Stage 6

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 begin to flatten and elongate, with the anterior head margin becoming apparent. The posterior portion of the embryo usually appears larger and more bulbous than the head prior to formation of the tail stripe, a muscular septum connecting the dorsal and ventral surfaces that bifurcates the posterior gut. Embryos remain unpigmented and immotile during S6. Differentiation of the definitive organ systems continues. Parenchymal epidermal progenitors are abundant and are more densely packed on the dorsal side of the embryo, and are often more numerous in the anterior. Fusiform muscle cells, once uniformly distributed across the sphere, become more densely packed on the presumptive dorsal side of the elongating animal. The gut cavity, still filled with yolk, is ovoid and largely unbranched. Differentiated gut cells expressing embryonic gut markers are detected through S6; gut cells expressing definitive gut markers increase during S6. The definitive pharynx develops beneath the degenerating temporary embryonic pharynx on the ventral side of the embryo, near the presumptive posterior pole. Aggregations of differentiating neurons accumulate along the anterior head margin, along the presumptive dorsoventral boundary, and constellations of nerve cells form the ventral nerve cords, beginning from the base of the cerebral ganglia and progressively proceeding posteriorly. Although eye progenitors are present and differentiating, the eyes are generally not visible in live specimens during S6.

 

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Images

Brightfield images of live Stage 6 (S6) embryos harvested for RNA-Sequencing (left), a paraffin embedded S6 embryo sectioned coronally and stained with hematoxylin and eosin (middle), and heat maps for S6 enriched transcripts (right). Black arrowhead: definitive pharynx. Scale bars: 100 µm.

 

Figure 1 – figure supplement 8. Mean centered expression and average RPKM profiles for S6 enriched transcripts. Organized by cluster membership presented shown in the heat map and S6 enriched transcript excel file (Figure 1 – source data 6). S2-S8: Stages 2-8. Y: Yolk.

 

 

 

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Download Supplemental Table

 

 

Figure 1 – source data 6. S6 enriched transcripts from pairwise and/or mixed stage reference comparisons
Criteria for flagging differentially expressed transcripts:

1) Pairwise comparison (S5 vs S6): adjusted p-value < 1e-5 S6 vs mixed reference comparison: adjusted p-value <1e-20 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 S6 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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