31 research outputs found
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
Re-assessing the validity of the Moral Sensitivity Questionnaire (MSQ):Two new scales for moral deliberation and paternalism
RATIONALE, AIMS, AND OBJECTIVES: The current study and previous research have called the six-component model of Lützen's 30-item Moral Sensitivity Questionnaire (MSQ) into question. For this reason, we re-examined the construct validity of this instrument. METHODS: In this cross-sectional study, which was based on a convenience sample of Dutch nurse practitioners (NPs) and physician assistants (PAs), we tested the validity of MSQ items using exploratory and confirmatory factor analyses (EFA and CFA, respectively). RESULTS: The EFA revealed a two-component model, which was then tested as a target model with CFA and was found to have good model fit. Some items were correlated with two uncorrelated latent constructs, which we labelled as "paternalistic" and "deliberate" attitudes towards patients. CONCLUSIONS: As in previous studies, the analyses in the current study, which was conducted among PAs and NPs, did not reveal six dimensions for the 30 items. Two new latent dimensions of moral sensitivity were psychometrically tested and confirmed. These two components relate to studies investigating ethical behaviour, and they can be used to describe the moral climate in healthcare organizations. The scales are indicators of the extent to which health professionals behave in a deliberate (sensitive) or paternalistic (insensitive) manner towards the opinions of patients within the context of medical decision-making
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
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
GSK-3 Activity Is Critical for the Orientation of the Cortical Microtubules and the Dorsoventral Axis Determination in Zebrafish Embryos
The formation of dorsal-ventral (D–V) axis is the earliest event that breaks the radial symmetry and determines the bilateral body plan of a vertebrate embryo, however, the maternal control of this process is not fully understood. Here, we discovered a new dorsalizing window of acute lithium treatment, which covers only less than 10 minutes after fertilization. Lithium treatment in this window was not able to reverse the ventralized phenotype in tokkeabi (tkk) mutant embryos, and its dorsalizing activity on wild-type embryos was inhibited by nocodazole co-treatment. These evidences indicate that the underlying mechanism is independent of a direct activation of Wnt/β-catenin signaling, but depends on the upstream level of the microtubule mediated dorsal determinant transport. In order to identify the target of lithium in this newly discovered sensitive window, GSK-3 inhibitor IX as well as the IMPase inhibitor L690, 330 treatments were performed. We found that only GSK-3 inhibitor IX treatment mimicked the lithium treatment in the dorsalizing activity. Further study showed that the parallel pattern of cortical microtubules in the vegetal pole region and the directed migration of the Wnt8a mRNA were randomized by either lithium or GSK-3 inhibitor IX treatment. These results thus revealed an early and critical role of GSK-3 activity that regulates the orientation of the cortical microtubules and the directed transport of the dorsal determinants in zebrafish embryos
MAPK pathway activation in pilocytic astrocytoma
Pilocytic astrocytoma (PA) is the most common tumor of the pediatric central nervous system (CNS). A body of research over recent years has demonstrated a key role for mitogen-activated protein kinase (MAPK) pathway signaling in the development and behavior of PAs. Several mechanisms lead to activation of this pathway in PA, mostly in a mutually exclusive manner, with constitutive BRAF kinase activation subsequent to gene fusion being the most frequent. The high specificity of this fusion to PA when compared with other CNS tumors has diagnostic utility. In addition, the frequency of alteration of this key pathway provides an opportunity for molecularly targeted therapy in this tumor. Here, we review the current knowledge on mechanisms of MAPK activation in PA and some of the downstream consequences of this activation, which are now starting to be elucidated both in vitro and in vivo, as well as clinical considerations and possible future directions