Object Segmentation and Ground Truth in 3D Embryonic Imaging

Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets

The clockfront and wavefront model revisited

The currently accepted interpretation of the clock and wavefront model of somitogenesis is that a posteriorly moving molecular gradient sequentially slows the rate of clock oscillations, resulting in a spatial readout of temporal oscillations. However, while molecular components of the clocks and wavefronts have now been identified in the pre-somitic mesoderm (PSM), there is not yet conclusive evidence demonstrating that the observed molecular wavefronts act to slow clock oscillations. Here we present an alternative formulation of the clock and wavefront model in which oscillator coupling, already known to play a key role in oscillator synchronisation, plays a fundamentally important role in the slowing of oscillations along the anterior–posterior (AP) axis. Our model has three parameters which can be determined, in any given species, by the measurement of three quantities: the clock period in the posterior PSM, somite length and the length of the PSM. A travelling wavefront, which slows oscillations along the AP axis, is an emergent feature of the model. Using the model we predict: (a) the distance between moving stripes of gene expression; (b) the number of moving stripes of gene expression and (c) the oscillator period profile along the AP axis. Predictions regarding the stripe data are verified using existing zebrafish data. We simulate a range of experimental perturbations and demonstrate how the model can be used to unambiguously define a reference frame along the AP axis. Comparing data from zebrafish, chick, mouse and snake, we demonstrate that: (a) variation in patterning profiles is accounted for by a single nondimensional parameter; the ratio of coupling strengths; and (b) the period profile along the AP axis is conserved across species. Thus the model is consistent with the idea that, although the genes involved in pattern propagation in the PSM vary, there is a conserved patterning mechanism across species

Modelling Oscillator synchronisation during vertebrate axis segmentation

he somitogenesis clock regulates the periodicity with which somites form in the posterior pre-somitic mesoderm. Whilst cell heterogeneity results in noisy oscillation rates amongst constituent cells, synchrony within the population is maintained as oscillators are entrained via juxtracine signalling mechanisms. Here we consider a population of phase-coupled oscillators and investigate how biologically motivated perturbations to the entrained state can perturb synchrony within the population. We find that the ratio of mitosis length to clock period can influence levels of desynchronisation. Moreover, we observe that random cell movement, and hence change of local neighbourhoods, increases synchronisation

Aberrant behaviours of reaction diffusion self-organisation models on growing domains in the presence of gene expression time delays

Turing’s pattern formation mechanism exhibits sensitivity to the details of the initial conditions suggesting that, in isolation, it cannot robustly generate pattern within noisy biological environments. Nonetheless, secondary aspects of developmental self-organisation, such as a growing domain, have been shown to ameliorate this aberrant model behaviour. Furthermore, while in-situ hybridisation reveals the presence of gene expression in developmental processes, the influence of such dynamics on Turing’s model has received limited attention. Here, we novelly focus on the Gierer–Meinhardt reaction diffusion system considering delays due the time taken for gene expression, while incorporating a number of different domain growth profiles to further explore the influence and interplay of domain growth and gene expression on Turing’s mechanism. We find extensive pathological model behaviour, exhibiting one or more of the following: temporal oscillations with no spatial structure, a failure of the Turing instability and an extreme sensitivity to the initial conditions, the growth profile and the duration of gene expression. This deviant behaviour is even more severe than observed in previous studies of Schnakenberg kinetics on exponentially growing domains in the presence of gene expression (Gaffney and Monk in Bull. Math. Biol. 68:99–130, 2006). Our results emphasise that gene expression dynamics induce unrealistic behaviour in Turing’s model for multiple choices of kinetics and thus such aberrant modelling predictions are likely to be generic. They also highlight that domain growth can no longer ameliorate the excessive sensitivity of Turing’s mechanism in the presence of gene expression time delays. The above, extensive, pathologies suggest that, in the presence of gene expression, Turing’s mechanism would generally require a novel and extensive secondary mechanism to control reaction diffusion patterning

Understanding the enhanced synchronization of delay-coupled networks with fluctuating topology

