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Background: Early during onychophoran development and prior to the formation of the germ band, a posterior tissue thickening forms the posterior pit. Anterior to this thickening forms a groove, the embryonic slit, that marks the anterior-posterior orientation of the developing embryo. This slit is by some authors considered the blastopore, and thus the origin of the endoderm, while others argue that the posterior pit represents the blastopore. This controversy is of evolutionary significance because if the slit represents the blastopore, then this would support the amphistomy hypothesis that suggests that a slit-like blastopore in the bilaterian ancestor evolved into protostomy and deuterostomy. Results: In this paper, we summarize our current knowledge about endoderm and mesoderm development in onychophorans and provide additional data on early endoderm- and mesoderm-determining marker genes such as Blimp, Mox, and the T-box genes. Conclusion: We come to the conclusion that the endoderm of onychophorans forms prior to the development of the embryonic slit, and thus that the slit is not the primary origin of the endoderm. It is thus unlikely that the embryonic slit represents the blastopore. We suggest instead that the posterior pit indeed represents the lips of the blastopore, and that the embryonic slit (and surrounding tissue) represents a morphologically superficial archenteron-like structure. We conclude further that both endoderm and mesoderm development are under control of conserved gene regulatory networks, and that many of the features found in arthropods including the model Drosophila melanogaster are likely derived.

Background The common house spider Parasteatoda tepidariorum represents an emerging new model organism of arthropod evolutionary and developmental (EvoDevo) studies. Recent technical advances have resulted in the first single-cell sequencing (SCS) data on this species allowing deeper insights to be gained into its early development, but mid-to-late stage embryos were not included in these pioneering studies. Results Therefore, we performed SCS on mid-to-late stage embryos of Parasteatoda and characterized resulting cell clusters by means of in-silico analysis (comparison of key markers of each cluster with previously published information on these genes). In-silico prediction of the nature of each cluster was then tested/verified by means of additional in-situ hybridization experiments with additional markers of each cluster. Conclusions Our data show that SCS data reliably group cells with similar genetic fingerprints into more or less distinct clusters, and thus allows identification of developing cell types on a broader level, such as the distinction of ectodermal, mesodermal and endodermal cell lineages, as well as the identification of distinct developing tissues such as subtypes of nervous tissue cells, the developing heart, or the ventral sulcus (VS). In comparison with recent other SCS studies on the same species, our data represent later developmental stages, and thus provide insights into different stages of developing cell types and tissues such as differentiating neurons and the VS that are only present at these later stages.

Background Formation of tissue-specific transcriptional programs underlies multicellular development, including dorsoventral (DV) patterning of the Drosophila embryo. This involves interactions between transcriptional enhancers and promoters in a chromatin context, but how the chromatin landscape influences transcription is not fully understood. Results Here we comprehensively resolve differential transcriptional and chromatin states during Drosophila DV patterning. We find that RNA Polymerase II pausing is established at DV promoters prior to zygotic genome activation (ZGA), that pausing persists irrespective of cell fate, but that release into productive elongation is tightly regulated and accompanied by tissue-specific P-TEFb recruitment. DV enhancers acquire distinct tissue-specific chromatin states through CBP-mediated histone acetylation that predict the transcriptional output of target genes, whereas promoter states are more tissue-invariant. Transcriptome-wide inference of burst kinetics in different cell types revealed that while DV genes are generally characterized by a high burst size, either burst size or frequency can differ between tissues. Conclusions The data suggest that pausing is established by pioneer transcription factors prior to ZGA and that release from pausing is imparted by enhancer chromatin state to regulate bursting in a tissue-specific manner in the early embryo. Our results uncover how developmental patterning is orchestrated by tissue-specific bursts of transcription from Pol II primed promoters in response to enhancer regulatory cues.

Organisms throughout the tree of life have evolved distinct ways to regulate gene expression. Some of these processes involve non-coding RNAs (ncRNAs), which are not translated but functional nonetheless. These ncRNAs are of utmost importance, with dysregulation of some causing severe developmental effects or even being lethal. In order to get a better fundamental understanding of gene regulation, and the ncRNAs that evolved to regulate gene expression, we study this in Amoebozoa. Members of this taxon vary greatly in lifestyle and organismal complexity. Some are strictly unicellular, free-living, whereas others, such as the social amoeba Dictyostelium discoideum can transition between unicellular and multicellular lifestyles. D. discoideum features a variety of small ncRNAs. Among these are the microRNAs. microRNAs have mostly been studied in plants and animals, where they are believed to have evolved convergently, and hypothesized to have played a role when these taxa evolved multicellular lifestyles. At what point the D. discoideum microRNAs evolved, how they function, and if they are involved in its multicellular lifestyle are fundamental questions addressed in this thesis. Here, we studied the evolution and function of microRNAs in a broad set of species belonging to Amoebozoa. We could identify microRNAs in all studied amoebae, and concluded that they are probably not involved in the evolution of multicellularity. To in detail investigate the evolution of microRNAs, we performed comparative genomics using D. discoideum and the close relative Dictyostelium firmibasis. For this, we sequenced, assembled and annotated the genome of the latter. At this point, our findings suggest that the microRNAs evolved several times in Amoebozoa, although we cannot rule out if they have a deep evolutionary history. The Class I RNAs are another type of ncRNAs. These, on the other hand, are only present in the social amoebae. They are hypothesized to regulate the transition fro

