Gene regulation /function card Anatomical network card Tassy et al., Figure S1: Navigation diagram of ANISEED Anatomical structure card Expression card Gene card Find a gene by its name Find a clone Cis-regulatory card Find Cis-reg regions by name Find genes by their GO annotation Find genes by their Interpro annotation Clone card Search articles by authors or title word Genome browser Blast Keyword search Find territories by their fates Find the progeny/precursors of a territory Find In situ patterns linked to a gene/clone Find genes expressed in a given territory Find in situ patterns linked to a regulatory region Find coexpressed genes at a given stage Digital Differential Display Find a molecular tool Find neighbours of a cell Find cells by their shapes Find asymmetric cleavages Find articles related to a given gene Find articles describing the specification of a given cell fate Article Card Find an anatomical territory on an embryo scheme Display the anatomical ontology for a given stage

Tassy et al., Figure S2: ANISEED menus

Tassy et al., Figure S3 Young adult 8-cell stage Early tailbud stage C.intestinalis, C. savignyi, P. mammillata H. roretzi, B. villosa B A C b8.19 Dorsal TNC ependymal cells Dorsal posterior sensory vesicle Dorsal visceral ganglion B7.5 Anterior ventral primary muscle lineage Trunk ventral cells B7.6 Posterior endodermal strand (B8.11 line) Germ cell precursors (B8.12 line) B7.5 Anterior ventral primary muscle lineage Trunk ventral cells Endoderm b8.19 Dorsal TNC ependymal cells Dorsal posterior sensory vesicle Dorsal visceral ganglion Posterior most secondary muscle lineage B7.6 Posterior endodermal strand (B8.11 line) Germ cell precursors (B8.12 line)

Tassy et al., Figure S4: Three-dimensional replicas of Ciona intestinalis embryos between the 64 and 128 cell stages. Early Gastrula (112-cell) Gastrula (128-cell) Cleavage (76-cell) Cleavage (64-cell)

Tassy et al., Figure S5:The NISEED gene annotation pipeline Gene ontology Functional prediction Motif prediction Interproscan Manual annotation Biological name BlastP Gene models Clustering Clones InparanoidInparanoid ParalogsOrthologs

Basal Promoter: Basal Promoter: : A cis-regulatory region composed of the TSS(s) and binding sites for TF_complexes of the basal transcription machinery. (SO: ) Enhancer: Enhancer: A short cis-regulatory region, located away from the basal promoter and that drives a simple expression pattern. (SO: ) Silencer : Silencer : A cis-regulatory region, which upon binding of sequence-specific transcription factors, represses the transcription of the gene or genes it controls. (SO: ) Insulator : Insulator : A cis-regulatory region that when located between a cis-regulatory module (CRM; enhancer or silencer) and a gene's promoter, prevents the CRM from modulating that gene’s expression (SO: ) Extended Promoter : A Extended Promoter : A 5’ flanking sequence, including basal promoter and cis-regulatory modules, and showing transcriptional activity Complex Region : Complex Region : A genomic region with transcriptional cis-regulatory activity, which can encompass 5’ flanking, intronic and 3’ flanking regions of a gene Untested region : Untested region : a genomic region which has not been experimentally tested. Usually defined as a root sequence including two or more non-overlapping elements (boxed in blue) tested in vivo. Inactive region: Inactive region: a genomic region which has no transcriptional cis-regulatory activity. (SO:010001) InsulatorEnhancerBasal prom.Silencer Gene Exon SilencerEnhancer Tassy et al., Figure S6: Classification of cis-regulatory sequences

Markedly unequal cleavage (index >25%) Weakly unequal cleavage (index >15%) Very strongly unequal cleavage (index >50%) A5.1 A5.2 a5.3 a5.4 B5.2 b5.3 b5.4 A6.2 A6.3 a6.5 a6.7 a6.8 b6.5 b6.6 b6.7 b6.8 A7.7 A7.5 A7.6 A7.8 B7.3 B7.4 B7.7 B7.5 B7.6 B7.8 a7.9 a7.10 a7.11 a7.12 a7.13 a Cell16-Cell32-Cell64-Cell112-Cell8-Cell a 8.17 B 8.5 B 8.6 a 8.18 a 8.19 a 8.20 a 8.25 a 8.26 b 8.19 b 8.20 b 8.21 b 8.22 b 8.25 b 8.26 AB Tassy et al., Figure S7: Unequal cleavages between fertilization and the onset of gastrulation B6.1 B6.2 B6.3 B6.4 b7.9 b7.10 b7.11 b7.12 b7.13 b7.14 B3 B4.1 A6.4 A6.1 b4.2 a6.6 a4.2 A3 A4.1 B5.1 b7.15 b7.16 A8.11 A8.12

Gene Ontology (Localization) Digital Differential Display (Expression) Biometry (Distance) Query integration by ID Lineage Extracellular proteins (815 gene models) Expressed at cleavage stages (348 gene models) Correct spatial expression (2 gene models) In Situ (Spatial expression) Vegetal neighbors at the 16-cell stage of mother of a6.5 (2 cell pairs) Mother of a6.5 cell pair at the 16-cell stage (2 cells) Tassy et al. Figure S8: In silico identification of the anterior neural inducer by sequential querying of heterogeneous data. Molecular queries Anatomical queries

Tassy et al., Figure S9: Automatic inference of regulatory interactions significantly extends the Ciona developmental Gene Regulatory Network. Number of interactions Developmental stage when interactions were analyzed.