Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens

Adaptations in cancer: a molecular and mechanistic rewiring

Cancer cells are constantly subject to complex stresses in the tumour microenvironment. These stresses encapsulate a wide array of challenging stimuli from physical and mechanical forces to interference in major mechanistic signalling cascades. The work in this thesis aims to tackle the question of cancer adaptations in two contexts. First, we investigate the impact of mechanical stress on cancer cells and how subcellular mitochondria redistribution leads to important metabolic and nuclear energetics cues. These cues in turn enable critical DNA damage repair processes which are necessary to maintain correct cell cycle progression. Second, we investigate the loss of ARF6, an important mediator of KRAS signalling in pancreatic cancer, whose loss is compensated through a mechanistic rewiring which activates TLR2 signalling. Dual inhibition of both ARF6 and TLR2 together almost entirely abolishes pancreatic cancer cell proliferation, migration, and spheroid formation in vitro, and tumour growth and metastasis in vivo. Together, the work in this thesis highlights important processes in the mechanical and mechanistic adaptive landscapes of cancer.Las células cancerosas están constantemente sometidas a complejos factores de estrés en el microambiente tumoral. Estos factores de estrés engloban una amplia variedad de estímulos que van desde fuerzas físicas y mecánicas hasta interferencias en las principales cascadas de señalización. El trabajo de esta tesis tiene como objetivo abordar la cuestión de las adaptaciones del cáncer en dos contextos. Primero, investigamos el impacto del estrés mecánico en las células cancerosas y cómo la redistribución subcelular mitocondrial conduce a importantes señales energéticas metabólicas y nucleares. Estas señales, a su vez, permiten procesos críticos de reparación del daño del ADN que son necesarios para mantener la correcta progresión del ciclo celular. En segundo lugar, investigamos la pérdida de ARF6, un mediador importante de la señalización de KRAS en el cáncer de páncreas, cuya pérdida se compensa a través de un mecanismo de adaptación que activa la señalización de TLR2. La inhibición simultanea de ARF6 y TLR2 inhibe prácticamente por completo la proliferación, migración y formación de esferoides en células de cáncer de páncreas in vitro, así como el crecimiento tumoral y la metástasis in vivo. En conjunto, el trabajo de esta tesis enfatiza adaptaciones de procesos mecánicos y cascadas de señalización del cáncer.Programa de Doctorat en Biomedicin

A role for the fusogen eff-1 in epidermal stem cell number robustness in Caenorhabditis elegans

Developmental patterning in Caenorhabditis elegans is known to proceed in a highly stereotypical manner, which raises the question of how developmental robustness is achieved despite the inevitable stochastic noise. We focus here on a population of epidermal cells, the seam cells, which show stem cell-like behaviour and divide symmetrically and asymmetrically over post-embryonic development to generate epidermal and neuronal tissues. We have conducted a mutagenesis screen to identify mutants that introduce phenotypic variability in the normally invariant seam cell population. We report here that a null mutation in the fusogen eff-1 increases seam cell number variability. Using time-lapse microscopy and single molecule fluorescence hybridisation, we find that seam cell division and differentiation patterns are mostly unperturbed in eff-1 mutants, indicating that cell fusion is uncoupled from the cell differentiation programme. Nevertheless, seam cell losses due to the inappropriate differentiation of both daughter cells following division, as well as seam cell gains through symmetric divisions towards the seam cell fate were observed at low frequency. We show that these stochastic errors likely arise through accumulation of defects interrupting the continuity of the seam and changing seam cell shape, highlighting the role of tissue homeostasis in suppressing phenotypic variability during development.Funding: Some C. elegans strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was funded by the European Research Council (ROBUSTNET-639485

A metabolic map of the DNA damage response identifies PRDX1 in the control of nuclear ROS scavenging and aspartate availability