Dysregulation of physiological and cellular processes underlies various pathological conditions, including cancer and inflammatory disorders. Unraveling the molecular mechanisms driving these processes is crucial. The aim of this thesis was to investigate the roles of evolutionarily conserved POU/Oct transcription factors using Drosophila melanogaster as a model organism. The thesis highlights the functions of Nubbin (Nub) protein isoforms (Nub-PB and Nub-PD) in the regulation of cellular proliferation and mitosis, epithelial regeneration, and innate immune responses. In paper I, we demonstrate that Nub-PB acts as a potent transcriptional activator of immune and stress response genes, while Nub-PD represses their expression, indicating transcriptional antagonism by these Nub isoforms. Overexpression of Nub-PB in midgut cells effectively cleared local infections. However, prolonged Nub-PB overexpression caused a hyperactive immune response, leading to pro-inflammatory reactions, apoptosis, and reduced adult lifespan. These findings emphasize the importance of Nub protein isoforms in fine-tuning immune responses. In Paper II, we generated and phenotypically characterized a Nub-PB-specific mutant revealing impaired gut morphology, disorganized visceral muscles, and aberrant lineage specification in the midgut. In addition, it displays impaired immune gene activation, shortened lifespan, and enhanced reactive oxygen species (ROS) expression, which correlates with increased numbers of gut microbiota, featuring an important role of Nub-PB in intestinal epithelium homeostasis. In Paper III, we show that Nub-PD is necessary for proper nuclear divisions in transcriptionally silent pre-blastoderm embryos. The Nub-PD protein is enriched around the mitotic spindles in metaphase, requiring intact spindle microtubules. Live imaging of mitotic divisions revealed that Nub-PD is involved in the maintenance of spindle organization and its dynamics. We also infer similar mitotic rol

All organisms have to carefully regulate their gene expression, not least during development. mRNA levels are often used as proxy for protein output; however, this approach ignores post-transcriptional effects. In particular, mRNA-protein correlation remains elusive for organisms that exhibit aggregative rather than clonal multicellularity. We addressed this issue by generating a paired transcriptomics and proteomics time series during the transition from uni-to multicellular stage in the social ameba Dictyostelium discoideum. Our data reveals that mRNA and protein levels correlate highly during unicellular growth, but decrease when multicellular development is initiated. This accentuates that transcripts alone cannot accurately predict protein levels. The dataset provides a useful resource to study gene expression during aggregative multicellular development. Additionally, our study provides an example of how to analyze and visualize mRNA and protein levels, which should be broadly applicable to other organisms and conditions.

The aggrandised advancement in utility of advanced day-to-day materials and nanomaterials has raised serious concern on their biocompatibility with human and other biotic members. In last few decades, understanding of toxicity of these materials has been given the centre stage of research using many in vitro and in vivo models. Zebrafish (Danio rerio), a freshwater fish and a member of the minnow family has garnered much attention due to its distinct features, which make it an important and frequently used animal model in various fields of embryology and toxicological studies. Given that fertilization and development of zebrafish eggs take place externally, they serve as an excellent model organism for studying early developmental stages. Moreover, zebrafish possess a comparable genetic composition to humans and share almost 70% of their genes with mammals. This particular model organism has become increasingly popular, especially for developmental research. Moreover, it serves as a link between in vitro studies and in vivo analysis in mammals. It is an appealing choice for vertebrate research, when employing high-throughput methods, due to their small size, swift development, and relatively affordable laboratory setup. This small vertebrate has enhanced comprehension of pathobiology and drug toxicity. This review emphasizes on the recent developments in toxicity screening and assays, and the new insights gained about the toxicity of drugs through these assays. Specifically, the cardio, neural, and, hepatic toxicology studies inferred by applications of nanoparticles have been highlighted.

Background: The classical concept of brain sex differentiation suggests that steroid hormones released from the gonads program male and female brains differently. However, several studies indicate that steroid hormones are not the only determinant of brain sex differentiation and that genetic differences could also be involved. Methods: In this study, we have performed RNA sequencing of rat brains at embryonic days 12 (E12), E13, and E14. The aim was to identify differentially expressed genes between male and female rat brains during early development. Results: Analysis of genes expressed with the highest sex differences showed that Xist was highly expressed in females having XX genotype with an increasing expression over time. Analysis of genes expressed with the highest male expression identified three early genes, Sry2, Eif2s3y, and Ddx3y. Discussion: The observed sex-specific expression of genes at early development confirms that the rat brain is sexually dimorphic prior to gonadal action on the brain and identifies Sry2 and Eif2s3y as early genes contributing to male brain development.

The herbicide linuron can cause endocrine disrupting effects in Xenopus tropicalis frogs, including offspring that were never exposed to the contaminant. The mechanisms by which these effects are transmitted across generations need to be further investigated. Here, we examined transgenerational alterations of brain and testis DNA methylation profiles paternally inherited from grandfathers developmentally exposed to an environmentally relevant concentration of linuron. Reduced representation bisulfite sequencing (RRBS) revealed numerous differentially methylated regions (DMRs) in brain (3060 DMRs) and testis (2551 DMRs) of the adult male F2 generation. Key genes in the brain involved in somatotropic ( igfbp4 ) and thyrotropic signaling ( dio1 and tg ) were differentially methylated and correlated with phenotypical alterations in body size, weight, hind limb length and plasma glucose levels, indicating that these methylation changes could be potential mediators of the trans- generational effects of linuron. Testis DMRs were found in genes essential for spermatogenesis, meiosis and germ cell development ( piwil1 , spo11 and tdrd9) ) and their methylation levels were correlated with the number of germ cells nests per seminiferous tubule, an endpoint of disrupted spermatogenesis. DMRs were also identified in several genes central for the machinery that regulates the epigenetic landscape including DNA methylation ( dnmt3a and mbd2) ) and histone acetylation ( hdac8 , ep300, , elp3, , kat5 and kat14), ), which may at least partly drive the linuron-induced transgenerational effects. The results from this genome-wide DNA methylation profiling contribute to better understanding of potential transgenerational epigenetic inheritance mechanisms in amphibians.

