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During embryonic development, orderly patterns of gene expression eventually assign each cell in the embryo its particular fate. For the anteroposterior axis of the Drosophila embryo, the first step in this process depends on a spatial gradient of the maternal morphogen Bicoid (Bcd). Positional information of this gradient is transmitted to downstr...
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... determine whether low AID expression was itself responsible for the low mutation rates in Ramos clone 1, this clone was stably transfected with either a vector expressing human AID (hAID) or an empty vector control. Mutation rates of typical transfected clones were then determined by sequencing unselected V regions after 1 or 2 months in culture (Table 1). Clones expressing low levels of AID (that is, clones C.1 and A.1) had very few mutations in the V region, whereas clones that expressed ,25-fold higher levels of AID mRNA (that is, clones A.2 and A.5) had many more V-region mutations (Fig. 1b and Table 1). ...
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... rates of typical transfected clones were then determined by sequencing unselected V regions after 1 or 2 months in culture (Table 1). Clones expressing low levels of AID (that is, clones C.1 and A.1) had very few mutations in the V region, whereas clones that expressed ,25-fold higher levels of AID mRNA (that is, clones A.2 and A.5) had many more V-region mutations (Fig. 1b and Table 1). Table 2 summarizes the mutational features of all the Ramos clones that expressed elevated levels of AID and shows that the rates and characteristics of the mutations in all of these clones were similar: there was a targeting bias of G/C nucleotides, transitions were slightly favoured over transversions, and ,35% of mutations were in G or C nucleotides within RGYW (A/G, G, C/T, A/T) or WRCY hot-spot sequences, motifs that are frequently targeted in SHM both in vivo and in vitro 7,8 ...
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... After fertilization, the Bicoid protein forms a concentration gradient across the anterior-posterior (A-P) axis of the embryos, providing positional information for downstream targets, including gap and pair-rule genes such as hunchback (hb), giant (gt), krüppel (Kr), and even-skipped (eve). These genes and others together constitute a complex network that determines segmentation 18 and scaling 19,20 along the A-P axis of the embryo. It has been shown that the patterning outcomes of the network directly respond to the gene dosage of bicoid, with a higher dosage causing a posterior shift of the cephalic furrow and increased frequency of segmentation defects in larval cuticles 21 . ...
Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the network’s regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8–15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.
... Taken together, our data suggest that most of the embryonic transcriptome is conserved and that the differential regulation of a relatively small fraction of the transcriptome confers heat tolerance. Drosophila embryogenesis has been shown to be unperturbed across a range of more benign temperatures, from 18°C to 29°C (Houchmandzadeh et al., 2002). Thus, it seems that a potential explanation for the pattern of overall transcriptional homogeneity across heat shock temperatures and among embryos from different geographic regions may be the highly conserved nature of early embryonic development (Davidson, 1986), such that developmental processes have evolved to be robust to environmental and mutational perturbations-i.e., embryonic transcriptomes are canalized (von Heckel et al., 2016;Waddington, 1942). ...
In organisms with complex life cycles, life stages that are most susceptible to environmental stress may determine species persistence in the face of climate change. Early embryos of Drosophila melanogaster are particularly sensitive to acute heat stress, yet tropical embryos have higher heat tolerance than temperate embryos, suggesting adaptive variation in embryonic heat tolerance. We compared transcriptomic responses to heat stress among tropical and temperate embryos to elucidate the gene regulatory basis of divergence in embryonic heat tolerance. The transcriptomes of tropical and temperate embryos were differentiated by the expression of relatively few genes, including genes involved in oxidative stress. But most of the transcriptomic response to heat stress was shared among all embryos. Further, embryos shifted the expression of thousands of genes and showed robust gene activation, demonstrating that, contrary to previous reports, early embryos are not transcriptionally silent. The involvement of oxidative stress genes in embryonic heat tolerance corroborates recent reports on the critical role of redox homeostasis in coordinating developmental transitions. By characterizing adaptive variation in the transcriptomic basis of embryonic heat tolerance, this study is a novel contribution to the literature on developmental physiology and genetics, which often lacks ecological and evolutionary context.
... We use the first hours of development in the fruit fly as an example, following spatial patterns of morphogen concentration as they flow through three layers of a genetic network, from maternal inputs to the gap genes to the pair rule genes [15][16][17]. In the spirit of earlier work [18][19][20][21][22] we analyze discrete positional markers, such as the stripes in pair rule-gene expression, and find that positions of these markers vary in proportion to the length of the embryo with better than 1% accuracy [23]. We then go beyond discrete markers, decomposing the information carried by graded patterns of gap gene expression into information about fractional or scaled position vs. information about the absolute position; we find that all the available information is about fractional position along the anterior-posterior axis. ...
... More precisely, if we fit these linear relations then intercepts are zero and slopes are equal to the mean fractional positions, as in Eq. (3), both results with error bars of less than 1% (Appendix C). This provides prima facie evidence for scaling of the pair-rule stripes, reinforcing the conclusions of earlier work [18][19][20][21]. ...
