Nonlinearity arising from noncooperative transcription factor binding enhances negative feedback and promotes genetic oscillations

We study the effects of multiple binding sites in the promoter of a genetic oscillator. We evaluate the regulatory function of a promoter with multiple binding sites in the absence of cooperative binding, and consider different hypotheses for how the number of bound repressors affects transcription rate. Effective Hill exponents of the resulting regulatory functions reveal an increase in the nonlinearity of the feedback with the number of binding sites. We identify optimal configurations that maximize the nonlinearity of the feedback. We use a generic model of a biochemical oscillator to show that this increased nonlinearity is reflected in enhanced oscillations, with larger amplitudes over wider oscillatory ranges. Although the study is motivated by genetic oscillations in the zebrafish segmentation clock, our findings may reveal a general principle for gene regulation.Comment: 11 pages, 8 figure

Repressor Dimerization in the Zebrafish Somitogenesis Clock

The oscillations of the somitogenesis clock are linked to the fundamental process of vertebrate embryo segmentation, yet little is known about their generation. In zebrafish, it has been proposed that Her proteins repress the transcription of their own mRNA. However, in its simplest form, this model is incompatible with the fact that morpholino knockdown of Her proteins can impair expression of their mRNA. Simple self-repression models also do not account for the spatiotemporal pattern of gene expression, with waves of gene expression shrinking as they propagate. Here we study computationally the networks generated by the wealth of dimerization possibilities amongst transcriptional repressors in the zebrafish somitogenesis clock. These networks can reproduce knockdown phenotypes, and strongly suggest the existence of a Her1–Her7 heterodimer, so far untested experimentally. The networks are the first reported to reproduce the spatiotemporal pattern of the zebrafish somitogenesis clock; they shed new light on the role of Her13.2, the only known link between the somitogenesis clock and positional information in the paraxial mesoderm. The networks can also account for perturbations of the clock by manipulation of FGF signaling. Achieving an understanding of the interplay between clock oscillations and positional information is a crucial first step in the investigation of the segmentation mechanism

Somitogenesis Clock-Wave Initiation Requires Differential Decay and Multiple Binding Sites for Clock Protein

Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM) to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms

Hairy switches and oscillators - reconstructing the zebrafish segmentation clock

Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’

Analysis of her1 and her7 Mutants Reveals a Spatio Temporal Separation of the Somite Clock Module

Somitogenesis is controlled by a genetic network consisting of an oscillator (clock) and a gradient (wavefront). The “hairy and Enhancer of Split”- related (her) genes act downstream of the Delta/Notch (D/N) signaling pathway, and are crucial components of the segmentation clock. Due to genome duplication events, the zebrafish genome, possesses two gene copies of the mouse Hes7 homologue: her1 and her7. To better understand the functional consequences of this gene duplication, and to determine possible independent roles for these two genes during segmentation, two zebrafish mutants her1hu2124 and her7hu2526 were analyzed. In the course of embryonic development, her1hu2124 mutants exhibit disruption of the three anterior-most somite borders, whereas her7hu2526 mutants display somite border defects restricted to somites 8 (+/−3) to 17 (+/−3) along the anterior-posterior axis. Analysis of the molecular defects in her1hu2124 mutants reveals a her1 auto regulatory feedback loop during early somitogenesis that is crucial for correct patterning and independent of her7 oscillation. This feedback loop appears to be restricted to early segmentation, as cyclic her1 expression is restored in her1hu2124 embryos at later stages of development. Moreover, only the anterior deltaC expression pattern is disrupted in the presomitic mesoderm of her1hu2124 mutants, while the posterior expression pattern of deltaC remains unaltered. Together, this data indicates the existence of an independent and genetically separable anterior and posterior deltaC clock modules in the presomitic mesdorm (PSM)

Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex

<p>Abstract</p> <p>Background</p> <p>The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick <it>hairy1 </it>gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of <it>hairy1 </it>oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners.</p> <p>Results</p> <p>Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both <it>sap18 </it>and <it>sin3a </it>genes during chick embryo development, using <it>in situ </it>hybridization experiments. We found that both <it>sap18 </it>and s<it>in3a </it>expression patterns co-localize <it>in vivo </it>with <it>hairy1 </it>expression domains in chick rostral presomitic mesoderm and caudal region of somites.</p> <p>Conclusion</p> <p>Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since <it>sap18 </it>and <it>sin3a </it>are not expressed in the PSM territory where <it>hairy1 </it>presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites.</p

Computational Models of the Notch Network Elucidate Mechanisms of Context-dependent Signaling