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development are still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS1 (HLS1) and PHYTOCHROME INTERACTING FACTOR4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins, or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima, and PIF4 and HLS1 expression. Analyses of plants expressing a F & ouml;rster resonance energy transfer-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signaling module to control differential cell elongation during hook formation. Loss of cell wall integrity suppresses gibberellic acid accumulation and HOOKLESS1 and PHYTOCHROME INTERACTING FACTOR4 expression, ultimately repressing apical hook formation in Arabid

Human development is complex and intricate, where the positions of cells, expression of key markers, and cell-cell interactions contribute to the development of various organs from different germ layers and the establishment of the body axis. Therefore, understanding human development within spatial and temporal aspects is crucial. Spatial and temporal aspects can be studiedthrough multiplexed imaging, which enables the assessment of multiple markers on the same tissue, offering critical insights into protein expressions in the cells and tissues. Within the scope of this thesis, we focused on the spatial and single-cell profiling of cell types during the first trimester of human development, both at the systemic and organ levels, using multiplexed imaging. Paper I of this thesis presents a spatial and single-cell map of the developing human lung in the first trimester. We used multiplexed imaging on post-conception week 6 to 13 lungs employing a 30-plex antibody panel and, as a result, analyzed nearly 1 million cells. We provide a spatially resolved cell type composition of the developing human lung, focusing on spatiotemporal changes in the cell types, such as immune cells, endothelial cells, lymphatic cells, and proliferative cell states. Key findings of the first paper are that the proliferation patterns in the epithelium reveal differences in the elongation of smaller and larger distal and proximal airways and the presence of some immune cells around arteries, highlighting location-function relationships. Additionally, this paper represents the first application of multiplexed imaging on the developing human lung. Paper II aimed to systematically investigate human development in whole embryos by focusing on cell types such as immune and endothelial cells. We analyzed human whole embryo tissues from week 3 to 5 using a 28-plex multiplexed antibody panel. A key finding of the paper is the appearance of liver immune cells as early as week 4 and differences in their, Människans utveckling är komplex och invecklad, där cellernas positioner, uttryck av viktiga proteinmarkörer och cell-cell interaktioner bidrar till etableringen av kroppens axel och till utvecklingen av organ från olika germinallager. Därför är spatiala och temporala aspekter avgörande för förståelsen av människans utveckling. Detta är möjligt att studera genom multiplexad bildteknik, vilket möjliggör samtidig bedömning av multipla markörer i samma vävnadsprov och erbjuder viktiga insikter kring proteinuttrycket i olika celler och vävnader. Inom ramen för denna avhandling fokuserade vi på spatial- och single-cellprofilering av olika celltyper under den första trimestern av människans utveckling, både på system- och organnivå, med hjälp av multiplexad bildteknik.Artikel I i denna avhandling presenterar en spatial och single-cellkarta av den mänskliga lungans utveckling under den första trimestern. Vi använde multiplexad bildteknik med en 30-plex antikroppspanel för vävnadsprover från lungor isolerade 6-13 veckor efter befruktningen och analyserade nästan 1 miljon celler. Vi tillhandahåller en karta över celltypskomposition med spatial upplösning under den mänskliga lungans utveckling, med fokus på spatio-temporala förändringar av olika celltyper, såsom immunceller, endotelceller och lymfatiska celler, och proliferativa celltillstånd. Nyckelfynden i den första artikeln är att proliferationsmönstren i epitelet avslöjar skillnader i förlängningen av mindre respektive större distala och proximala luftvägar, och att närvaron av vissa immun-celler runt artärerna belyser förhållandet mellan lokalisation och funktion. Dessutom representerar denna artikel den första tillämpningen av multiplexad bildteknik för att studera den mänskliga lungans utveckling. Artikel II syftade till att systematiskt undersöka människans utveckling i hela embryon, med fokus på celltyper som immunceller och endotelceller. Vi analyserade hela mänskliga embryovävnader isolerade vid 3-5 veckor med hjä, QC 2024-04-12

Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development.