Data de publicació electrònica: 01-06-2023While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential for the resolution of DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Further analysis identified that Peroxiredoxin 1, PRDX1, contributes to the DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it reduces DNA damage-induced nuclear reactive oxygen species. Moreover, PRDX1 loss lowers aspartate availability, which is required for the DNA damage-induced upregulation of de novo nucleotide synthesis. In the absence of PRDX1, cells accumulate replication stress and DNA damage, leading to proliferation defects that are exacerbated in the presence of etoposide, thus revealing a role for PRDX1 as a DNA damage surveillance factor.AM and CC were funded by the Austrian Science Fund (grant number P 33024 awarded to JIL). The Loizou lab is funded by an ERC Synergy Grant (DDREAMM Grant agreement ID: 855741). The Sdelci lab's contributions to this study were funded by an ERC Starting Grant (ERC-StG-852343-EPICAMENTE). This work was funded, in part, by a donation from Benjamin Landesmann. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. CeMM is funded by the Austrian Academy of Sciences. MGVH acknowledges funding from R35CA242379, the Lustgarten Foundation, the Ludwig Center at MIT, and the MIT Center for Precision Cancer Medicine

The <i>icb38</i> mutation represents a loss of function mutation in <i>lin-22</i>.

<p>(A) Illustration of the <i>lin-22(icb38)</i> mutation, which is a 3,329 bp deletion removing part of the distal <i>lin-22</i> promoter (2,371 bp upstream of the <i>lin-22</i> ATG). The deletion also removes part of the third exon and 3′ UTR of the upstream gene Y54G2A.3. The deleted part is replaced by a 1,733 bp insertion consisting of exon 7 and parts of introns 6 and 7 of the downstream gene <i>mca-3</i>. The position of other <i>lin-22</i> alleles described in the manuscript is shown on the wild-type sequence. (B) Quantification of the number of PDE neurons (<i>dat-1</i>∷<i>GFP</i> foci) in the EMS-derived <i>lin-22(icb-38)</i> mutant and CRISPR-derived <i>lin-22</i> mutants (<i>n</i> ≥ 30). Reference sample is <i>egIs1</i> containing only the marker. (C) Quantification of seam cell number in <i>lin-22(icb38)</i> and other CRISPR-derived <i>lin-22</i> mutants (<i>n</i> ≥ 30). Note an increase in seam cell number variance in <i>lin-22</i> mutants depicted with red stars. Black stars show statistically significant changes in the mean with one-way ANOVA followed by the Dunnett test, and red stars depict changes in variance with a Levene’s median test (in both cases, **** corresponds to <i>P</i> value < 0.0001). Error bars show mean ± SEM (B) or mean ± SD (C). Numerical data used for Fig 2B, C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; EMS, ethyl methanesulfonate; GFP, green fluorescent protein; PDE, post-deirid; scm, seam cell marker; UTR, untranslated region.</p

Quantification of <i>lin-22</i> expression in the seam.

<p>(A) Transgenic animals carrying transcriptional reporters consisting of various fragments of upstream of <i>lin-22</i> sequences fused to GFP. From top to bottom: full <i>lin-22</i> endogenous promoter, distal <i>lin-22</i> promoter region that is deleted in <i>lin-22(icb38)</i>, proximal <i>lin-22</i> promoter present in <i>lin-22(icb38)</i>, distal <i>lin-22</i> promoter with deleted CR1, and CR1 only driving expression of GFP. White arrows indicate expression in the seam cells; white arrowheads expression in the hypodermis and green arrowheads expression in intestinal cells. (B) Quantification of expression pattern for each transcriptional reporter (<i>n</i> ≥ 35 animals). (C) Representative smFISH images showing <i>lin-22</i> expression (black spots correspond to mRNAs and seam cells are labelled in green due to <i>scm</i>∷<i>GFP</i> expression) in wild-type V cells after the symmetric L2 division (top), the L2 asymmetric division (middle), and late after the L2 asymmetric division (bottom). (D) Quantification of <i>lin-22</i> spots per seam cell in wild-type and <i>lin-22(icb38)</i> animals at the late L1 stage (<i>n</i> ≥ 10 cells per genotype). (E) Quantification of <i>lin-22</i> spots in wild-type, <i>lin-22(ot267)</i>, <i>lin-22(ot269)</i>, and <i>lin-22(icb49)</i> mutants in pools of H cells and V cells at the late L1 stage (<i>n</i> ≥ 41). (F-G) Comparison of number of <i>lin-22</i> spots between wild-type and the <i>elt-1(ku491)</i> mutant (F) or the <i>egl-18(ok290)</i> mutant, (G) both at the late L1 stage in pools of H and V cells (<i>n</i> ≥ 49). Black stars show statistically significant changes in the mean with a <i>t</i> test or one-way ANOVA as follows: * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, **** <i>P</i> < 0.0001. Reference samples for comparisons in E, F, G are the control samples depicted in black. Scale bars in A and C are 100 μm and 10 μm, respectively. Error bars show mean ± SEM (D, F, G) or mean ± SD (E). Numerical data used for Fig 3B, D, E, F, G can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. CR1, conserved region 1; GFP, green fluorescent protein; L1, first larval stage; L2, second larval stage; smFISH, single molecule fluorescent in situ hybridization.</p

