Towards a quantitative understanding of chytrid cellular development

The current understanding of fungal developmental biology is almost entirely derived from dikaryan hyphae and yeast, neglecting the diversity and prevalence of other major fungal lineages. The ‘chytrids’ (phylum Chytridiomycota) are a predominantly unicellular group of fungi pervasive throughout aquatic environments. As prominent saprotrophs, parasites, and pathogens, chytrids are integral to biogeochemical cycling in aquatic ecosystems. Additionally, they retain ancestral cellular characteristics present in the last common ancestor of branching (i.e hyphal and rhizoidal) fungi, making chytrids powerful models to study the evolution of fungal-specific innovations. Despite the evolutionary and ecological importance of chytrids, their basic cell biology and development remains poorly resolved. This fundamental gap must be closed if a proper appreciation for chytrids is to be achieved. To address this, this thesis aimed to present a quantitative picture of chytrid development and identify shifts in biology across the life cycle. Using Rhizoclosmatium globosum as a model species for chytrid biology, this thesis set out to 1) establish an experimental toolkit for chytrid developmental biology, 2) identify the cellular and molecular drivers of the chytrid life cycle, and 3) quantify the development of the rhizoid, all of which were achieved. The combination of 3D electron microscopy reconstructions and transcriptomic profiling achieved a holistic developmental atlas for the chytrid life cycle shedding light on lipid metabolism, vacuolisation, and zoospore development in R. globosum, and revealing the chytrid apophysis to be a functionally delineated structure governed by intracellular trafficking. Live-cell confocal microscopy and reconstruction of developing rhizoids demonstrated that rhizoid growth was analogous to hyphal morphogenesis, adaptive to resource availability, and capable of spatiotemporal functional differentiation. Overall, this thesis achieved a quantitative characterisation of chytrid development, uncovered previously hidden cellular complexities important for ecological and evolutionary chytrid biology, and will provide a solid foundation for future investigations into chytrid biology

Chytrid rhizoid morphogenesis resembles hyphal development in multicellular fungi and is adaptive to resource availability

Key to the ecological prominence of fungi is their distinctive cell biology, our understanding of which has been principally based on dikaryan hyphal and yeast forms. The early-diverging Chytridiomycota (chytrids) are ecologically important and a significant component of fungal diversity, yet their cell biology remains poorly understood. Unlike dikaryan hyphae, chytrids typically attach to substrates and feed osmotrophically via anucleate rhizoids. The evolution of fungal hyphae appears to have occurred from rhizoid-bearing lineages and it has been hypothesized that a rhizoid-like structure was the precursor to multicellular hyphae. Here, we show in a unicellular chytrid, Rhizoclosmatium globosum, that rhizoid development exhibits striking similarities with dikaryan hyphae and is adaptive to resource availability. Rhizoid morphogenesis exhibits analogous patterns to hyphal growth and is controlled by β-glucan-dependent cell wall synthesis and actin polymerization. Chytrid rhizoids growing from individual cells also demonstrate adaptive morphological plasticity in response to resource availability, developing a searching phenotype when carbon starved and spatial differentiation when interacting with particulate organic matter. We demonstrate that the adaptive cell biology and associated developmental plasticity considered characteristic of hyphal fungi are shared more widely across the Kingdom Fungi and therefore could be conserved from their most recent common ancestor

The architecture of cell differentiation in choanoflagellates and sponge choanocytes.

Although collar cells are conserved across animals and their closest relatives, the choanoflagellates, little is known about their ancestry, their subcellular architecture, or how they differentiate. The choanoflagellate Salpingoeca rosetta expresses genes necessary for animal development and can alternate between unicellular and multicellular states, making it a powerful model for investigating the origin of animal multicellularity and mechanisms underlying cell differentiation. To compare the subcellular architecture of solitary collar cells in S. rosetta with that of multicellular 'rosette' colonies and collar cells in sponges, we reconstructed entire cells in 3D through transmission electron microscopy on serial ultrathin sections. Structural analysis of our 3D reconstructions revealed important differences between single and colonial choanoflagellate cells, with colonial cells exhibiting a more amoeboid morphology consistent with higher levels of macropinocytotic activity. Comparison of multiple reconstructed rosette colonies highlighted the variable nature of cell sizes, cell-cell contact networks, and colony arrangement. Importantly, we uncovered the presence of elongated cells in some rosette colonies that likely represent a distinct and differentiated cell type, pointing toward spatial cell differentiation. Intercellular bridges within choanoflagellate colonies displayed a variety of morphologies and connected some but not all neighbouring cells. Reconstruction of sponge choanocytes revealed ultrastructural commonalities but also differences in major organelle composition in comparison to choanoflagellates. Together, our comparative reconstructions uncover the architecture of cell differentiation in choanoflagellates and sponge choanocytes and constitute an important step in reconstructing the cell biology of the last common ancestor of animals

Multiscale correlative imaging of horse and zebra placental villi

International audienceDespite having a single evolutionary origin, the mammalian placenta exhibits wide interspecific morphological and structural variation. Equine (horses and their kin) placentas display branching villi which sit in apposition with maternal tissue and represent the site of fetomaternal nutrient and waste exchange. Three-dimensional imaging techniques have recently identified and quantified novel structures in human placental villi, however similar tools have yet to be broadly applied to other species. Such approaches have the potential to both expand our understanding of comparative placentation and better resolve the structural composition of the studied taxa. Using scanning electron microscopy (SBF-SEM) of horse and zebra placenta, we demonstrated the presence of stromal macrovesicles previously only observed in human placental villi. Here, we also present a workflow for correlative threedimensional imaging of equine placental villi by combining x-ray microtomography (microCT) and SBF-SEM. This allows calculation of the total surface area of the equine placenta including microvilli. Through this workflow, we quantify the villus structure across multiple orders of magnitude in horse and zebra placentas. These morphometric data, including volume, surface area, and branching angle, help us to better resolve equine placental organization and contribute towards a holistic understanding of equine placental function

The architecture of cell differentiation in choanoflagellates and sponge choanocytes

Although collar cells are conserved across animals and their closest relatives, the choanoflagellates, little is known about their ancestry, their subcellular architecture, or how they differentiate. The choanoflagellate Salpingoeca rosetta expresses genes necessary for animal development and can alternate between unicellular and multicellular states, making it a powerful model for investigating the origin of animal multicellularity and mechanisms underlying cell differentiation. To compare the subcellular architecture of solitary collar cells in S. rosetta with that of multicellular ‘rosette’ colonies and collar cells in sponges, we reconstructed entire cells in 3D through transmission electron microscopy on serial ultrathin sections. Structural analysis of our 3D reconstructions revealed important differences between single and colonial choanoflagellate cells, with colonial cells exhibiting a more amoeboid morphology consistent with higher levels of macropinocytotic activity. Comparison of multiple reconstructed rosette colonies highlighted the variable nature of cell sizes, cell–cell contact networks, and colony arrangement. Importantly, we uncovered the presence of elongated cells in some rosette colonies that likely represent a distinct and differentiated cell type, pointing toward spatial cell differentiation. Intercellular bridges within choanoflagellate colonies displayed a variety of morphologies and connected some but not all neighbouring cells. Reconstruction of sponge choanocytes revealed ultrastructural commonalities but also differences in major organelle composition in comparison to choanoflagellates. Together, our comparative reconstructions uncover the architecture of cell differentiation in choanoflagellates and sponge choanocytes and constitute an important step in reconstructing the cell biology of the last common ancestor of animals

Placental evolution from a three-dimensional and multiscale structural perspective

The placenta mediates physiological exchange between the mother and fetus. In placental mammals, all placentas are descended from a single common ancestor and functions are conserved across species; however, the placenta exhibits radical structural diversity. The selective pressures behind this structural diversity are poorly understood. Traditionally, placental structures have largely been investigated by grouping them into qualitative categories. Assessing the placenta on this basis could be problematic when inferring the relative 'efficiency' of a placental configuration to transfer nutrients from mother to fetus. We argue that only by considering placentas as 3D biological structures, integrated across scales, can the evolutionary questions behind their enormous structural diversity be quantitatively determined. We review the current state of placental evolution from a structural perspective, detail where 3D imaging and computational modelling have been used to gain insight into placental function, and outline an experimental roadmap to answer evolutionary questions from a multiscale 3D structural perspective. Our approach aims to shed light on placental evolution, and can be transferred to evolutionary investigations in any organ system.</p

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue

International audienceThree-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities

3D structural quantification of equine placental villi through multiscale correlative imaging: -

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