Medfödda hjärtfel är globalt en av de vanligaste defekterna vid födsel, trots det är den underliggande mekanismen och dess sjukdomsförlopp fortfarande inte välkända. Flera hundra sjukdomsgener har identifierats vilket gör sjukdomen inte bara komplex, men också heterogen. För att kunna avancera i förebyggande syfte, i patient-diagnostik och behandling behövs en djupare förståelse av dess etiologi. Noterbart är att många patienter med medfödda hjärtfel också upplever samsjuklighet som utvecklingsneurologiska störningar, neurodegenerativa sjukdomar samt psykiska hälsoproblem. I den här studien undersöktes tre potentiella sjukdomsgener: ANK3, COPZ1 och DCTN1 för deras roll i både hjärtat och hjärnans utveckling i zebrafisk som experimentell modell. Detta gjordes genom att göra gen-knockouts på generna individuellt med hjälp av F0 Crispant CRISPR/Cas9 tekniken i följd av in-situ hybridisering, för att undersöka potentiella laterala defekter i hjärtat samt bildanalys för att mäta storleken på hjärnan. Alla tre gener verkade orsaka en signifikant ökning av hjärtfel genom att laterala defekter observerades, där två av generna också visade en signifikant minskad hjärtfrekvens. Angående hjärnan så verkade två av generna orsaka en mindre hjärna medan en av generna orsakade en förstorad hjärna. Dessa resultat ger en preliminär inblick i deras medverkan i hjärtat och hjärnans utveckling, dock behövs vidare studier inte bara för att stärka resultaten men också för att kunna grundligt säkerställa korrelationen till samsjukligheten med utvecklingsneurologiska störningar, neurodegenerativa sjukdomar samt psykiska hälsoproblem., Congenital heart defects (CHD) are amongst the most common birth defects globally, yet the underlying mechanism and the disease trajectory of it is still poorly understood. Several hundred disease genes have been discovered, making it not only complex but also heterogeneous. In order to advance in prevention, patient diagnosis and treatment, a deeper understanding of its etiology is needed. Notably, many patients with CHD experience comorbidities such as neurodevelopmental- and neurodegenerative disorders as well as mental health issues. In this project, three candidate disease genes: ANK3, COPZ1 and DCTN1 were chosen to further investigate their role in both heart- and brain development in zebrafish as the experimental model. This was done by knocking out the genes individually using the F0 Crispant CRISPR/Cas9 method followed by whole mount in situ hybridization (WISH), to assess potential defective lateral phenotypes of the heart and image analysis to measure the size of the brain. All three genes seemed to cause a significant increase in overall heart defects by showing laterality defects whereas two of them also showed significantly slower heart rate. As for the brain, two of the genes seemed to cause a smaller brain, whereas one gene seemed to cause an enlarged brain. These findings give a preliminary insight into their involvement in the heart- and brain development, however, further research is necessary to not only strengthen the findings but to also more thoroughly establish the correlation to the comorbidity of the neurodevelopmental, neurodegenerative disorders and mental health issues.

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation. One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

In a great partnership, the Federation of European Neuroscience Societies (FENS) and the Hertie Foundation organized the FENS-Hertie 2022 Winter School on 'Neuro-immune interactions in health and disease'. The school selected 27 PhD students and 13 postdoctoral fellows from 20 countries and involved 14 faculty members experts in the field. The Winter School focused on a rising field of research, the interactions between the nervous and both innate and adaptive immune systems under pathological and physiological conditions. A fine-tuned neuro-immune crosstalk is fundamental for healthy development, while disrupted neuro-immune communication might play a role in neurodegeneration, neuroinflammation and aging. However, much is yet to be understood about the underlying mechanisms of these neuro-immune interactions in the healthy brain and under pathological scenarios. In addition to new findings in this emerging field, novel methodologies and animal models were presented to foment research on neuro-immunology. The FENS-Hertie 2022 Winter School provided an insightful knowledge exchange between students and faculty focusing on the latest discoveries in the biology of neuro-immune interactions while fostering great academic and professional opportunities for early-career neuroscientists from around the world., Funding Agencies|Federation of European Neuroscience Societies; Gemeinnutzige Hertie-Stiftung; Fonds de Recherche du Quebec - Sante; International Brain Research Organization; Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro; Zardi Gori Foundation; Cariplo Foundation; Nuffield Department of Clinical Neurosciences, University of Oxford; Knut and Alice Wallenberg foundation; Swedish Brain Foundation; Swedish Research Council; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior

Neural networks are the functional building blocks of the central nervous system. To better understand how these networks develop and operate, we turned to the zebrafish locomotor network, with a focus on subtypes of interneurons expressing dmrt3a and wt1a. These neurons first gained interest when a mutation in the Dmrt3 gene was found to be responsible for Icelandic horses’ ability to perform additional gaits, indicating a flexibility within their locomotor central pattern generator. In zebrafish, the Dmrt3 population is known to be commissural, inhibitory and involved in escape behaviors and left-right alternation during locomotion. We characterized the locomotor behavior at embryonic, larval and juvenile stages in dmrt3a mutants. A strong phenotype was observed in larval escape behavior, showing reduced top speed while the animals spent more time accelerating. While the phenotype subdued as the animals developed, juveniles still maintained a lower maximum locomotor speed. To get a more detailed understanding of the observed phenotypes, an experimental setup was established combining dual ventral root recordings with calcium imaging and various sensory stimuli to induce diverse locomotor outputs in fictively behaving larva. Implementing this method, we investigated the function of Dmrt3 and Wt1 expressing interneurons in escape behaviors and found that knock-down of Dmrt3a disturbed the fast phase of tail evoked escapes, while knock-down of Wt1a lead to aberrant looming evoked escapes, indicating sub-functionalization. Finally, calcium imaging was employed to reveal the activity of Dmrt3 neurons at a population level. The fraction of active cells steadily increased during development and small clusters of correlated Dmrt3 interneuron ensembles were observed within a segment. This work provides insights into how parallel motor networks are orchestrated to generate a flexible behavioral output, revealing fundamental principles extending to the workings of our own b

Teeth, scales, fin spines, and other odontodes are hot topics in evolutionary biology due to their great variation in number, shape, and anatomical localization in vertebrates as a result of different types of adaptations. Structural adaptation arose through variations in the composition of the enamel/enameloid and dentin layers that build up the mineralized components of teeth and other odontodes. These variations are known from both fossil and extant groups of vertebrates and rely on differences in the proteins secreted by the specialized odontode cells, ameloblasts, and odontoblasts. In this chapter, we focus on recent genomic and gene expression data to better understand the evolution of gene families engaged in the processes of secretion, mineralization, and maturation of odontode matrices. Our focus will be structural matrix proteins (collagenous and non-collagenous), calcium-interacting proteins necessary for mineralization, and proteins regulating matrix degradation over the course of tissue maturation.

