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

On the 3D nature of the Magpie (Aves: Pica pica) functional hindlimb anatomy during the take-off jump

Take-off is a critical phase of flight, and many birds jump to take to the air. Although the actuation of the hindlimb in terrestrial birds is not limited to the sagittal plane, and considerable non-sagittal plane motion has been observed during take-off jumps, how the spatial arrangement of hindlimb muscles in flying birds facilitates such jumps has received little attention. This study aims to ascertain the 3D hip muscle function in the magpie (Pica pica), a bird known to jump to take-off. A musculoskeletal model of the magpie hindlimb was developed using μCT scans (isotropic resolution of 18.2 μm) to derive bone surfaces, while the 3D muscle path definition was further informed by the literature. Function was robustly characterized by determining the 3D moment-generating capacity of 14 hip muscles over the functional joint range of motion during a take-off leap considering variations across the attachment areas and uncertainty in dynamic muscle geometry. Ratios of peak flexion-extension (FE) to internal-external rotation (IER) and abduction-adduction (ABD) moment-generating capacity were indicators of muscle function. Analyses of 972 variations of the 3D muscle paths showed that 11 of 14 muscles can act as either flexor or extensor, while all 14 muscles demonstrated the capacity to act as internal or external rotators of the hip with the mean ratios of peak FE to IER and ABD moment-generating capacity were 0.89 and 0.31, respectively. Moment-generating capacity in IER approaching levels in the FE moment-generating capacity determined here underline that the avian hip muscle function is not limited to the sagittal plane. Together with previous findings on the 3D nature of hindlimb kinematics, our results suggest that musculoskeletal models to develop a more detailed understanding of how birds orchestrate the use of muscles during a take-off jump cannot be restricted to the sagittal plane

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

Illumination of x-rays: the usual lights exposed

Aims: Faced with pressure of work and limited resource, doctors frequently use the nearest available light to examine x-ray films. The aim of this study was to examine the relative intensities of hospital light sources, and to compare these with the light intensity guidelines (1500 to 3000 candelas) of the British Institute of Radiology. Methods: The relative intensities of seven hospital light sources were examined using a standard light meter at a constant 30 cm from the source. A control group of 10 individual consultant's x-ray viewing boxes were compared with six other ward based light sources. Results: Only two light sources approached the British Institute of Radiology light intensity x-ray viewing criteria: the x-ray viewing boxes of consultant radiologists with a median light intensity of 3503 candelas (χ(2)=13.3, df 1; p=0.0001), and daylight from north facing windows with a median of 1464 candelas when overcast (χ(2)=8.571, df 1; p=0.003) and 4669 candelas in sunshine (χ(2)= 6.364, df 1; p=0.0001). Conclusion: Few hospital light sources met the British Institute of Radiology guidelines. The long held high regard of artists for northern light appears justified even in the environment of a British district general hospital