Context-dependent gain and loss of variability in <i>lin-22(icb38)</i> mutants.

<p>(A) Quantification of brood size in wild-type (<i>n</i> = 13) and <i>lin-22(icb38)</i> mutants (<i>n</i> = 15). Black bars show mean ± SEM. (B) Quantification of P3.p division frequency in wild-type and <i>lin-22(icb38)</i> animals. Note that almost all mutant animals show division of P3.p. (C) Model showing <i>lin-22</i> interactions discovered in this study (orange), while previously known interactions are shown with dashed grey lines. These new interactions may not be direct. Seam cell number variability is increased in <i>lin-22</i> mutants due to loss and gain of symmetric divisions. Stochastic loss of symmetric divisions at the L2 stage generates more neuroblasts at the expense of seam cells. Stochastic gain of symmetric divisions towards the seam cell fate mostly at the L3/L4 stage generates more seam cells. Cell-to-cell variability in Wnt pathway activation correlates with phenotypic variability. Numerical data used for Fig 7A, B can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. L2, second larval stage; L3, third larval stage; L4, fourth larval stage.</p

Gene expression changes associated with loss and gain of daughter cell fate symmetry in <i>lin-22</i> mutants.

<p>(A-B) Representative smFISH images and quantification of wild-type and <i>lin-22(icb38)</i> animals at the L2 symmetric division stage using an <i>elt-1</i> probe. (A) Comparable amounts of <i>elt-1</i> spots are detected in wild-type V cell daughters and more spots in the posterior H2 daughter. In <i>lin-22(icb38)</i> animals, more spots are detected in the posterior V daughters than the anterior (marked by arrowheads) and even numbers in the 2 H2.p daughters (arrow points to anterior H2.p daughter cell). (B) Quantification of <i>elt-1</i> expression in H2.p daughters (<i>n</i> > 9) and pools of V.p daughter cells (<i>n</i> > 41) of wild-type and <i>lin-22(icb38)</i> animals at the L2 symmetric division stage. (C) <i>mab-5</i> expression expands to posterior daughters of V1–V4 cells (arrowheads) in <i>lin-22(icb38)</i> animals, reminiscent of the expression in the posterior V5 cell in wild-type (arrow). (D-F) <i>egl-18</i> smFISH images and quantification of wild-type and <i>lin-22(icb38)</i> animals. (D) Quantification of <i>egl-18</i> smFISH spots in the H2.p cell daughters at the L2 stage (<i>n</i> ≥ 21). (E) Images depicting <i>egl-18</i> expression at the L3 stage. Note expression in anterior daughter cells in <i>lin-22(icb38)</i> animals (arrowheads). (F) Quantification of <i>egl-18</i> smFISH spots in V1-V4 cells (<i>n</i> ≥ 68) of wild-type and <i>lin-22(icb38)</i> animals at the L2 asymmetric division stage. (G) Representative <i>eff-1</i> smFISH images of wild-type and <i>lin-22(icb38)</i> animals at the L3 asymmetric division stage. Note absence of signal in the most anterior of the 4 daughter cells in <i>lin-22(icb38)</i> animals (arrowheads). H2 consists of 4 cells that have arisen due to symmetric division at the L2 stage. (H) Quantification of seam cell number in wild-type (<i>n</i> = 39), <i>lin-22(icb38)</i> (<i>n</i> = 29), <i>eff-1(hy21)</i> (<i>n</i> = 31), and <i>lin-22(icb38)</i>; <i>eff-1(hy21)</i> (<i>n</i> = 35) animals. Note that the <i>eff-1(hy21)</i> does not show a significant difference in seam cell numbers compared to wild-type, but the double mutant does in comparison to both parental strains. Black stars show statistically significant changes in the mean with a <i>t</i> test or one-way ANOVA /Dunnett’s test. Scale bars in A, C, E, and G are 10 μm; black spots correspond to mRNAs and green labels the seam cell nuclei. Error bars in B, D, F, H show mean ± SD. Numerical data used for Fig 5B, D, F, H can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. GFP, green fluorescent protein; L2, second larval stage; L2sym, symmetric first division at the L2 stage; L3, third larval stage; SCM, seam cell marker; smFISH, single molecule fluorescent in situ hybridization.</p