The Notch signaling pathway controls numerous cell fate decisions during development and adulthood through diverse mechanisms. Thus, whereas it functions as an oscillator during somitogenesis, it can mediate an all-or-none cell fate switch to influence pattern formation in various tissues during development. Furthermore, while in some contexts continuous Notch signaling is required, in others a transient Notch signal is sufficient to influence cell fate decisions. However, the signaling mechanisms that underlie these diverse behaviors in different cellular contexts have not been understood. Notch1 along with two downstream transcription factors hes1 and RBP-Jk forms an intricate network of positive and negative feedback loops, and we have implemented a systems biology approach to computationally study this gene regulation network. Our results indicate that the system exhibits bistability and is capable of switching states at a critical level of Notch signaling initiated by its ligand Delta in a particular range of parameter values. In this mode, transient activation of Delta is also capable of inducing prolonged high expression of Hes1, mimicking the “ON” state depending on the intensity and duration of the signal. Furthermore, this system is highly sensitive to certain model parameters and can transition from functioning as a bistable switch to an oscillator by tuning a single parameter value. This parameter, the transcriptional repression constant of hes1, can thus qualitatively govern the behavior of the signaling network. In addition, we find that the system is able to dampen and reduce the effects of biological noise that arise from stochastic effects in gene expression for systems that respond quickly to Notch signaling

Number of active transcription factor binding sites is essential for the Hes7 oscillator

BACKGROUND: It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. RESULTS: We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. CONCLUSION: Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which case Hirata's delay system does not explain the experimental results. Recent experiments by Chen et al. seem to support the former hypothesis, but the discussion is still open

Investigating the localization of an avian hairy homolog (c-Hairy1) protein

Dissertação de mest., Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Univ. do Algarve, 2013The embryo segmental pattern is first established with the formation of somites. Somites are embryonic segments of vertebrates, periodically formed in a strict temporal precision, which has been believed to be governed by a biological clock, called the ‘segmentation clock’. In the mid-70s, the “Clock and Wavefront Model” was proposed, predicting the existence of an intracellular clock or oscillator in the presomitic mesoderm (PSM) cells. Several years later, Palmeirim et. al., identified the first molecular evidence of this clock by discovering and characterizing the oscillatory expression of c-hairy1 in the chicken embryo paraxial PSM, which strikingly matched the period of somite formation (90 min). This present work aims to characterize the chicken Hairy1 protein, and to study its expression both in early embryo stages and chicken embryonic fibroblasts (CEFs). Bioinformatic tools have predicted the biochemical properties, primary and secondary structure, post transcriptional modifications and subcellular localization of c-Hairy1 protein. By western blot were established the optimal working conditions of the customized monoclonal antibody, as well as the expression of the protein in both chick embryos and CEFs. The protein distribution and its subcellular localization in CEFs was assessed by immunofluorescence. Results of western blot have demonstrated sensitivity of the antibody, although its specificity for c-Hairy1 protein remains debatable. Even more, both immunofluorescence and bioinformatics analysis showed c-Hariy1 to be localized both in nucleus and cytoplasm. Interestingly, it was also demonstrated that the nucleus:cytoplasm ratio distribution varied between cells. These work’s findings suggest that c-Hairy1 protein holds much investigation potential, and the optimization of the antibody working conditions enables its use for further studies

Post-transcriptional regulation in the developing embryo

Embryonic development is critically reliant on well-defined spatial and temporal patterns of gene expression. These patterns are often achieved through the regulation of gene expression at the mRNA level. This form of regulation is commonly referred to as post-transcriptional regulation and is frequently mediated by RNA binding proteins (RBPs) and regulatory sequences located in the untranslated regions (UTRs) of the mRNAs. The mechanisms that underlie these post-transcriptional regulation phenomena have been the focus of an increasing level of attention in recent years. However, their specific roles in embryogenesis, and their relative importance to the different processes that take place in the developing embryo, still require further investigation. In this thesis we focused our attention on post-transcriptional regulation mechanisms that operate in the developing zebrafish embryo, and investigated their importance to embryogenesis from two perspectives: the perspective of a post-transcriptional regulator – the Quaking A RBP – and the perspective of a set of regulatory sequences – the fgf8a alternative 3’UTRs. Quaking A belongs to the STAR family of RBPs, which has been implicated in several late developmental processes. Using a loss-of-function approach, we uncovered evidence for two previously undescribed functions for Quaking A, namely, in posterior body shaping and in the establishment of internal organ laterality. Furthermore, in our search for potential mRNA targets of Quaking A we came across the cell adhesion molecule Cadherin 11, which also appears to contribute to the establishment of internal organ laterality. Our investigation of the fgf8a alternative 3’UTRs, revealed that the most abundant 3’UTR for this gene mediates a strong translational repression, when compared to a more sparsely used alternative 3’UTR, which supports a higher translation efficiency. By inducing a shift in the selection efficiency of the associated polyadenylation sites, we observed a temporally and spatially specific impact of fgf8a 3’UTR usage on embryogenesis, in particular at late stages during sensory system development. In addition, we identified a previously undescribed role for Fgf signalling in the initial stages of superficial retinal vascularization. In conclusion, our investigation of Quaking A revealed two previously undescribed roles for this RBP in embryogenesis, thus adding to the current view of STAR proteins, as major regulators of a considerable diversity of developmental processes. In addition, our study of the fgf8a alternative 3’UTRs revealed that within the wide range of developmental processes that involve the fgf8a gene, only a specific subset appears to rely critically on the regulation of the relative abundances of these 3’UTRs. Overall, these findings highlight the importance of addressing post-transcriptional regulation mechanisms to fully understand gene and pathway functions in embryonic development