An Amphioxus Gli Gene Reveals Conservation of Midline Patterning and the Evolution of Hedgehog Signalling Diversity in Chordates

Background. Hedgehog signalling, interpreted in receiving cells by Gli transcription factors, plays a central role in the development of vertebrate and Drosphila embryos. Many aspects of the signalling pathway are conserved between these lineages, however vertebrates have diverged in at least one key aspect: they have evolved multiple Gli genes encoding functionally-distinct proteins, increasing the complexity of the hedgehog-dependent transcriptional response. Amphioxus is one of the closest living relatives of the vertebrates, having split from the vertebrate lineage prior to the widespread gene duplication prominent in early vertebrate evolution. Principal findings. We show that amphioxus has a single Gli gene, which is deployed in tissues adjacent to sources of hedgehog signalling derived from the midline and anterior endoderm. This shows the duplication and divergence of the Gli family, and hence the origin of vertebrate Gli functional diversity, was specific to the vertebrate lineage. However we also show that the single amphioxus Gli gene produces two distinct transcripts encoding different proteins. We utilise three tests of Gli function to examine the transcription regulatory capacities of these different proteins, demonstrating one has activating activity similar to Gli2, while the other acts as a weak repressor, similar to Gli3. Conclusions. These data show that the vertebrates and amphioxus have evolved functionally-similar repertoires of Gli proteins using parallel molecular routes; vertebrates via gene duplication and divergence, and amphioxus via alternate splicing of a single gene. Our results demonstrate that similar functional complexity of intercellular signalling can be achieved via different evolutionary pathways

Protochordate Zic genes define primitive somite compartments and highlight molecular changes underlying neural crest evolution

The vertebrate Zic gene family encodes C2H2 zinc finger transcription factors closely related to the Gli proteins. Zic genes are expressed in multiple areas of developing vertebrate embryos, including the dorsal neural tube where they act as potent neural crest inducers. Here we describe the characterization of a Zic ortholog from the amphioxus Branchiostoma floridae and further describe the expression of a Zic ortholog from the ascidian Ciona intestinalis. Molecular phylogenetic analysis and sequence comparisons suggest the gene duplications that formed the vertebrate Zic family were specific to the vertebrate lineage. In Ciona maternal CiZic/Ci-macho1 transcripts are localized during cleavage stages by asymmetric cell division, whereas zygotic expression by neural plate cells commences during neurulation. The amphioxus Zic ortholog AmphiZic is expressed in dorsal mesoderm and ectoderm during gastrulation, before being eliminated first from midline cells and then from all neurectoderm during neurulation. After neurulation, expression is reactivated in the dorsal neural tube and dorsolateral somite. Comparison of CiZic and AmphiZic expression with vertebrate Zic expression leads to two main conclusions. First, Zic expression allows us to define homologous compartments between vertebrate and amphioxus somites, showing primitive subdivision of vertebrate segmented mesoderm. Second, we show that neural Zic expression is a chordate synapomorphy, whereas the precise pattern of neural expression has evolved differently on the different chordate lineages. Based on these observations we suggest that a change in Zic regulation, specifically the evolution of a dorsal neural expression domain in vertebrate neurulae, was an important step in the evolution of the neural crest

Scanning electron microscopy and synchrotron radiation x-ray tomographic microscopy of 330 million year old charcoalified seed fern fertile organs

Abundant charcoalified seed fern (pteridosperm) pollen organs and ovules have been recovered from Late Viséan (Mississippian 330 Ma) limestones from Kingswood, Fife, Scotland. To overcome limitations of data collection from these tiny, sometimes unique, fossils, we have combined low vacuum scanning electron microscopy on uncoated specimens with backscatter detector and synchrotron radiation X-ray tomographic microscopy utilizing the Materials Science and TOMCAT beamlines at the Swiss Light Source of the Paul Scherrer Institut. In combination these techniques improve upon traditional cellulose acetate peel sectioning because they enable study of external morphology and internal anatomy in multiple planes of section on a single specimen that is retained intact. The pollen organ Melissiotheca shows a basal parenchymatous cushion bearing more than 100 sporangia on the distal face. Digital sections show the occurrence of pollen in some sporangia. The described ovule is new and has eight integumentary lobes that are covered in spirally arranged glandular hairs. Virtual longitudinal sections reveal the lobes are free above the pollen chamber. Results are applied in taxonomy and will subsequently contribute to our understanding of the former diversity and evolution of ovules, seeds, and pollen organs in the seed ferns, the first seed-bearing plants to conquer the land

Deciphering the fossil record of early bilaterian embryonic development in light of experimental taphonomy

