Oliver Tills

Studying the altered timing of physiological events during development: it's about time…or is it?

The investigation of the altered timing of developmental events is key to understanding evolution. Most empirical investigations of event timing are biased towards studying morphological variation. Recent reviews, however, have attempted to marshal the evidence for the importance of altered timing of physiological events, focusing on such timing shifts between species (physiological heterochrony) and within species (physiological heterokairy). Here we update these reviews. We firstly take a comparative developmental physiology approach to explore how recent studies have furthered our understanding of the links between physiological event timing shifts at different levels of biological organisation (i.e. individual, population and species). The alternative strategy of concentrating effort on one model system is then considered, in particular focussing on substantial recent advances in our understanding of fetal haemoglobin expression in humans. We conclude that, while the fetal haemoglobin model may be appropriate as a model for some questions, it can never be the model study system. We also discuss the different quantitative analyses available for investigating event timing alterations. We consider the efficacy of the terms heterochrony and heterokairy.

A genetic basis for intraspecific differences in developmental timing

Heterochrony, altered developmental timing between ancestors and their descendents, has been proposed as a pervasive evolutionary feature and recent analytical approaches have confirmed its existence as an evolutionary pattern. Yet, the mechanistic basis for heterochrony remains unclear and, in particular, whether intraspecific variation in the timing of developmental events generates, or has the potential to generate, future between‐species differences. Here we make a key step in linking heterochrony at the inter‐ and intraspecific level by reporting an association between interindividual variation in both the absolute and relative timing (position within the sequence of developmental events) of key embryonic developmental events and genetic distance for the pond snail, Radix balthica. We report significant differences in the genetic distance of individuals exhibiting different levels of dissimilarity in their absolute and relative timing of developmental events such as spinning activity, eyespot formation, heart ontogeny, and hatching. This relationship between genetic and developmental dissimilarity is consistent with there being a genetic basis for variation in developmental timing and so suggests that intraspecific heterochrony could provide the raw material for natural selection to produce speciation.

Parent–offspring similarity in the timing of developmental events: an origin of heterochrony?

Understanding the link between ontogeny (development) and phylogeny (evolution) remains a key aim of biology. Heterochrony, the altered timing of developmental events between ancestors and descendants, could be such a link although the processes responsible for producing heterochrony, widely viewed as an interspecific phenomenon, are still unclear. However, intraspecific variation in developmental event timing, if heritable, could provide the raw material from which heterochronies originate. To date, however, heritable developmental event timing has not been demonstrated, although recent work did suggest a genetic basis for intraspecific differences in event timing in the embryonic development of the pond snail, Radix balthica. Consequently, here we used high-resolution (temporal and spatial) imaging of the entire embryonic development of R. balthica to perform a parent–offspring comparison of the timing of twelve, physiological and morphological developmental events. Between-parent differences in the timing of all events were good predictors of such timing differences between their offspring, and heritability was demonstrated for two of these events (foot attachment and crawling). Such heritable intraspecific variation in developmental event timing could be the raw material for speciation events, providing a fundamental link between ontogeny and phylogeny, via heterochrony.

A novel application of motion analysis for detecting stress responses in embryos at different stages of development

BackgroundMotion analysis is one of the tools available to biologists to extract biologically relevant information from image datasets and has been applied to a diverse range of organisms. The application of motion analysis during early development presents a challenge, as embryos often exhibit complex, subtle and diverse movement patterns. A method of motion analysis able to holistically quantify complex embryonic movements could be a powerful tool for fields such as toxicology and developmental biology to investigate whole organism stress responses. Here we assessed whether motion analysis could be used to distinguish the effects of stressors on three early developmental stages of each of three species: (i) the zebrafish Danio rerio (stages 19 h, 21.5 h and 33 h exposed to 1.5% ethanol and a salinity of 5); (ii) the African clawed toad Xenopus laevis (stages 24, 32 and 34 exposed to a salinity of 20); and iii) the pond snail Radix balthica (stages E3, E4, E6, E9 and E11 exposed to salinities of 5, 10 and 15). Image sequences were analysed using Sparse Optic Flow and the resultant frame-to-frame motion parameters were analysed using Discrete Fourier Transform to quantify the distribution of energy at different frequencies. This spectral frequency dataset was then used to construct a Bray-Curtis similarity matrix and differences in movement patterns between embryos in this matrix were tested for using ANOSIM.ResultsSpectral frequency analysis of these motion parameters was able to distinguish stage-specific effects of environmental stressors in most cases, including Xenopus laevis at stages 24, 32 and 34 exposed to a salinity of 20, Danio rerio at 33 hpf exposed to 1.5% ethanol, and Radix balthica at stages E4, E9 and E11 exposed to salinities of 5, 10 and 15. This technique was better able to distinguish embryos exposed to stressors than analysis of manual quantification of movement and within species distinguished most of the developmental stages studied in the control treatments.ConclusionThis innovative use of motion analysis incorporates data quantifying embryonic movements at a range of frequencies and so provides an holistic analysis of an embryo’s movement patterns. This technique has potential applications for quantifying embryonic responses to environmental stressors such as exposure to pharmaceuticals or pollutants, and also as an automated tool for developmental staging of embryos.