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.

Animals use the Polycomb system to epigenetically repress developmental genes. The repression requires trimethylation of lysine 27 of histone H3 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2), but the dynamics of this process is poorly understood. To bridge the gap, we developed a computational model that forecasts H3K27 methylation in Drosophila with high temporal resolution and spatial accuracy of contemporary experimental techniques. Using this model, we show that pools of methylated H3K27 in dividing cells are defined by the effective concentration of PRC2 and the replication frequency. We find that the allosteric stimulation by preexisting H3K27me3 makes PRC2 better in methylating developmental genes as opposed to indiscriminate methylation throughout the genome. Applied to Drosophila development, our model argues that, in this organism, the intergenerationally inherited H3K27me3 does not “survive” rapid cycles of embryonic chromatin replication and is unlikely to transmit the memory of epigenetic repression to the offspring. Our model is adaptable to other organisms, including mice and humans.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed spatial transcriptomics of variable, diversity, and joining (VDJ) sequences (Spatial VDJ), a method that maps B cell and T cell receptor sequences in human tissue sections. Spatial VDJ captures lymphocyte clones that match canonical B and T cell distributions and amplifies clonal sequences confirmed by orthogonal methods. We found spatial congruency between paired receptor chains, developed a computational framework to predict receptor pairs, and linked the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity and lineage trajectories within their anatomical niche. Thus, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, providing a platform to harness clonal sequences for therapy., QC 20240102

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre–oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development., QC 20240103

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods., QC 20231025

The combination of estrus synchronization and superovulation (SS) treatments causes alterations in ovarian and endometrial gene expression patterns, resulting in abnormal follicle and oocyte growth, fertilization, and embryo development. However, the impact of combined SS treatments on the transcriptome of the surviving embryos remains unidentified. In this study, we examined gene expression changes in day 6 blastocysts that survived a brief regimen of synchronization treatment combined with superovulation. The sows were included in one of three groups: SS7 group (n = 6), sows were administered Altrenogest (ALT) 7 days from the day of weaning and superovulated with eCG 24 h after the end of ALT treatment and hCG at the onset of estrus; SO group (n = 6), ALT nontreated sows were superovulated with eCG 24 h postweaning and hCG at the onset of estrus; control group (n = 6), weaned sows displaying natural estrus. Six days after insemination, the sows underwent a surgical intervention for embryo collection. Transcriptome analysis was performed on blastocyst-stage embryos with good morphology. Differentially expressed genes (DEGs) between groups were detected using one-way ANOVA with an un-adjusted p-value < 0.05 and a fold change 1.5. The effect of SO treatment on the number of altered pathways and DEGs within each pathway was minimal. Only four pathways were disrupted comprising only a total of four altered transcripts, which were not related to reproductive functions or embryonic development. On the other hand, the surviving blastocysts subjected to SS7 treatments exhibited moderate gene expression changes in terms of DEGs and fold changes, with seven pathways disrupted containing a total of 10 transcripts affected. In this case, the up-regulation of certain pathways, such as the metabolic pathway, with two up-regulated genes associated with reproductive functions, namely RDH10 and SPTLC2, may suggest suboptimal embryo quality, while the down-r, Funding Agencies|MCIN/AEI/ERDF a way of making Europe, Madrid, Spain [RTI2018-093525-B-I00]; Fundacion Seneca, Murcia, Spain [19892/GERM/15]; Swedish Research Council FORMAS, Stockholm, Sweden [2019-00288]

In common with other plant species, the garden pea (Pisum sativum) produces the auxin indole-3-acetic acid (IAA) from tryptophan via a single intermediate, indole-3-pyruvic acid (IPyA). IPyA is converted to IAA by PsYUC1, also known as Crispoid (Crd). Here, we extend our understanding of the developmental processes affected by the Crd gene by examining the phenotypic effects of crd gene mutations on leaves, flowers, and roots. We show that in pea, Crd/PsYUC1 is important for the initiation and identity of leaflets and tendrils, stamens, and lateral roots. We also report on aspects of auxin deactivation in pea.

The mechanisms underlying the construction of an air-liquid interface in respiratory organs remain elusive. Here, we use live imaging and genetic analysis to describe the morphogenetic events generating an extracellular lipid lining of the Drosophila airways required for their gas filing and animal survival. We show that sequential Rab39/Syx1A/Syt1-mediated secretion of lysosomal acid sphingomyelinase (Drosophila ASM [dASM]) and Rab11/35/Syx1A/Rop-dependent exosomal secretion provides distinct components for lipid film assembly. Tracheal inactivation of Rab11 or Rab35 or loss of Rop results in intracellular accumulation of exosomal, multi-vesicular body (MVB)-derived vesicles. On the other hand, loss of dASM or Rab39 causes luminal bubble-like accumulations of exosomal membranes and liquid retention in the airways. Inactivation of the exosomal secretion in dASM mutants counteracts this phenotype, arguing that the exosomal secretion provides the lipid vesicles and that secreted lysosomal dASM organizes them into a continuous film. Our results reveal the coordinated functions of extracellular vesicle and lysosomal secretions in generating a lipid layer crucial for airway gas filling and survival.

The histone deacetylase HDAC3 is associated with the NCoR/SMRT co-repressor complex, and its canonical function is in transcriptional repression, but it can also activate transcription. Here, we show that the repressor and activator functions of HDAC3 can be genetically separated in Drosophila. A lysine substitution in the N terminus (K26A) disrupts its catalytic activity and activator function, whereas a combination of substitutions (HEBI) abrogating the interaction with SMRTER enhances repressor activity beyond wild type in the early embryo. We conclude that the crucial functions of HDAC3 in embryo development involve catalytic-dependent gene activation and non-enzymatic repression by several mechanisms, including tethering of loci to the nuclear periphery.

The fish fauna of the Baltic Sea reflects its 9 KY history of Arctic and temperate conditions and is a mixture of species that have invaded from the Atlantic and the continental watersheds. In spite of the challenging environmental conditions, such as low salinity in the entire Baltic Sea and varying temperature conditions, limiting the possibilities for successful reproduction, the number of species is comparably high. Except for the Baltic Ice Lake and certain stages of the cold Yoldia Sea and freshwater Ancylus Lake, the fish fauna of the Baltic Sea, as recorded by archaeological and historical notes, has to a large extent remained unchanged. Some freshwater and cold-water species such as Arctic char may have disappeared while others, such as fourhorned sculpin and eelpout, have adapted and persist as “ice age relicts.” There are few viable introductions of novel species; the round goby may be the most conspicuous invasive species. The extinction rate is still low; the loss of sturgeon and the common skate within the HELCOM (the Helsinki Commission) area is due to fishing, and, for the riverine sturgeon, due to damming. Since the formation of the Baltic Sea, fishing has played an essential role in supplying coastal settlements and their hinterlands and in trading. The archaeological and historical records have indicated fishing conducted with varying intensity using different methods. Herring fishing has been a significant economic driver from the Middle Ages onward. Recent archaeological findings indicate that organized fishing was established at the outer archipelagos along the present Swedish east coast on predominately herring and cod archipelagos for self-sufficiency shortly before or during the Viking Age, and later to engage in barter. The fact that the cod abundance has sometimes been sufficient for letting cod fishing be the most important fishery in the northern Baltic Proper alongside the fishery on herring may indicate that the eastern cod stock had r

In male mice, the transcription factor A-MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A-MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A-MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiate the production of piRNAs from A-MYB-dependent older loci, ensuring the self-propagation of pachytene piRNAs. A-MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals.

Endocrine disrupting chemicals (EDCs) are ubiquitous and their adverse impact on nature, wildlife and humans is extensively researched. We are constantly exposed to EDCs, such as the widespread and extensively researched Bisphenol A, as well as its substitute Bisphenol F, which is coming into wider use, even though it is much less is researched and limited information is available about its endocrine effects. Realistically, we are exposed to mixtures rather than single substances. In the Swedish Environmental Longitudinal, Mother and Child, Asthma and allergy (SELMA) study, the co-exposure of EDCs was assessed. Based on the SELMA cohort data, a study identified a mixture of EDCs, Mixture N1, which is associated with delayed language development. In recent years, it has been hypothesized that epigenetic alterations are one of the underlying mechanisms for the effect of EDC exposures. For example, EDC induced changes in DNA Methylation of the promoter region of a gene might lead to altered gene expression, which can result in adverse health effects. Several studies already indicate an impact of the formerly introduced chemicals/mixtures on the DNA methylation on genes such as NR3C1 and GRIN2B in animals. However, limited research is available on the impact on NR3C1 and GRIN2B in the early human brain, which is of interest since both genes are crucial for the development of the brain and altered gene expression often leads to adverse effects. This study aimed to investigate the impact of BPA, BPF and Mixture N1 on NR3C1 and GRIN2B in the developing human brain as well as establish a protocol for differentiation of human stem cells into neuroprogenitor cells that express GRIN2B and NR3C1. In the end stem cells were differentiated in vitro into neural progenitor cells (NPCs) using the protocol of Hosseini et al. (2020). During the differentiation, the cells were exposed to different concentrations of the former mentioned chemicals. Afterwards, RNA and DNA were extracted

Lymphatic endothelial cells (LECs) migrate across body to form a branched network, which is crucial for fluid drainage and immune cell trafficking of the tissues. However, the molecular mechanisms behind the spatiotemporal regulation and fine-tuning of LEC migration remain largely unknown. The rapid development and the available transgenic tools in zebrafish allow to study these processes in unprecedented resolution and provide new insights into cellular and molecular dynamics regulating lymphangiogenesis. This study aims to dissect the lymphatic development of zebrafish, with a focus of LEC migration and its regulation in the trunk. Proper cell migration requires signalling and guidance cue from the environment and tissue-tissue interactions. In paper I, we identified arterial mural cells as a novel source of growth factor and chemokine essential for the migration and robust formation of the lymphatic network. This is important as understanding the sources of guiding molecules can help optimizing the formation and repair of lymphatic vessels in pathological conditions. VEGFC-VEGFR3 is an essential signalling required throughout lymphangiogenesis. However, it still remains unclear how pro-lymphangiogenic cues are interpreted by LECs to induce differential behaviours. In paper II, we investigated the secondary sprouting which is dependent on VEGFC-VEGFR3 signalling. We characterized the cell migrating behaviours and analysed the molecular signatures of the sprouts. Furthermore, we identified Ca2+ activities are required for proper sprouting and potentially serves as modulator for VEGFC-VEGFR3 signalling. In paper III, we investigated LEC proliferation driven by VEGFC-VEGFR3 signalling at different development stages. We identified three key timepoints of LEC expansion as well as the novel molecular factors regulating the proliferation. Together we demonstrated the how cell cycle machinery is driven by VEGFC-VEGFR3 signalling. In paper IV, we identified enhancers s

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including mu CT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SR mu CT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2(-/-) mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2(-/-) mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2(-/-) zebrafish, and demonstrate the promise of DICE-PPC-SR mu CT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.