The developmental basis of variability in <i>lin-22</i> mutants.

<p>(A) The upper panel shows wild-type seam cell lineages, while the bottom panel indicates the most frequently occurring errors in <i>lin-22(icb38)</i> mutants (<i>n</i> = 14 independent complete lineages). The developmental errors are grouped for simplicity in 4 main classes and presented as a function of the developmental stage. The percentages refer to occurrence of these errors within the total number of relevant cell lineages. Note that the errors described are not independent, so they can occur within the same animal and even within the same lineage (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s010" target="_blank">S4 Fig</a>). (B) Heat map showing the frequency of errors per cell lineage and developmental stage (<i>n</i> = 14 lineages). Blue depicts errors leading to gain of terminal cell number due to gain of symmetric division, and red depicts errors leading to loss of symmetric division. (C-D) Quantification of seam cell number (C) and number of PDE neurons (D) in wild-type (<i>n</i> ≥ 36) and <i>lin-22(icb38)</i> animals (<i>n</i> ≥ 30) treated with control or <i>lin-28</i> RNAi. Black stars show statistically significant changes in the mean with a <i>t</i> test (<i>P</i> < 0.0001). Error bars show mean ± SD (C) or mean ± SEM in (D). Numerical data used for Fig 4B, C, D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. L2, second larval stage; L3, third larval stage; L4, fourth larval stage; PDE, post-deirid; RNAi, RNA interference.</p

Recovery and mapping of mutants with variable seam cell number.

<p>(A) Cartoon illustrating the seam cell lineages in WT. Fluorescent images show expression of 10 <i>scm</i>∷<i>GFP</i> (<i>wIs51</i>) positive cells at the L1 (above) and 16 cells at the early adult stage (below). (B) Design of the genetic screen to recover mutants with a Vsc phenotype as opposed to Msc or Lsc, based on selection of extreme seam cell number at the F2 generation. Control represents representative data for JR667 (<i>wIs51</i>) strain, black bar shows mean ± SD. (C) Relationship between SD and mean scn. Each point represents an independently recovered mutant from our EMS screen. Control strain JR667 is depicted in blue and the <i>vsc1</i> mutant in red. (D) <i>vsc1</i> mutants show variable seam cell numbers (SD = 0.33, <i>n</i> = 278 animals for control JR667, and SD = 1.87, <i>n</i> = 563 for <i>vsc1</i> mutants). Note that only 1 animal shows extreme seam cell counts in this experiment in WT. Error bar shows mean ± SD and red stars depict statistically significant change in variance in relationship to control with a Levene’s median test (<i>P</i> < 0.0001). Numerical data used for Fig 1, B, C, D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002429#pbio.2002429.s003" target="_blank">S2 Data</a>. SD, standard deviation; EMS, ethyl methanesulfonate; GFP, green fluorescent protein; L1, first larval stage; Vsc, variable seam cell number phenotype; Lsc, less seam cells phenotype; Msc, more seam cells phenotype; SCM, seam cell marker; scn, seam cell number; WT, wild-type.</p