The Role of Regulated mRNA Stability in Establishing Bicoid Morphogen Gradient in Drosophila Embryonic Development

The Bicoid morphogen is amongst the earliest triggers of differential spatial pattern of gene expression and subsequent cell fate determination in the embryonic development of Drosophila. This maternally deposited morphogen is thought to diffuse in the embryo, establishing a concentration gradient which is sensed by downstream genes. In most model based analyses of this process, the translation of the bicoid mRNA is thought to take place at a fixed rate from the anterior pole of the embryo and a supply of the resulting protein at a constant rate is assumed. Is this process of morphogen generation a passive one as assumed in the modelling literature so far, or would available data support an alternate hypothesis that the stability of the mRNA is regulated by active processes? We introduce a model in which the stability of the maternal mRNA is regulated by being held constant for a length of time, followed by rapid degradation. With this more realistic model of the source, we have analysed three computational models of spatial morphogen propagation along the anterior-posterior axis: (a) passive diffusion modelled as a deterministic differential equation, (b) diffusion enhanced by a cytoplasmic flow term; and (c) diffusion modelled by stochastic simulation of the corresponding chemical reactions. Parameter estimation on these models by matching to publicly available data on spatio-temporal Bicoid profiles suggests strong support for regulated stability over either a constant supply rate or one where the maternal mRNA is permitted to degrade in a passive manner

A Precise Bicoid Gradient Is Nonessential during Cycles 11–13 for Precise Patterning in the Drosophila Blastoderm

Background: During development, embryos decode maternal morphogen inputs into highly precise zygotic gene expression. The discovery of the morphogen Bicoid and its profound effect on developmental programming in the Drosophila embryo has been a cornerstone in understanding the decoding of maternal inputs. Bicoid has been described as a classical morphogen that forms a concentration gradient along the antero-posterior axis of the embryo by diffusion and initiates expression of target genes in a concentration-dependent manner in the syncytial blastoderm. Recent work has emphasized the stability of the Bicoid gradient as a function of egg length and the role of nuclear dynamics in maintaining the Bicoid gradient. Bicoid and nuclear dynamics were observed but not modulated under the ideal conditions used previously. Therefore, it has not been tested explicitly whether a temporally stable Bicoid gradient prior to cellularization is required for precise patterning. Principal Findings: Here, we modulate both nuclear dynamics and the Bicoid gradient using laminar flows of different temperature in a microfluidic device to determine if stability of the Bicoid gradient prior to cellularization is essential for precise patterning. Dramatic motion of both cytoplasm and nuclei was observed prior to cellularization, and the Bicoid gradient was disrupted by nuclear motion and was highly abnormal as a function of egg length. Despite an abnormal Bicoid gradient during cycles 11–13, Even-skipped patterning in these embryos remained precise. Conclusions: These results indicate that the stability of the Bicoid gradient as a function of egg length is nonessential during syncytial blastoderm stages. Further, presumably no gradient formed by simple diffusion on the scale of egg length could be responsible for the robust antero-posterior patterning observed, as severe cytoplasmic and nuclear motion would disrupt such a gradient. Additional mechanisms for how the embryo could sense its dimensions and interpret the Bicoid gradient are discussed

Considerations for a European animal welfare standard to evaluate adverse phenotypes in teleost fish