The lymphatic vascular network is composed of a series of blind ended vessels, and is involved in physiological functions such as fluid homeostasis, lipid metabolism and immune trafficking. Despite lymphatic endothelial cells being derived from multiple organs, they share common molecular denominators, including the expression of the transcription factor Prox1. However, many of the molecular mechanisms involved in the acquisition and maintenance of lymphatic identity remains to be uncovered. The aim of my thesis is to investigate how transcription factors and chromatin re-arrangements are involved in the acquisition and maintenance of lymphatic identity. In Paper I, the role of the transcription factor mafbb and its ability to partially compensate for the loss of its paralog mafba is investigated, leading to the description of topologically distinct requitements for LECs development in zebrafish. In Paper II, an evolutionary conserved prox1a enhancer is shown to be necessary for correct lymphangiogenesis in mouse, and its ablation is shown to cause the transition of LECs to hematopoietic-like cells. In Paper III, the effects of partial and total prox1 loss in zebrafish are investigated from a transcriptomic and epigenetic perspective, revealing an important role for prox1 in establishing LEC identity by suppressing genes associated with VEC fate. In Paper IV, the cis-regulatory landscape of prox1a is characterised identifying a number of lymphatic enhancers driving expression in either all LECs in the embryo or in anatomically distinct subsets of lymphatic vasculature. Finally, in Paper V, the chromatin organisation signature of LECs is explored, finding new candidate genes and enhancers active in the lymphatic endothelium. In summary, my doctoral studies investigated the different levels of regulation of cell identity involved in LECs differentiation. My work focused on chromatin organisation, enhancer activity, transcription factors and differential gene expressi

Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right noses, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, i.e., cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

A length–frequency sample (n = 295) from a fossil population of the Ordovician trilobite Triarthrus eatoni Hall, 1838, assembled and analyzed by J. L. Cisne in 1973 is here reexamined using methods of length–frequency analysis commonly used in fishery science and marine biology. Theoretical considerations and the empirical data at hand suggest that the growth of T. eatoni was not “linear,” but asymptotic, as is the growth of most Recent marine invertebrates. The parameters of the von Bertalanffy growth function (L∞ = 41 mm, K = 0.29 yr−1) suggest that T. eatoni, which apparently lived in a challenging environment, grew somewhat more slowly than the extant marine isopod Ceratoserolis trilobitoides (Eights, 1833), used here as Recent analogue to T. eatoni. This trilobite probably lived up to 10 years, rather than the suggested 4 years, and its mortality rate was 15%–20% per year rather than 30%–40% per year. These represent the first estimates of trilobite absolute growth characteristics using methods known to accurately model growth in extant water-breathing ectotherms. These provide a baseline for trilobite growth that can be used to make inferences about growth in other species. The approach used here may also be applied to other trilobites for which suitable length–frequency data exist.

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain -contain-ing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hor-mone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remark-ably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in medi-ating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

During development, the lymphatic vasculature forms as a second, new vascular network derived from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is the first step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet downstream mechanisms remain to be elucidated. We present a single cell transcriptomic atlas of lymphangiogenesis in zebrafish revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a VEC-LEC fate reversion during differentiation. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new transcriptional targets and regulatory regions of the genome downstream of Prox1 in LEC maintenance, as well as showing that Prox1 specifies LEC fate primarily by limiting blood vascular and hematopoietic fate. This extensive single cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development1-3. Here we describe a novel enhancer element that is important for regulating the expression of Prox1 in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1 expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.

Background Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane-bound receptors such as Uncoordianted-5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. Results Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated-5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. Conclusion Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages.

Drosophila insulators were the first DNA elements found to regulate gene expression by delimiting chromatin contacts. We still do not know how many of them exist and what impact they have on the Drosophila genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains uncertain. To bridge these gaps, we mapped chromatin contacts in Drosophila cells lacking the key insulator proteins CTCF and Cp190. With this approach, we found hundreds of insulator elements. Their study indicates that Drosophila insulators play a minor role in the overall genome folding but affect chromatin contacts locally at many loci. Our observations argue that Cp190 promotes cobinding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. Our insulator catalog provides an important resource to study mechanisms of genome folding.

Endocrine-disrupting chemicals (EDCs) are known to cause endocrine disruption (ED), developmental neurotoxicity (DNT), infertility and impaired embryo development. EDCs do therefore impose a threat to humans, wildlife and the environment. The present study investigated the effects of the reference compounds dihydrotestosterone, estradiol, benzo(a)pyrene, rosiglitazone, as well as the EDCs bisphenol F and perfluorooctanesulfonic acid at various concentrations on zebrafish embryos. The scientific questions of the study were to investigate how these environmental pollutants impact the development of zebrafish, what their molecular mechanisms are and what the link between ED and DNT is. Zebrafish embryos were exposed for 5 days to the compounds and various parameters on development were collected at different time points. The expression of 41 genes (qPCR) related to ED and DNT, and the levels of 23 steroids (LC-MS/MS) were determined. Gene correlations were determined with Pearson’s correlation test and paired t-tests were used to determine significantly altered gene activities. The significant gene expression changes were further related to the pathways of steroids in order to connect how gene activity impacted steroid levels. Exposure to estradiol, dihydrotestosterone and bisphenol F induced cyp19a1b expression which can affect personality traits. Perfluorooctanesulfonic acid interferes with thyroid hormone transport by binding to TTR causing profound effects on neurodevelopmental processes and cognitive functions. The compounds influenced genes that can disrupt endocrine systems which can cause neurodevelopmental impairments.