Experimental analyses of decay in a tunicate deuterostome and three lophotrochozoans indicate that the controls on decay and preservation of embryos, identified previously based on echinoids, are more generally applicable. Four stages of decay are identified regardless of the environment of death and decay. Embryos decay rapidly in oxic and anoxic conditions, although the gross morphology of embryos is maintained for longer under anoxic conditions. Under anoxic reducing conditions, the gross morphology of the embryos is maintained for the longest period of time, compatible with the timescale required for bacterially mediated mineralization of soft tissues. All four stages of decay were encountered under all environmental conditions, matching the spectrum of preservational qualities encountered in all fossil embryo assemblages. The preservation potential of embryos of deuterostomes and lophotrochozoans is at odds with the lack of such embryos in the fossil record. Rather, the fossil record of embryos, as sparse as it is, is dominated by forms interpreted as ecdysozoans, cnidarians, and stem-metazoans. The dearth of deuterostome and lophotrochozoan embryos may be explained by the fact that ecdysozoans, at least, tend to deposit their eggs in the sediment rather than through broadcast spawning. However, fossil embryos remain very rare and the main controlling factor on their fossilization may be the unique conspiracy of environmental conditions at a couple of sites. The preponderance of fossilized embryos of direct developers should not be used in evidence against the existence of indirect development at this time in animal evolutionary history

Cellular and Subcellular Structure of Neoproterozoic Animal Embryos

Stereoblastic embryos from the Doushantuo Formation of China exhibit occasional asynchronous cell division, with diminishing blastomere volume as cleavage proceeded. Asynchronous cell division is common in modern embryos, implying that sophisticated mechanisms for differential cell division timing and embryonic cell lineage differentiation evolved before 551 million years ago. Subcellular structures akin to organelles, coated yolk granules, or lipid vesicles occur in these embryos. Paired reniform structures within embryo cells may represent fossil evidence of cells about to undergo division. Embryos exhibit no evidence of epithelial organization, even in embryos composed of ?1000 cells. Many of these features are compatible with metazoans, but the absence of epithelialization is consistent only with a stem-metazoan affinity for Doushantuo embryos

Quantitative microCT imaging of a whole equine placenta and its blood vessel network

Placental structure is linked to function across morphological scales. In the placenta, changes to gross anatomy, such as surface area, volume, or blood vessel arrangement, are associated with suboptimal physiological outcomes. However, quantifying each of these metrics requires different laborious semi-quantitative methods. Here, we demonstrate how, with minimal sample preparation, whole-organ computed microtomography (microCT) can be used to calculate gross morphometry of the equine placenta and a range of additional metrics, including branching morphometry of placental vasculature, non-destructively from a single dataset. Our approach can be applied to quantify the gross structure of any large mammalian placenta.</p

Embryo fossilization is a biological process mediated by microbial biofilms

Fossilized embryos with extraordinary cellular preservation appear in the Late Neoproterozoic and Cambrian, coincident with the appearance of animal body fossils. It has been hypothesized that microbial processes are responsible for preservation and mineralization of organic tissues. However, the actions of microbes in preservation of embryos have not been demonstrated experimentally. Here, we show that bacterial biofilms assemble rapidly in dead marine embryos and form remarkable pseudomorphs in which the bacterial biofilm replaces and exquisitely models details of cellular organization and structure. The experimental model was the decay of cleavage stage embryos similar in size and morphology to fossil embryos. The data show that embryo preservation takes place in 3 distinct steps: (i) blockage of autolysis by reducing or anaerobic conditions, (ii) rapid formation of microbial biofilms that consume the embryo but form a replica that retains cell organization and morphology, and (iii) bacterially catalyzed mineralization. Major bacterial taxa in embryo decay biofilms were identified by using 16S rDNA sequencing. Decay processes were similar in different taphonomic conditions, but the composition of bacterial populations depended on specific conditions. Experimental taphonomy generates preservation states similar to those in fossil embryos. The data show how fossilization of soft tissues in sediments can be mediated by bacterial replacement and mineralization, providing a foundation for experimentally creating biofilms from defined microbial species to model fossilization as a biological process

Reverse roll-coating flow: a computational investigation towards high-speed defect free coating

A finite element Taylor–Galerkin pressure-correction algorithm is employed to simulate a high-speed defect-free roll-coating flow, which substantiates a coating process with a free meniscus surface. Findings are applicable across a wide range of coating sectors in optimisation of coating performance, which targets adaptive and intelligent process control. Industrially, there is a major drive towards using new material products and raising coating line-speeds, to address increased efficiency and productivity. This study has sought to attack these issues by developing an effective predictive toolset for high-speed defect-free coatings. Here, time-stepping/finite element methods are deployed to model this free-surface problem that involves the transfer of a coating fluid from a roller to a substrate (of prescribed wet-film thickness). This procedure is used in conjunction with a set of constitutive equations capable of describing the relevant fluid-film rheology in appropriate detail. Quantities of pressure, lift and drag have been calculated streamwise across the flow domain, and streamline patterns reveal a large recirculating vortex around the meniscus region. Such pressure distributions across the domain display a positive peak which decreases as nip-gap size increases. Further analysis has been conducted, mimicking the presence of a wetting line, whilst varying boundary conditions at the nip. Observation has shown that such inclusion would serve as a relief mechanism to the positive peak pressures generated around the nip zone. Here, through an elasto-hydrodynamic formulation, the elastic deformation of a rubber roll cover (elastomer) has also been introduced, which offers fresh insight into the process with respect to nip-flow behaviour, and allows for the analysis of both positive and negative nip-gaps