significant performance improvements. This becomes more notorious when the number of cells is low or the geometry of the cell graph is highly complex.

developmental biology↗

bioRxiv2023

Gap junctions in Turing-type periodic feather pattern formation

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogenous two-dimensional field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating non-protein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found seven of the twelve investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation, including patches of smooth skin and buds of irregular sizes. Later, after the primary feather arrays were laid out, inhibition of gap junctional intercellular communication in the ex vivo skin explant culture allowed the emergence of new feather buds in temporal waves at specific spatial locations relative to the existing primary buds. The results suggest that gap junctional communication may facilitate the propagation of long-distance inhibitory signals. Thus, the removal of GJ activity would enable the emergence of new feather buds if the local environment is competent and the threshold to form buds is reached. We propose Turing-based computational simulations that can predict the appearance of these ectopic bud waves. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

developmental biology↗

bioRxiv2023

Inhibition of cAMP signaling prevents congenital heart defects counteracting oxidative stress in Pde2A deficient embryos

BackgroundPhosphodiesterases (PDEs) are the enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) playing a key role in the homeostasis of these two second messengers. PDE2A is a dual-specific PDE that breaks down both cAMP and cGMP and can be activated by cGMP. It appears peculiar that the Pde2A-deficient (Pde2A-/-) mouse model is embryonically lethal, likely due to a strongly reduced size of liver and to a severe anemia. In addition, the heart of Pde2A-/- embryos shows ventricular and atrial septum defects, hypertrabeculation, heart dilatation and non-compaction defects. We recently highlighted a direct relationship between Pde2A impairment, consequent increase of cAMP and the onset of mouse congenital heart defects (CHDs), however the molecular mechanisms underlining the heart defects remain unknown. MethodsTranscriptome analysis of Pde2A-/- embryonic heart was performed by RNA sequencing and the most altered genes were also analyzed by quantitative real time PCR. In vivo treatment with drugs acting on cAMP signaling (Metoprolol and H89) and oxidative stress (N-Acetyl-Cysteine, NAC) were carried out on pregnant Pde2A+/- female. Histological, biochemical, and molecular analyses were then performed on embryonic hearts. ResultsWe found a significant modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in hearts from Pde2A-/- embryos. Metoprolol and H89 administration were able to prevent heart dilatation and hypertabeculation in Pde2A-/-embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Partial rescue of cardiac defects was observed by using the antioxidant NAC, indicating oxidative stress like one of the molecular mechanisms underpinning the CHDs. ConclusionsWe identified specific biological processes, molecules and cell signaling that can be targeted by selected drugs with consequent beneficial effects for cAMP-dependent CHDs. Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LICongenital Heart Defects are the most frequent heart birth defects including septal defects, hypertrabeculation and non-compacted myocardium. C_LIO_LIPde2A hydrolyses the cAMP and cGMP second messengers. C_LIO_LIPde2A-deficient mice are embryonic lethal and show cAMP-dependent Congenital Heart Defects. C_LI What New Information Does This Article Contribute?O_LIWe identified several novel pathways altered in hearts of Pde2A-/- embryos. C_LIO_LIWe demonstrated that drugs lowering cAMP levels rescued specific CHDs in Pde2A-/- embryos. C_LIO_LIWe discovered that antioxidants are beneficial for CHDs in Pde2A-/- embryos. C_LI The significance of this work relay in molecular discoveries and pharmacological approaches to treat CHDs by using a mouse model that recapitulate the major congenital heart defects. Among the pathways involved in specific defects associated with CHDs, the transcriptome analysis revealed an impairment of genes of the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in Pde2A-/- hearts. The scientific community will have open access to the RNA-seq data that can be utilized to further understand the congenital cardiac pathology and clarify the molecular implication in selected defects such as septal and ventricular wall defects. Up to date CHDs, when possible and if identified in time, are mostly treated trough surgery. The identification of drugs blunting the cAMP signaling response or reducing oxidative stress pathways will be useful for setting therapeutic approaches to alleviate CHDs.

developmental biology↗

bioRxiv2023

How human-derived brain organoids are built differently from brain organoids derived of genetically-close relatives: A multi-scale hypothesis

How genes affect tissue scale organization remains a longstanding biological puzzle. As experimental efforts are underway to solve this puzzle via quantification of gene expression, chromatin organization, cellular structure, and tissue structure, computational modeling efforts remain far behind. To help accelerate the computational modeling efforts, we review two recent publications, the first on a cellular-based model for tissues and the second on a model of a cell nucleus that includes a lamina shell and chromatin. We then address how the two models can be combined to ultimately test multiscale hypotheses linking the chromatin scale and the tissue scale. To be concrete, we turn to an in vitro system for the brain known as a brain organoid. We provide a multiscale hypothesis to distinguish structural differences between brain organoids built from induced-pluripotent human stem cells and from induced-pluripotent gorilla and chimpanzee stem cells. Recent experiments discover that a cell fate transition from neuroepithelial cells to radial glial cells includes a new intermediate state that is delayed in human-derived brain organoids as compared to their genetically-close relatives, which significantly narrows and lengthens the cells on the apical side [1]. Additional experiments revealed that the protein ZEB2 plays a major role in the emergence of this new intermediate state with ZEB2 mRNA levels peaking at the onset of the emergence [1]. We postulate that the enhancement of ZEB2 expression driving this intermediate state is potentially due to chromatin reorganization. More precisely, there exists critical strain triggering the reorganization that is higher for human-derived stem cells, thereby resulting in a delay. Such a hypothesis can readily be tested experimentally within individual cells and within brain organoids as well as computationally to help work towards solving the gene-to-tissue organization puzzle.

developmental biology↗

bioRxiv2024

Early mouse ovarian follicles develop into a thecal cell network that generates androgens during peri-puberty

The earliest growing mouse follicles, wave 1, rapidly develop in the ovarian medulla, while the great majority, wave 2, are stored for later use as resting primordial follicles in the cortex. Wave 1 follicles are known to mostly undergo atresia, a fate sometimes associated with the persistence of steroidogenic theca cells, but this connection is poorly understood. We characterized wave 1 follicle biology using tissue clearing, lineage tracing and scRNAseq to clarify their contributions to offspring and to steroidogenesis. Wave 1 follicles lineage marked by E16.5 FoxL2 expression in granulosa cells, reach preantral stages containing theca cell layers by 2 weeks. Atresia begins about a week later, during which 80-100% of wave 1 follicles degrade their oocytes, turn over most granulosa cells, but retain theca cells which expand in number together with interstitial gland cells in the medulla. During puberty (5 weeks), these cells ultrastructurally resemble steroidogenic cells and highly express androgen biosynthetic genes. Unexpectedly, the FoxL2 lineage tag also marked about 400 primordial follicles, located near the medullar-cortical boundary, that become the earliest-activated wave 2 follicles. These "boundary" or "wave 1.5" follicles generate 70-100% of the earliest mature oocytes, while fewer than 26 wave 1 follicles with oocytes survive. Consistent with their largely distinct fates in steroid or oocyte production, granulosa cells of antral wave 1 and wave 2 follicles differentially express multiple genes, including Wnt4 and Igfbp5. SIGNIFICANCE STATEMENTWe studied the ovaries of juvenile mice 2-6 weeks (wk) of age using transcriptomics and cell lineage to characterize the earliest developing groups of follicles. Wave 1 follicles begin growing at birth in the ovarian medulla, but few produce mature oocytes. Instead, most undergo partial atresia and expand steroidogenic cells expressing androgenic genes at puberty. A newly identified primordial follicle subset located at the medulla-cortex boundary are the earliest to activate and grow. Their oocytes give rise to most early offspring, while no more than a few derive from surviving wave 1 follicles. Our results highlight the importance of spatially localized follicle subgroups and show that follicular waves aid reproduction in distinct ways, some of which involve substantial atresia and cellular remodeling.

developmental biology↗

bioRxiv2018

Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependant pigment progenitors in zebrafish

Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, parade, exhibit ectopic pigment cells localised to the ventral trunk. We show that parade encodes Endothelin receptor Aa, expressed in the blood vessels. Using chemical genetics, coupled with analysis of cell fate studies, we show that the ectopic pigment cells derive from APSCs. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.\n\nLay AbstractPigment patterns are crucial for the many aspects of animal biology, for example, providing camouflage, enabling mate selection and protecting against UV irradiation. These patterns are generated by one or more pigment cell-types, localised in the skin, but derived from specialised stem cells (adult pigment stem cells, APSCs). In mammals, such as humans, but also in birds and fish, these APSCs derive from a transient population of multipotent progenitor cells, the neural crest. Formation of the adult pigment pattern is perhaps best studied in the zebrafish, where the adult pigment pattern is formed during a metamorphosis beginning around 21 days of development. The APSCs are set-aside in the embryo around 1 day of development, but then remain inactive until that metamorphosis, when they become activated to produce the adult pigment cells. We know something of how the cells are set-aside, but what signals maintain them in an inactive state is a mystery. Here we study a zebrafish mutant, called parade, which shows ectopic pigment cells in the embryo. We clone the parade gene, identifying it as ednraa encoding a component of a cell-cell communication process, which is expressed in blood vessels. By characterising the changes in the neural crest and in the pigment cells formed, and by combining this with an innovative assay identifying drugs that prevent the ectopic cells from forming, we deduce that the ectopic cells in the larva derive from precocious activation of APSCs to form pigment cells. We propose that a novel population of APSCs are associated with the blood vessels, that these are held in a quiescent state by signals coming from these vessels, and that these signals depend upon ednraa. Together this opens up an exciting opportunity to identify the signals maintaining APSC quiescence in zebrafish.