... In summary, stripes and boundaries of gene expression in the early fly embryo provide discrete positional markers, and the absolute positions of these markers are in all cases proportional to the length of the embryo. This is consistent with previous observations [18][19][20][21], but the precision of the scaling that we observe here is surprising. This suggests that the underlying genetic network exhibits true scale invariance, which we now test using the information decomposition [Eq. ...
The body plan of the fruit fly is determined by the expression of just a handful of genes. We show that the spatial patterns of expression for several of these genes scale precisely with the size of the embryo. Concretely, discrete positional markers such as the peaks in striped patterns have absolute positions along the anterior-posterior axis that are proportional to embryo length, with better than 1% accuracy. Further, the information (in bits) that graded patterns of expression provide about position can be decomposed into information about fractional or scaled position and information about absolute position or embryo length; all of the available information is about scaled position, again with ~1% accuracy. These observations suggest that the underlying genetic network exhibits scale invariance in a deeper mathematical sense. Taking this mathematical statement seriously requires that the network dynamics have a zero mode, which connects to many other observations on this system.
... Development of a Drosophila embryo is a highly precise and coordinated process, occurring with little variability despite intrinsic and extrinsic noise and perturbations (Arias and Hayward, 2006;Houchmandzadeh et al., 2002). Proper levels of essential genes and correct positioning of expression patterns are regulated by short non-coding DNA sequences known as enhancers (Banerji et al., 1981). ...
It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.
... This is a more difficult case than the twocuts scenario modeled in many planarian papers, because in the case of two cuts it is easy to visualize a morphogen gradient that sets the pattern of the fragment. However, in the case of one cut, the cells on either side of the cut were direct neighbors (in effect, at the exact same positional information level 58,59 ) and yet have directly opposite anatomical fates (head and tail respectively). This shows that the wound identity cannot be set locally and must arise as the result of long-range communication 5 ; those are the dynamics being modeled here. ...
... A negative flux J < 0 (corresponding to an outflux of SHH from the FP to the NC), on the other hand, shifts the peak concentration dorsally, to the interior of the FP (Fig. 2B). The concentration profile obtained with outflux strongly resembles the reported profiles of SHH in the NT [8] and Bicoid (Bcd) in the early Drosophila embryo [28]. The maximum can be found by requiring that dc/dx = 0. ...
... which is valid in good approximation, considering the reported shape of the Bcd and SHH profiles [8,28]. The subsequent results are qualitatively unaffected by this simplification. ...
During development, cells need to make fate decisions according to their position and the developmental timepoint. Morphogen gradients provide positional information, but how timing is controlled has remained elusive. In the mouse neural tube, morphogen signalling is transient and peaks shortly before the onset of differentiation. Here, we show that in morphogen gradients with constant decay length, cells experience transient, biphasic concentration profiles if the morphogen source expands in parallel with the uniformly growing tissue. This novel patterning paradigm offers a simple mechanism for the simultaneous control of position and time by morphogen gradients, and might apply in many patterning systems, as uniform growth is observed widely in development.
... One candidate mechanism for producing thermal plasticity and different reaction norms of wing spot size in D. guttifera is that the rearing temperature affects the mechanism of determining spot size by Wingless morphogen. The extracellular distribution of morphogens is considered or shown to be plastic to the surrounding temperature (Houchmandzadeh et al. 2002;Eldar et al. 2004;Barkai and Shilo 2009). In the context of color pattern formation in wings, it is considered that changes in the distribution of signaling molecules such as Wingless will alter the outcome patterning (Martin and Reed 2014;Martin and Courtier-Orgogozo 2017;Özsu et al. 2017). ...
Thermal plasticity of melanin pigmentation patterns in Drosophila species has been studied as a model to investigate developmental mechanisms of phenotypic plasticity. The developmental process of melanin pigmentation patterns on wings of Drosophila is divided into two parts, prepattern specification during the pupal period and wing vein-dependent transportation of melanin precursors after eclosion. Which part can be affected by thermal changes? To address this question, we used polka-dotted melanin spots on wings of Drosophila guttifera , whose spot areas are specified by wingless morphogen. In this research, we reared D. guttifera at different temperatures to test whether wing spots show thermal plasticity. We found that wing size becomes larger at lower temperature and that different spots have different reaction norms. Furthermore, we changed the rearing temperature in the middle of the pupal period and found that the most sensitive developmental periods for wing size and spot size are different. The results suggest that the size control mechanisms for the thermal plasticity of wing size and spot size are independent. We also found that the most sensitive stage for spot size was part of the pupal period including stages at which wingless is expressed in the polka-dotted pattern. Therefore, it is suggested that temperature change might affect the prepattern specification process and might not affect transportation through wing veins.
... x C x C e (1) approximate the shape of measured morphogen gradients very well (Kicheva et al., 2007;Gregor et al., 2007;Yu et al., 2009;Wartlick et al., 2011;Cohen et al., 2015;Mateus et al., 2020). For such gradients, the mean readout position to exceed that of their readouts (Houchmandzadeh et al., 2002;Gregor et al., 2007;Zagorski et al., 2017). ...
... Several theories have been proposed to explain the high readout precision despite inevitable noise and variation in morphogen gradients and their readout processes. They include temporal and spatial averaging, self-enhanced morphogen turnover, the use of opposing gradients, dynamic readouts, and cell-cell signalling (Houchmandzadeh et al., 2002;Gregor et al., 2007;Lander et al., 2009;Iwasa, 2009, 2011;Tkačik et al., 2015;Zagorski et al., 2017;Erdmann et al., 2009;Sokolowski and Tkačik, 2015;Ellison et al., 2016;Mugler et al., 2016;Reyes et al., 2022 preprint). In zebrafish, where cells are rather motile, cell sorting and competition can further enhance boundary precision (Xiong et al., 2013;Akieda et al., 2019;Tsai et al., 2020). ...
... Previously, these mechanisms have been mainly analysed on the level of the morphogen readouts-typically transcription factors (TFs)-which are averaged by diffusion between nuclei (Houchmandzadeh et al., 2002;Bialek and Setayeshgar, 2005;Gregor et al., 2007;Erdmann et al., 2009;Sokolowski and Tkačik, 2015;Ellison et al., 2016;Mugler et al., 2016). This is easily possible in a syncytium, as present in the early Drosophila embryo, but the role of TF diffusion in increasing patterning precision has remained controversial (Jaeger and Verd, 2020). ...
Tissue patterning during embryonic development is remarkably precise. In this paper, we numerically determine the impact of the cell diameter, gradient length, and the morphogen source on the variability of morphogen gradients. In this way, we show that the positional error increases with the gradient length relative to the size of the morphogen source, and with the square root of the cell diameter and the readout position. We provide theoretical explanations for these relationships, and show thatthey enable high patterning precision over developmental time for readouts that scale with expanding tissue domains, as observed in the Drosophila wing disc. Our analysis suggests that epithelial tissues generally achieve higher patterning precision with small cross-sectional cell areas. An extensive survey of measured apical cell areas shows that they are indeed small in developing tissues that are patterned by morphogen gradients. Enhanced precision may thus have led to the emergence of pseudostratification in epithelia, a phenomenon for which the evolutionary benefit had so far remained elusive.
... In the French flag model of patterning, morphogen concentration levels determine tissue domains of different cell fates [2]. While development is highly reproducible, reported gradient shapes differ substantially between embryos [3][4][5][6][7]-a seemingly contradictory situation. How a high patterning precision is achieved in spite of considerable noise in morphogen kinetics is therefore an active field of research. ...
... Self-enhanced decay has A B C D Fig. 1: Molecular noise induces variability in gradient shape and readout positions. A Inter-cellular and interembryonic variability in the morphogen production, decay and diffusion rates leads to differently shaped gradients (colors), which attain a concentration threshold C θ at different readout positions x θ,1 ...x θ, 3 . Their standard deviation is termed the positional error. ...
Robust embryonic development requires pattern formation with high spatial accuracy. In epithelial tissues that are patterned by morphogen gradients, the emerging patterns achieve levels of precision that have recently been explained by a simple one-dimensional reaction-diffusion model with kinetic noise. Here, we show that patterning precision is even greater if transverse diffusion effects are at play in such tissues. The positional error, a measure for spatial patterning accuracy, decreases in wider tissues but then saturates beyond a width of about ten cells. This demonstrates that the precision of gradient-based patterning in two- or higher-dimensional systems can be even greater than predicted by 1D models, and further attests to the potential of noisy morphogen gradients for high-precision tissue patterning.
... For example, the ability to perturb regulatory networks at a specific time point is essential for understanding development. 3,4 Several inducible Gal4-UAS systems using small chemicals or heat as the inducers provide high degrees of temporal control over the expression of transgenes and enable the experimenter to modulate the level and persistence of transgene expression by varying the concentrations of the chemicals or the intensity and duration of the heat shock. 5−10 However, these systems suffer from the potential biological side effects of the inducers and the slow OFF kinetics of gene expression caused by the long time required for the chemical inducers to be degraded or metabolized. ...
The light-regulated Gal4-UAS system has offered new ways to control cellular activities with precise spatial and temporal resolution in zebrafish and Drosophila. However, the existing optogenetic Gal4-UAS systems suffer from having multiple protein components and a dependence on extraneous light-sensitive cofactors, which increase the technical complexity and limit the portability of these systems. To overcome these limitations, we herein describe the development of a novel optogenetic Gal4-UAS system (ltLightOn) for both zebrafish and Drosophila based on a single light-switchable transactivator, termed GAVPOLT, which dimerizes and binds to gene promoters to activate transgene expression upon blue light illumination. The ltLightOn system is independent of exogenous cofactors and exhibits a more than 2400-fold ON/OFF gene expression ratio, allowing quantitative, spatial, and temporal control of gene expression. We further demonstrate the usefulness of the ltLightOn system in regulating zebrafish embryonic development by controlling the expression of lefty1 by light. We believe that this single-component optogenetic system will be immensely useful in understanding the gene function and behavioral circuits in zebrafish and Drosophila.