An Annotated Draft Genome for Radix auricularia (Gastropoda, Mollusca)

Molluscs are the second most species-rich phylum in the animal kingdom, yet only 11 genomes of this group have been published so far. Here, we present the draft genome sequence of the pulmonate freshwater snail Radix auricularia. Six whole genome shotgun libraries with different layouts were sequenced. The resulting assembly comprises 4,823 scaffolds with a cumulative length of 910 Mb and an overall read coverage of 72×. The assembly contains 94.6% of a metazoan core gene collection, indicating an almost complete coverage of the coding fraction. The discrepancy of ∼690 Mb compared with the estimated genome size of R. auricularia (1.6 Gb) results from a high repeat content of 70% mainly comprising DNA transposons. The annotation of 17,338 protein coding genes was supported by the use of publicly available transcriptome data. This draft will serve as starting point for further genomic and population genetic research in this scientifically important phylum.

Embryonic transcriptome of the brackishwater amphipod Gammarus chevreuxi

Environmental change can dramatically alter the development of aquatic organisms. While the effect of such change on physiological and morphological ontogenies is becoming clearer, the molecular mechanisms underpinning them are largely unexplored. Characterizing these mechanisms is often limited by the lack of molecular resources. We have applied Illumina HiSeq sequencing to RNA isolated from different developmental stages of the brackishwater amphipod Gammarus chevreuxi. Over 52.6M paired-end reads were assembled de novo into 172,081 contigs, representing 118,812 potential genes. The assembly generated constitutes a reference embryonic transcriptome for an ecologically-important aquatic shredder species. This resource will contribute to our understanding of the mechanisms underpinning the development of physiological function through functional, comparative and quantitative expression studies. It will also allow the identification of candidate biomarkers for assessing the impact of environmental stressors in estuarine systems.

Combining Motion Analysis and Microfluidics – A Novel Approach for Detecting Whole-Animal Responses to Test Substances

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.

An embryonic transcriptome of the pulmonate snail Radix balthica.

The pond snail, Radix balthica (Linnaeus 1758), is an emerging model species within ecological developmental biology. While its development has been characterised in detail, genomic resources for embryonic stages are lacking. We applied Illumina MiSeq RNA-seq to RNA isolated from pools of embryos at two points during development. Embryos were cultured in either the presence or absence of predator kariomones to increase the diversity of the transcripts assembled. Sequencing produced 47.2M paired-end reads, assembled into 54,360 contigs of which 73% were successfully annotated. This transcriptome provides an invaluable resource to build a mechanistic understanding of developmental plasticity.

Short-term acclimation in adults does not predict offspring acclimation potential to hypoxia.

The prevalence of hypoxic areas in coastal waters is predicted to increase and lead to reduced biodiversity. While the adult stages of many estuarine invertebrates can cope with short periods of hypoxia, it remains unclear whether that ability is present if animals are bred and reared under chronic hypoxia. We firstly investigated the effect of moderate, short-term environmental hypoxia (40% air saturation for one week) on metabolic performance in adults of an estuarine amphipod, and the fitness consequences of prolonged exposure. We then reared the offspring of hypoxia-exposed parents under hypoxia, and assessed their oxyregulatory ability under declining oxygen tensions as juveniles and adults. Adults from the parental generation were able to acclimate their metabolism to hypoxia after one week, employing mechanisms typically associated with prolonged exposure. Their progeny, however, did not develop the adult pattern of respiratory regulation when reared under chronic hypoxia, but instead exhibited a poorer oxyregulatory ability than their parents. We conclude that species apparently hypoxia-tolerant when tested in short-term experiments, could be physiologically compromised as adults if they develop under hypoxia. Consequently, we propose that the increased prevalence of hypoxia in coastal regions will have marked effects in some species currently considered hypoxia tolerant.

Developmental Plasticity and Heterokairy

There is a resurgence of interest in using phenotypic plasticity, ‘the environmentally sensitive production of alternative phenotypes by given genotypes’, as a framework in the study of evolutionary biology. The term developmental plasticity describes a more specific strand of investigation dealing with how alterations to developmental processes and outcomes shape such environmentally induced variation. Nested within developmental plasticity is the notion of heterokairy, the potential of a single genotype to alter the timing of a developmental event (e.g. onset of a particular structure, function or components of that function), in response to an environmental signal or influence. Here we make a case for using the term heterokairy as a way of focusing on altered timing across different biological disciplines, and we suggest a road map for such an approach. Heterokairy as an interdisciplinary term could be used to (a) bring together the substantial knowledge currently available of environmentally sensitive, genetic and hormonal control of the timing of developmental transitions, (b) embed the study of altered timing of developmental events within developmental plasticity and (c) highlight the role that plasticity can play in adaptive evolution, particularly in response to global environmental change.