No abstract available

Master equation simulation analysis of immunostained Bicoid morphogen gradient

<p>Abstract</p> <p>Background</p> <p>The concentration gradient of Bicoid protein which determines the developmental pathways in early <it>Drosophila </it>embryo is the best characterized morphogen gradient at the molecular level. Because different developmental fates can be elicited by different concentrations of Bicoid, it is important to probe the limits of this specification by analyzing intrinsic fluctuations of the Bicoid gradient arising from small molecular number. Stochastic simulations can be applied to further the understanding of the dynamics of Bicoid morphogen gradient formation at the molecular number level, and determine the source of the nucleus-to-nucleus expression variation (noise) observed in the Bicoid gradient.</p> <p>Results</p> <p>We compared quantitative observations of Bicoid levels in immunostained <it>Drosophila </it>embryos with a spatially extended Master Equation model which represents diffusion, decay, and anterior synthesis. We show that the intrinsic noise of an autonomous reaction-diffusion gradient is Poisson distributed. We demonstrate how experimental noise can be identified in the logarithm domain from single embryo analysis, and then separated from intrinsic noise in the normalized variance domain of an ensemble statistical analysis. We show how measurement sensitivity affects our observations, and how small amounts of rescaling noise can perturb the noise strength (Fano factor) observed. We demonstrate that the biological noise level in data can serve as a physical constraint for restricting the model's parameter space, and for predicting the Bicoid molecular number and variation range. An estimate based on a low variance ensemble of embryos suggests that the steady-state Bicoid molecular number in a nucleus should be larger than 300 in the middle of the embryo, and hence the gradient should extend to the posterior end of the embryo, beyond the previously assumed background limit. We exhibit the predicted molecular number gradient together with measurement effects, and make a comparison between conditions of higher and lower variance respectively.</p> <p>Conclusion</p> <p>Quantitative comparison of Master Equation simulations with immunostained data enabled us to determine narrow ranges for key biophysical parameters, which for this system can be independently validated. Intrinsic noise is clearly detectable as well, although the staining process introduces certain limits in resolution.</p

Molecularly defined diffuse leptomeningeal glioneuronal tumor (DLGNT) comprises two subgroups with distinct clinical and genetic features

Diffuse leptomeningeal glioneuronal tumors (DLGNT) represent rare CNS neoplasms which have been included in the 2016 update of the WHO classification. The wide spectrum of histopathological and radiological features can make this enigmatic tumor entity difficult to diagnose. In recent years, large-scale genomic and epigenomic analyses have afforded insight into key genetic alterations occurring in multiple types of brain tumors and provide unbiased, complementary tools to improve diagnostic accuracy. Through genome-wide DNA methylation screening of &gt; 25,000 tumors, we discovered a molecularly distinct class comprising 30 tumors, mostly diagnosed histologically as DLGNTs. Copy-number profiles derived from the methylation arrays revealed unifying characteristics, including loss of chromosomal arm 1p in all cases. Furthermore, this molecular DLGNT class can be subdivided into two subgroups [DLGNT methylation class (MC)-1 and DLGNT methylation class (MC)-2], with all DLGNT-MC-2 additionally displaying a gain of chromosomal arm 1q. Co-deletion of 1p/19q, commonly seen in IDH-mutant oligodendroglioma, was frequently observed in DLGNT, especially in DLGNT-MC-1 cases. Both subgroups also had recurrent genetic alterations leading to an aberrant MAPK/ERK pathway, with KIAA1549:BRAF fusion being the most frequent event. Other alterations included fusions of NTRK1/2/3 and TRIM33:RAF1, adding up to an MAPK/ERK pathway activation identified in 80% of cases. In the DLGNT-MC-1 group, age at diagnosis was significantly lower (median 5 vs 14 years, p &lt; 0.01) and clinical course less aggressive (5-year OS 100, vs 43% in DLGNT-MC-2). Our study proposes an additional molecular layer to the current histopathological classification of DLGNT, of particular use for cases without typical morphological or radiological characteristics, such as diffuse growth and radiologic leptomeningeal dissemination. Recurrent 1p deletion and MAPK/ERK pathway activation represent diagnostic biomarkers and therapeutic targets, respectively—laying the foundation for future clinical trials with, e.g., MEK inhibitors that may improve the clinical outcome of patients with DLGNT

Stable, Precise, and Reproducible Patterning of Bicoid and Hunchback Molecules in the Early Drosophila Embryo

Precise patterning of morphogen molecules and their accurate reading out are of key importance in embryonic development. Recent experiments have visualized distributions of proteins in developing embryos and shown that the gradient of concentration of Bicoid morphogen in Drosophila embryos is established rapidly after fertilization and remains stable through syncytial mitoses. This stable Bicoid gradient is read out in a precise way to distribute Hunchback with small fluctuations in each embryo and in a reproducible way, with small embryo-to-embryo fluctuation. The mechanisms of such stable, precise, and reproducible patterning through noisy cellular processes, however, still remain mysterious. To address these issues, here we develop the one- and three-dimensional stochastic models of the early Drosophila embryo. The simulated results show that the fluctuation in expression of the hunchback gene is dominated by the random arrival of Bicoid at the hunchback enhancer. Slow diffusion of Hunchback protein, however, averages out this intense fluctuation, leading to the precise patterning of distribution of Hunchback without loss of sharpness of the boundary of its distribution. The coordinated rates of diffusion and transport of input Bicoid and output Hunchback play decisive roles in suppressing fluctuations arising from the dynamical structure change in embryos and those arising from the random diffusion of molecules, and give rise to the stable, precise, and reproducible patterning of Bicoid and Hunchback distributions

Spatial Bistability Generates hunchback Expression Sharpness in the Drosophila Embryo

During embryonic development, the positional information provided by concentration gradients of maternal factors directs pattern formation by providing spatially dependent cues for gene expression. In the fruit fly, Drosophila melanogaster, a classic example of this is the sharp on–off activation of the hunchback (hb) gene at midembryo, in response to local concentrations of the smooth anterior–posterior Bicoid (Bcd) gradient. The regulatory region for hb contains multiple binding sites for the Bcd protein as well as multiple binding sites for the Hb protein. Some previous studies have suggested that Bcd is sufficient for properly sharpened Hb expression, yet other evidence suggests a need for additional regulation. We experimentally quantified the dynamics of hb gene expression in flies that were wild-type, were mutant for hb self-regulation or Bcd binding, or contained an artificial promoter construct consisting of six Bcd and two Hb sites. In addition to these experiments, we developed a reaction–diffusion model of hb transcription, with Bcd cooperative binding and hb self-regulation, and used Zero Eigenvalue Analysis to look for multiple stationary states in the reaction network. Our model reproduces the hb developmental dynamics and correctly predicts the mutant patterns. Analysis of our model indicates that the Hb sharpness can be produced by spatial bistability, in which hb self-regulation produces two stable levels of expression. In the absence of self-regulation, the bistable behavior vanishes and Hb sharpness is disrupted. Bcd cooperative binding affects the position where bistability occurs but is not itself sufficient for a sharp Hb pattern. Our results show that the control of Hb sharpness and positioning, by hb self-regulation and Bcd cooperativity, respectively, are separate processes that can be altered independently. Our model, which matches the changes in Hb position and sharpness observed in different experiments, provides a theoretical framework for understanding the data and in particular indicates that spatial bistability can play a central role in threshold-dependent reading mechanisms of positional information

The Formation of the Bicoid Morphogen Gradient Requires Protein Movement from Anteriorly Localized mRNA

New quantitative data show that the Bicoid morphogen gradient is generated from a dynamic localized source and that protein gradient formation requires protein movement along the anterior-posterior axis

Canalization of Gene Expression and Domain Shifts in the Drosophila Blastoderm by Dynamical Attractors

The variation in the expression patterns of the gap genes in the blastoderm of the fruit fly Drosophila melanogaster reduces over time as a result of cross regulation between these genes, a fact that we have demonstrated in an accompanying article in PLoS Biology (see Manu et al., doi:10.1371/journal.pbio.1000049). This biologically essential process is an example of the phenomenon known as canalization. It has been suggested that the developmental trajectory of a wild-type organism is inherently stable, and that canalization is a manifestation of this property. Although the role of gap genes in the canalization process was established by correctly predicting the response of the system to particular perturbations, the stability of the developmental trajectory remains to be investigated. For many years, it has been speculated that stability against perturbations during development can be described by dynamical systems having attracting sets that drive reductions of volume in phase space. In this paper, we show that both the reduction in variability of gap gene expression as well as shifts in the position of posterior gap gene domains are the result of the actions of attractors in the gap gene dynamical system. Two biologically distinct dynamical regions exist in the early embryo, separated by a bifurcation at 53% egg length. In the anterior region, reduction in variation occurs because of stability induced by point attractors, while in the posterior, the stability of the developmental trajectory arises from a one-dimensional attracting manifold. This manifold also controls a previously characterized anterior shift of posterior region gap domains. Our analysis shows that the complex phenomena of canalization and pattern formation in the Drosophila blastoderm can be understood in terms of the qualitative features of the dynamical system. The result confirms the idea that attractors are important for developmental stability and shows a richer variety of dynamical attractors in developmental systems than has been previously recognized

Fast Homozygosity Mapping and Identification of a Zebrafish ENU-Induced Mutation by Whole-Genome Sequencing

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish