Stage 5

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

 

Spherical embryos continue to grow via yolk ingestion, reaching maximal size by the end of Stage 5 (~400-800 µm in diameter). Blastomeres fill the parenchyma, forming a dense collection of cells blanketing the sphere. Yolk-laden gut cells generate a continuous, honeycomb-like lattice encircling the central ingested yolk mass. Many early embryo enriched transcripts expressed in S4 blastomeres are downregulated during S5. Progenitors for many of the definitive organs, including the epidermis, nervous system, muscle, gut, pharynx, protonephridia, eyes and primordial germ cells first appear during Stage 5, marking the onset of organogenesis. Pluripotent neoblasts and adult lineage progenitors arise in the blastomere population during S5.

 

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Images

Brightfield images of live Stage 5 (S5) embryos harvested for RNA-Sequencing (left), a paraffin embedded S5 embryo sectioned and stained with hematoxylin and eosin (middle), and heat maps for S5 enriched transcripts (right). Yellow arrowhead: temporary embryonic pharynx. Scale bars: 100 µm.

 

Figure 1 – figure supplement 7. Mean centered expression and average RPKM profiles for S5 enriched transcripts. Organized by cluster membership presented shown in the heat map and S5 enriched transcript excel file (Figure 1 – source data 5). S2-S8: Stages 2-8. Y: Yolk.

 

 

 

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Download Supplemental Table

 

 

Figure 1 – source data 5. S5 enriched transcripts from pairwise and/or mixed stage reference comparisons
Criteria for flagging differentially expressed transcripts:

1) Pairwise comparison (S4 vs S5): adjusted p-value < 1e-5 S5 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 S5 4) Transcript has ≥ 1 ORF 5) Transcripts derived from transposase or retroviruses were omitted.

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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