Selection pressure is a constant force in evolution, pushing birds and their wings towards an optimal shape and structure, were increasing performance, and minimizing the costs is central. But even though the science of aerodynamics can provide calculations of the optimal construction for the wing in different situations this rarely directly correspond to what is observed in nature. Since the optima are not the same for all birds due to different specifications and ecology this optimum becomes harder to determine and different functions can even have different optima, resulting in selection conflict. In the genius of birds there is an immense variation between species and their wings in everything from size, shape, and function. The aim of this study is to investigate how wing morphology over a large phylogeny of bird species correlates to migration and habitat/ecology. Many studies have been done focusing on the effect of migration on the wing morphology, and some have been done focusing on other parameters such as display or daily usage. But by including the bird’s ecology and habitat related information with migration and morphology and looking at the selection from a broad perspective, can we uncover something more? The morphology of the wing cannot provide a perfect optimum for all circumstances since they require different specifications. What then, has the largest impact on the wing’s morphology? And does the relative length of the tail provide any correlations with its habitat and performance? 1185 birds of 137 species were caught at Ottenby, Öland and information on age, weight, sex, and tail length was collected for each individual bird. Photographs were taken of the back of the bird with the left wing outstretched 90 degrees from the body and analyzed in ImageJ to calculate aspect ratio and wing loading. Data on migration distance, foraging behavior, diet, and habitat density was then added for each of the species. Mean values of all parameters was cal

Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdha and Ncam2, encode cell adhesion molecules. In Hnrnpab knockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3′ UTR of Pcdha leads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.

The mammalian brain comprises a large number and diversity of neurons and non-neural cells. Most of these cell types derive from a single sheet of neuroepithelium (NE) that contains a limited pool of neural stem cells (NSCs). NSCs in the developing brain are under complex regulation to achieve maximum daughter cell type diversity. In this thesis, we investigate how NSCs are regulated spatially and temporally by a critical transcription factor (TF), PRDM16, in the developing mouse brain. In the first paper, we study the mechanisms that guide specification of an essential brain structure, the Choroid plexus (ChP). The ChP localizes at the brain dorsal midline, and the cell identity of the ChP epithelium depends on the induction by Bone morphogenetic protein (BMP) morphogens. NSCs at the presumptive ChP exit cell cycle, lose neural potential and subsequently obtain the ChP epithelial cell identity. We found that PRDM16 is required for ChP epithelium specification, as proliferative NSCs failed to acquire quiescence in the Prdm16 mutant brain. To investigate the molecular mechanisms controlling NSC proliferation and quiescence, we determined transcriptional states of proliferative versus quiescent NSCs and profiled genomic binding patterns for the BMP signaling effectors SMAD4 and pSMAD1/5/8. We found that BMP signaling suppresses cell proliferation genes in NSCs, and this activity requires PRDM16 co-binding to the enhancers of these target genes. One of the PRDM16-SMAD co-regulated genes is the Wnt pathway ligand Wnt7b. Using a single-cell-resolution fluorescent in situ technique, we confirm that Wnt7band Wnt activity are upregulated in Prdm16 mutant ChP epithelial cells and that the increased Wnt activity correlates with NSC over-proliferation. Together, our work reveals the mechanism by which BMP signaling induces NSC quiescence and defines a regulatory circuit composed of BMP, Wnt signaling and PRDM16 in the specification of the ChP epithelium. NSCs sequentiall

The degree to which developmental biases affect trait evolution is subject to much debate. Here, we first quantify fluctuating asymmetry as a measure of developmental variability, i.e., the propensity of developmental systems to create some phenotypic variants more often than others, and show that it predicts phenotypic and standing genetic variation as well as deep macroevolutionary divergence in wing shape in sepsid flies. Comparing our data to the findings of a previous study demonstrates that developmental variability in the sepsid fly Sepsis punctum strongly aligns with mutational, standing genetic, and macroevolutionary variation in the Drosophilidae--a group that diverged from the sepsid lineage ca. 64 My ago. We also find that developmental bias in S. punctum wing shape aligns with the effects of allometry, but less so with putatively adaptive thermal plasticity and population differentiation along latitude. Our findings demonstrate that developmental bias in fly wings predicts evolvability and macroevolutionary trajectories on a much greater scale than previously appreciated but also suggest that causal explanations for such alignments may go beyond simple constraint hypotheses.

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhanc

During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demon-strated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the het-erogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical loca-tions, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural ret-inoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 & mu;g flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Mullerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Mullerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 & mu;g/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Mullerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

Conserved genomic sequences disrupted in humans may underlie uniquely human phenotypic traits. We identified and characterized 10,032 human-specific conserved deletions (hCONDELs). These short (average 2.56 base pairs) deletions are enriched for human brain functions across genetic, epigenomic, and transcriptomic datasets. Using massively parallel reporter assays in six cell types, we discovered 800 hCONDELs conferring significant differences in regulatory activity, half of which enhance rather than disrupt regulatory function. We highlight several hCONDELs with putative human-specific effects on brain development, including HDAC5, CPEB4, and PPP2CA. Reverting an hCONDEL to the ancestral sequence alters the expression of LOXL2 and developmental genes involved in myelination and synaptic function. Our data provide a rich resource to investigate the evolutionary mechanisms driving new traits in humans and other species.