This data was collected to verify the spatial localization of human fetal tooth germ cell types identified via sn-RNA-seq. Using this and other data, we have proposed a more precise developmental timeline and trajectory for human tooth development.

Description of methods used for collection/generation of data: We used a RNAscope™ HiPlex12 Reagent Kit (488, 550, 650, 750) v2 Standard Assay (Advanced Cell Diagnostics, Inc.) and probes against 13 transcripts VWDE, SALL1, FGF4, IGFBP5, FGF10, PRRX1, FBN2, ENAM, PCDH7, SOX5, KRT5, and either DSPP or LGR6. 

Probe/Channel Key:

• Round 1
  • DAPI R1C1
  • VWDE R1C2
  • SALL1 R1C3
  • FGF4 R1C4
  • IGFBP5 R1C5
• Round 2
  • DAPI R2C1
  • DSPP R2C2
  • LGR6 R2C2
  • FGF10 R2C3
  • PRRX1 R2C4
  • FBN2 R2C5
• Round 3
  • DAPI R3C1
  • PCDH7 R3C3
  • SOX5 R3C4
  • KRT5 R3C5

Fresh frozen human fetal tooth germ tissue sections were stained according to the ACD protocol: https://acdbio.com/sites/default/files/UM%20324409%20RNAscope%20HiPlex%20v2%20User%20Manual%20%28488%2C%20550%2C%20650%2C750%29.pdf and imaged using a Nikon Ti2 with an Aura light engine and BrightLine Sedat filter set optimized for DAPI, FITC, TRITC, Cy5 & Cy7 (LED-DA/FI/TR/Cy5/Cy7-5X5M-A-000) for samples 1-4 or using a Yokogawa CSU-X1 spinning disk confocal microscope with a Celesta light engine and ORCA-Fusion scientific CMOS camera for sample 5.

Methods for processing the data: DAPI channel images for imaging rounds two and three were aligned to the DAPI image for imaging round one using the BigDataViewer > BigWarp plugin in Fiji. The resultant landmark tables were used in a custom .groovy script to align the DAPI, FITC, Cy3, Cy5, and Cy7 images from the three rounds of imaging in Fiji. Aligned images were background corrected and converted to a hyperstack in Fiji.

Describe any quality-assurance procedures performed on the data: Thresholding determined using positive and negative control sections

Instrument- or software-specific information needed to interpret the data: Fiji (ImageJ2 v2.3.0).