Stage 4

Developmental timeline and staging designations for Smed embryogenesis. Timeline: days (d) post egg capsule deposition at 20˚C. Gray bars indicate time windows for RNA-Sequencing samples. Double headed arrows: time windows for Stages (S) S1-S8.

 


 

▻ Overview

▻ Stage Description

▻ Images

▻ Download Supplemental Table

▻ In Situ Hybridization Data

▻ Sequences

 


 

Overview

 

 

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Stage Description

 

Blastomeres continue to divide asynchronously in the embryonic wall. The temporary embryonic pharynx ingests yolk cells into the gut cavity of spherical embryos, ranging in size from ~300-500 µm in diameter. Islands of differentiating gut cells appear within and beneath the parenchyma. It is not known whether the embryonic gut created during stage 4 is a temporary or definitive tissue.

 

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Images

Brightfield images of live Stage 4 (S4) embryos harvested for RNA-Sequencing (left), a paraffin embedded S4 embryo sectioned and stained with hematoxylin and eosin (middle), and heat maps for S4 enriched transcripts (right). Yellow arrowhead: temporary embryonic pharynx. Scale bars: 100 µm.

 

Figure 1 – figure supplement 6. Mean centered expression and average RPKM profiles for S4 enriched transcripts. Organized by cluster membership presented shown in the heat map and S4 enriched transcript excel file (Figure 1 – source data 4). S2-S8: Stages 2-8. Y: Yolk.

 

 

 

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Download Supplemental Table

 

 

Figure 1 – source data 4. S4 enriched transcripts from pairwise and/or mixed stage reference comparisons
Criteria for flagging differentially expressed transcripts:

1) Pairwise comparison (S3 vs S4): adjusted p-value < 1e-5 S4 vs mixed reference comparison: adjusted p-value <1e-5 2) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only) 3) Average scaled RPKM value ≥ 1.0 for S4 4) Transcript has ≥ 1 ORF 5) Transcripts derived from transposase or retroviruses were omitted, as were S4 enriched transcripts also upregulated in yolk in the mixed reference comparison.

Tabs in this excel file contain 1) pairwise comparison data (if applicable), 2) mixed stage reference comparison data, 3) cluster membership, average RPKM values across embryogenesis (Y-S8), and in C4 and SX adults, as well as best BLASTx hits (E < 0.001) versus the NR, Swiss-Prot, C. elegans, D. melanogaster, D. rerio, X. tropicalis, M. musculus and H. sapiens REF-Seq databases, 4) GO analysis: Manually curated and categorized Biological Process (BP) GO IDs and 5) GO analysis: unabridged results. S2-S8: Stages 2-8. Y: Yolk. C4: C4 asexual adults. SX: Mature sexual adults

 

 

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In Situ Hybridization Data

Smed IDGene NameAliasExpression PatternWISH Image
Click to see image symbols and abbreviations
Abbreviation or symbolDefinition
Ooral hemisphere
Aaboral hemisphere
Ddorsal
Vventral
Llateral
black arrowheadembryonic pharynx
red arrowheaddefinitive pharynx
black arrowsprimitive gut
yellow arrowsprimitive ectoderm cells
cyan arrowsbrain
cyan arrowheadsnerve cords
blue arrowheadseye progenitors (trail cells)
purple arrowheadseyes
scale bar100 µm

 

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Please provide a type, feature name or genus

Sequences

smed_20140614 transcript sequences for genes validated by in situ hybridization (above).

 

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