We study the dynamics of networks with coupling delay, from which the connectivity changes over time. The synchronization properties are shown to depend on the interplay of three time scales: the internal time scale of the dynamics, the coupling delay along the network links and time scale at which the topology changes. Concentrating on a linearized model, we develop an analytical theory for the stability of a synchronized solution. In two limit cases the system can be reduced to an “effective” topology: In the fast switching approximation, when the network fluctuations are much faster than the internal time scale and the coupling delay, the effective network topology is the arithmetic mean over the different topologies. In the slow network limit, when the network fluctuation time scale is equal to the coupling delay, the effective adjacency matrix is the geometric mean over the adjacency matrices of the different topologies. In the intermediate regime the system shows a sensitive dependence on the ratio of time scales, and specific topologies, reproduced as well by numerical simulations. Our results are shown to describe the synchronization properties of fluctuating networks of delay-coupled chaotic maps

Prospective Power Calculations for the Four Lab Study of A Multigenerational Reproductive/Developmental Toxicity Rodent Bioassay Using A Complex Mixture of Disinfection By-Products in the Low-Response Region

In complex mixture toxicology, there is growing emphasis on testing environmentally representative doses that improve the relevance of results for health risk assessment, but are typically much lower than those used in traditional toxicology studies. Traditional experimental designs with typical sample sizes may have insufficient statistical power to detect effects caused by environmentally relevant doses. Proper study design, with adequate statistical power, is critical to ensuring that experimental results are useful for environmental health risk assessment. Studies with environmentally realistic complex mixtures have practical constraints on sample concentration factor and sample volume as well as the number of animals that can be accommodated. This article describes methodology for calculation of statistical power for non-independent observations for a multigenerational rodent reproductive/developmental bioassay. The use of the methodology is illustrated using the U.S. EPA’s Four Lab study in which rodents were exposed to chlorinated water concentrates containing complex mixtures of drinking water disinfection by-products. Possible experimental designs included two single-block designs and a two-block design. Considering the possible study designs and constraints, a design of two blocks of 100 females with a 40:60 ratio of control:treated animals and a significance level of 0.05 yielded maximum prospective power (~90%) to detect pup weight decreases, while providing the most power to detect increased prenatal loss

Use of a food frequency questionnaire in American Indian and Caucasian pregnant women: a validation study

BACKGROUND: Food frequency questionnaires (FFQs) have been validated in pregnant women, but few studies have focused specifically on low-income women and minorities. The purpose of this study was to examine the validity of the Harvard Service FFQ (HSFFQ) among low-income American Indian and Caucasian pregnant women. METHODS: The 100-item HSFFQ was administered three times to a sample of pregnant women, and two sets of 24-hour recalls (six total) were collected at approximately 12 and 28 weeks of gestation. The sample included a total of 283 pregnant women who completed Phase 1 of the study and 246 women who completed Phase 2 of the study. Deattenuated Pearson correlation coefficients were used to compare intakes of 24 nutrients estimated from the second and third FFQ to average intakes estimated from the week-12 and week-28 sets of diet recalls. RESULTS: Deattenuated correlations ranged from 0.09 (polyunsaturated fat) to 0.67 (calcium) for Phase 1 and from 0.27 (sucrose) to 0.63 (total fat) for Phase 2. Average deattenuated correlations for the two phases were 0.48 and 0.47, similar to those reported among other groups of pregnant women. CONCLUSION: The HSFFQ is a simple self-administered questionnaire that is useful in classifying low-income American Indian and Caucasian women according to relative dietary intake during pregnancy. Its use as a research tool in this population may provide important information about associations of nutrient intakes with pregnancy outcomes and may help to identify groups of women who would benefit most from nutritional interventions

SARS-CoV-2 B.1.617 mutations L452 and E484Q are not synergistic for antibody evasion

SARS-CoV-2 B.1.617系統（俗称「インド株」）のL452R変異とE484Q変異は 中和抗体感受性の低下において、相加的な抵抗性を示さない. 京都大学プレスリリース. 2021-08-24.The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that two key mutations seen in the receptor binding domain L452R and E484Q would have additive effects on evasion of neutralising antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine elicited neutralising antibodies by L452R and E484Q but lack of synergistic loss of sensitivity

SARS-CoV-2 B.1.617 Mutations L452R and E484Q Are Not Synergistic for Antibody Evasion.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine-elicited neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity