Capicua controls Toll/IL-1 signaling targets independently of RTK regulation.

The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here, we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-κB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.

Signaling pathways in mammalian preimplantation development: Linking cellular phenotypes to lineage decisions.

The first stages of mammalian development, before implantation of the embryo in the maternal uterus, result in the establishment of three cell populations in the blastocyst: trophectoderm, epiblast, and primitive endoderm. These events involve only a small number of cells, and are initiated by morphological differences among them related to cell adhesion and polarity. Much attention has been paid to the master transcription factors that are critical for establishing and maintaining early lineage choices. Nevertheless, a large body of work also reveals that additional molecular mechanisms are involved. Here, we provide an updated view of the role of different signaling pathways in the first stages of mouse development, and how their cross-talk and interplay determine the initial lineage decisions occurring in the blastocyst. We will also discuss how these pathways are critical for translating cellular phenotypes, the product of the morphogenetic events occurring at these stages, into transcriptional responses and expression of lineage-specifying transcription factors. Developmental Dynamics 246:245-261, 2017. © 2016 Wiley Periodicals, Inc.

Mirror represses pipe expression in follicle cells to initiate dorsoventral axis formation in Drosophila.

Dorsoventral (DV) axis formation in Drosophila begins with selective activation of EGFR, a receptor tyrosine kinase (RTK), in dorsal-anterior (DA) ovarian follicle cells. A critical event regulated by EGFR signaling is the repression of the sulfotransferase-encoding gene pipe in dorsal follicle cells, but how this occurs remains unclear. Here we show that Mirror (Mirr), a homeodomain transcription factor induced by EGFR signaling in DA follicle cells, directly represses pipe expression by binding to a conserved element in the pipe regulatory region. In addition, we find that the HMG-box protein Capicua (Cic) supports pipe expression in ventral follicle cells by repressing Mirr in this region. Interestingly, this role of Cic resembles its function in regulating anteroposterior (AP) body patterning, where Cic supports gap gene expression in central regions of the embryo by repressing Tailless, a repressor induced by RTK signaling at the embryonic poles. Thus, related RTK-Cic repressor circuits regulate the early stages of Drosophila DV and AP body axis formation.

RTK signaling modulates the Dorsal gradient.

The dorsoventral (DV) axis of the Drosophila embryo is patterned by a nuclear gradient of the Rel family transcription factor, Dorsal (Dl), that activates or represses numerous target genes in a region-specific manner. Here, we demonstrate that signaling by receptor tyrosine kinases (RTK) reduces nuclear levels and transcriptional activity of Dl, both at the poles and in the mid-body of the embryo. These effects depend on wntD, which encodes a Dl antagonist belonging to the Wingless/Wnt family of secreted factors. Specifically, we show that, via relief of Groucho- and Capicua-mediated repression, the Torso and EGFR RTK pathways induce expression of WntD, which in turn limits Dl nuclear localization at the poles and along the DV axis. Furthermore, this RTK-dependent control of Dl is important for restricting expression of its targets in both contexts. Thus, our results reveal a new mechanism of crosstalk, whereby RTK signals modulate the spatial distribution and activity of a developmental morphogen in vivo.

Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila.

RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

A protocol to study three-dimensional genome structure in individual mutant preimplantation mouse embryos.

Hi-C studies the three-dimensional structure of the genome by detecting genome-wide chromatin regions that are in spatial proximity within the nucleus. We developed single-blastocyst Hi-C in mutant mouse embryos to genotype them on sequence. We describe steps for embryo fixation and nuclei permeabilization, after which chromatin is digested and re-ligated having incorporated a biotin-labeled nucleotide at the ligation junction. After cross-link reversal, we then detail purification of immobilized chimeric DNA ligations, library generation, sequencing, and genome-wide analysis of interactions. For complete details on the use and execution of this protocol, please refer to Andreu et al. (2022).1.

Influence of physical fitness components on personality factors and risk perception of children and adolescents: a cross-sectional study.

