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1 VOLUME: ARTICLE NUMBER: 3 In the formt provided y the uthors nd unedited. Developmentl constrints shpe the evolution of the nemtode mid-developmentl trnsition Hrel Zlts nd Iti Yni,3 Fculty of Biology, Technion Isrel Institute of Technology, Hif, Isrel Institute for Computtionl Medicine, NYU School of Medicine, USA 3 Corresponding uthor: Iti.Yni@nyumc.org SUPPLEMENTARY FIGURE CAPTIONS Figure S: Quntifying iologicl nd technicl gene expression vrition., Person correltion het mp for the iologicl replictes, the MA 5 strin time courses. The het mp represents correltions for the dynmiclly expressed genes (see Methods)., Boxplots indicting the correltions etween smples of the sme stge cross the strins (lue) nd MA 5 qudruplictes (yellow). c, Flowchrt representing the steps for computing the evolvility score in this study. Figure S: Clustering the dt using sliding window nlysis., The expression pttern for ech of the 55 sliding window clusters shown in Figure S., to find the corresponding stge represented within ech sliding window, we looked for highly expressed genes in ech stge with ech window. The het mp shows this enrichment. White circles indicte the inferred windows of stge-specific expression (lso see Methods). NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.
2 Figure S3: Controlling for technicl vrition., Het mp representing the expression pttern of rndomly sorted genes., E S distriution when gene order is rndom, s presented in. c-g, To test whether the result is ised y the numer of genes expressed in ech stge, we used k-mens clustering to produce distinct gene expression sugroups. c, Men expression pttern of the expression clusters we used for this nlysis. From ech sugroup, we rndomly selected genes, nd repeted the sliding window nlysis on these genes. We found similr results for the MA strins (d, e) providing further evidence tht mid-emryogenesis is highly conserved cross MA strins, while in the replictes (f, g) the signl is low nd insignificnt (Kolmogorov Smirnov test, P< -4 ). Figure S4: Compring whole emryo dt with seprte lineges cross emryonic time revels n emryonic checkpoint t mid-development., Experimentl design of the in vitro lineges experiment 33, where lstomeres were seprted nd llowed to proliferte individully, Trnscriptome correltion het mp etween whole emryos (y-xis) from this experiment nd the sum of in vitro lineges (x-xis), where stges were defined y E cell linege divisions. Note the high correspondence proceeding up to the phylotypic stge in whole emryos, nd then diminishes. For exmple, the stge smple in the in vitro experiment (n overnight smple) is most similr to whole-emryo stge 4, rther to the lrve stge. NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.
3 Supplementry Tle : Gene Ontology terms enriched in the integrtion genes group. SUPPLEMENTARY INFORMATION Gene ontolgy term p-vlue emryonic development ending in irth or egg htching.94e-8 reproduction 3.6E-6 nemtode lrvl development 6.59E-9 ATP inding.6e-8 growth.97e-8 protein inding 7.33E-8 nucleic cid inding 6.7E-7 hermphrodite genitli development.9e-6 receptor-medited endocytosis.3e-6 zinc ion inding 7.33E-6 nucleotide inding.e-5 positive regultion of growth rte.e-5 inding.6 DNA inding.7 cytokinesis.84 trnsltion. Hedgehog singling.33 protein mino cid phosphoryltion.6 protein serine/threonine kinse ctivity.74 ctlytic ctivity.78 protein kinse ctivity.8 cell division.869 determintion of dult life spn.979 structurl constituent of riosome.5 RNA interference.678 regultion of trnscription, DNA-dependent.35 morphogenesis of n epithelium.447 emryonic development.4776 oxidtion reduction.4839 molting cycle, collgen nd cuticulin-sed cuticle.656 intrcellulr protein trnsport.6665 structurl constituent of cuticle.6683 trnsport.683 serine-type endopeptidse inhiitor ctivity.7577 RNA inding.8648 trnscription fctor ctivity.6 inductive cell migrtion.973 locomotion.35 oxidoreductse ctivity.3633 trnsmemrne trnsport.55 strited.6558 metolic process.973 mitotic spindle orgniztion.889 GTP inding.5 cell communiction.33 signl trnsduction.34 protein trnsport.347 helicse ctivity.569 NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.
4 SUPPLEMENTARY INFORMATION Supplementry Figure R.5 Correltion MA lines Replictes c Reference strin : the men expression in the strins for ech gene: Expression dt from muttion-ccumultion strins N N Temporlly rrnge genes in ech strin using ZAVIT Assign indices for ech gene in ech strin Gene index ref Strins Clculte the distnce for ech gene etween ech strin nd the reference Clculte the men distnce for ech gene - Evolvilty Score NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved. 4
5 Supplementry figure Expression (stndrdized) most correlted with sliding window cluster NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.
6 Supplementry Figure 3 Expression level (stndrdized) Evolvilty (E S ) c d e f g Expression level (stndrdized) Expression level (stndrdized) - Divergent Conserved Expression level (stndrdized) - Divergent Conserved Evolvilty (E S ) Evolvilty (E S ) NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.
7 Supplementry Figure 4 AB E MS C P3... Whole emryo (this study) R In vitro lineges (Hshimshony et l., 5) E 4 cells cells E 4E E+ 8E 4E+ over-night 8E++ 8E+ Sum of in vitro lineges NATURE ECOLOGY & EVOLUTION DOI:.38/s Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.
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