developmental biology↗

bioRxiv2021

Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive distal myopathy with rimmed vacuoles that typically manifests in late adolescence/early adulthood. GNE encodes an enzyme that is the rate-limiting step in sialic acid biosynthesis which is necessary for proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study we aimed to address this knowledge gap by querying underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem cell (iPSC) model. Muscle and skin biopsies were acquired from two patients with GNE myopathy that presented with distinct histopathological features. Control and patient-specific iPSCs were derived from skin fibroblasts and differentiated down a skeletal muscle lineage in a three-stage process analogous to muscle development and muscle regeneration. Initial studies revealed: 1) the ability of patient-derived GNE iPSC clones to recapitulate key characteristics of the human pathology including TDP-43 accumulation and evidence of dysregulated autophagy, and 2) a striking defect in myogenic progression of the more severe GNE iPSC clone. Single-cell RNA sequencing time course studies were then performed to more rigorously explore myogenesis defects. Cluster-based bioinformatics analyses revealed clear differences between control and GNE iPSC-derived muscle precursor cells (iMPCs). On a transcriptional level, late stage GNE iMPCs resembled that of early stage control iMPCs, confirming stalled myogenic progression on a molecular level. Comparative expression and pathway studies revealed EIF2 signaling as a top signaling pathway altered in GNE iMPCs. In summary, we report a novel in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a likely novel contributing mechanism to the etiology of GNE myopathy. SUMMARY STATEMENTDevelopment of a novel cell-based model of GNE myopathy, utilizing GNE patient-derived samples, which recapitulates human disease characteristics, uncovered myogenic differentiation defects, and can elucidate possible mechanistic contributors to the disease.

developmental biology↗

bioRxiv2020

Simplified artificial blood feeding and oral infection method for mosquitoes

Mosquitoes such as Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are vectors of many pathogens that greatly affect humankind. The maintenance of these mosquitoes in laboratory permit different studies that can help understanding their biology, as well as the vector-pathogen interaction. In addition to sugar meals, the blood feeding is essential for maintenance of the reproductive cycle in several vectors. The main blood sources for many mosquito colonies are direct feeding on live animal or the use of human/animal blood through artificial feeders. However, this latter process has some disadvantages, as artificial feeders can be very laborious for assembly and decontamination. Based on these observations, a simplified technique for feeding and artificial infection was developed with cotton-pads soaked (CS) and blood or blood and viral supernatant to simulate an artificial infection. The efficiency of the CS technique was investigated through the number of mosquitoes fed/infected, when compared to their respective control group. The CS technique, with blood at room temperature, promoted a feeding rate of 61.4% for Ae. albopictus, 70.8% for Cx. quinquefasciatus and 17% for Ae. aegypti. The control group (Hemotec-feeding) presented 47.9%, 16.5% and 59.1% of feeding success, respectively. To improve the CS technique for Ae. aegypti mosquitoes, the procedure was then performed with blood at 38{degrees}C, which was possible to observe a feeding rate of 47.3%, in comparison to 53.2% for the control group (Hemotec). When using the CS technique for artificial infection with Mayaro virus, more than 80% of infection was observed for Ae. aegypti and 100% for Ae. albopictus. In the traditional infection technique (glass feeder), the infection rate was 90% (Ae. aegypti) and 96.6% (Ae. albopictus). For Cx. quinquefasciatus, the infection was positive only with the CS technique, resulting in 1 (5%) mosquito infected with Mayaro virus. Our results suggest that this simplified technique of low-cost feeding and easy assembly, offers good results for feeding (maintenance of colonies) and artificial infection of different species of mosquitoes.

developmental biology↗