OBJECTIVES: To examine the associations of cardiorespiratory fitness (VO2 max) and muscular strength with indicators related to the risk scale, such as perceived competence, sensation seeking, competitiveness, risk taking and risk perception in sports. DESIGN: Cross-sectional study. SETTING: High schools from the Region of Murcia (Spain). PARTICIPANTS: Three-hundred-and-seventeen adolescents participated (mean age: 13.69±1.2 years old). PRIMARY AND SECONDARY OUTCOME MEASURES: Body mass, body height, Course-Navette test, upper limb strength and psychoeducational factors that determine the propensity towards sports accidents in school children, the Sports Accident Propensity Scale were evaluated. It was performance t-test for independent samples, stepwise multiple linear regression models and a multiple mediation analysis. RESULTS: The analysis showed significant differences with respect to sex in height, VO2 max, handgrip strength and in all factors of the questionnaire (p=0.02-<0.01). Adolescents who presented greater VO2 max, strength in the handgrip test and age showed a higher score in factors 1 and 3. Higher scores in factor 2 were associated with better VO2 max and strength in handgrip test. Youngers and better values of strength in the handgrip showed higher score in factors 4 and 5. The mediation analysis with two mediating variables (handgrip strength and VO2 max) showed a significant indirect effect. When handgrip strength and VO2 max were included in the equations, the association between sex and each factor ceased to be significant. CONCLUSION: This study highlights the potential benefits of muscular strength (handgrip) and VO2 max in the perceived risk scale, and the variable of age on this. TRIAL REGISTRATION NUMBER: Clinical trial: NCT05544370 (pre-results).

Chromatin dynamics through mouse preimplantation development revealed by single molecule localisation microscopy.

Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.

Transitions in cell potency during early mouse development are driven by Notch.

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naïve pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

EGFR-dependent downregulation of Capicua and the establishment of Drosophila dorsoventral polarity.

Dorsoventral (DV) axis formation in Drosophila begins during oogenesis through the graded activation of the EGF receptor (EGFR)-Ras-MAPK signaling pathway in the follicle cell layer of the egg chamber. EGFR signaling, which is higher in dorsal follicle cells, represses expression of the sulfotransferase-encoding gene pipe, thereby delimiting a ventral domain of Pipe activity that is critical for the subsequent induction of ventral embryonic fates. We have characterized the transcriptional circuit that links EGFR signaling to pipe repression: in dorsal follicle cells, the homeodomain transcription factor Mirror (Mirr), which is induced by EGFR signaling, directly represses pipe transcription, whereas in ventral follicle cells, the HMG-box protein Capicua (Cic) supports pipe expression by repressing mirr. Although Cic is under negative post-transcriptional regulation by Ras-MAPK signaling in different contexts, the relevance of this mechanism for the interpretation of the EGFR signal during DV pattern formation remains unclear. Here, we consider a model where EGFR-mediated downregulation of Cic modulates the spatial distribution of Mirr protein in lateral follicle cells, thereby contributing to define the position at which the pipe expression border is formed.

Gene regulation by MAPK substrate competition.

Developing tissues are patterned by coordinated activities of signaling systems, which can be integrated by a regulatory region of a gene that binds multiple transcription factors or by a transcription factor that is modified by multiple enzymes. Based on a combination of genetic and imaging experiments in the early Drosophila embryo, we describe a signal integration mechanism that cannot be reduced to a single gene regulatory element or a single transcription factor. This mechanism relies on an enzymatic network formed by mitogen-activated protein kinase (MAPK) and its substrates. Specifically, anteriorly localized MAPK substrates, such as Bicoid, antagonize MAPK-dependent downregulation of Capicua, a repressor that is involved in gene regulation along the dorsoventral axis of the embryo. MAPK substrate competition provides a basis for ternary interaction of the anterior, dorsoventral, and terminal patterning systems. A mathematical model of this interaction can explain gene expression patterns with both anteroposterior and dorsoventral polarities.