Imaris fluorescence quantification and modeling

Flat-fielded image stacks were imported from Fiji into Imaris (Imaris V9.2.1, Bitplane Inc). A baseline subtraction was then performed for each probe, using a cutoff value specific for each probe and fluorophore combination. For all probes, except Hes7, the baseline cutoff was adjusted until signal from a tissue known not to express the gene was no longer detectable (e.g. neural epithelium for Tbx6; Nowotschin et al., 2012; Chapman et al., 1996). For Hes7, this cutoff was determined by using embryos that were homozygous for a Hes7 null allele (Bessho, 2001) that lacked sequences complementary to the HCR probes; the cutoff was chosen that resulted in no signal in these mutants (Figure 5—figure supplement 5).

The Surface model tool was used to build surfaces for each expression domain to be quantified except for Fgf8 and Hes7, which were quantified using a surface derived from the Tbx6 expression domain. Quantification of Spry2, Spry4 and Etv4 were determined by measurement of fluorescence intensity per cubic micrometer within the volume the respective gene expression domain. Surface models were generated using a surface detail value of 3 µM, absolute intensity setting, and an absolute intensity threshold cutoff that was set to exclude background signal in tissues known not to express the gene being modeled. Once established for each probe, the same intensity threshold cutoff values were used for generating surfaces for all embryos. For generating Tbx6 surfaces, a range of absolute intensity threshold cutoffs were used to achieve a final surface volume between 9.0e6 and 1.1e7 µm³. Values for mRNA expression represent the intensity sum within the volumetric model divided by the volume of the model.

In images where NICD was immunostained, the surface creation tool was used. The mesoderm was selected, eliminating the ectodermal and endodermal tissue, on alternating z-planes through the entirety of the z-stack. The resulting pattern was interpolated for intervening planes. Values for NICD expression represent the intensity sum within the volumetric model divided by the volume of the model.

Spot diameter was set to 4 µM with a point spread function value of 8 µM, local background subtraction was used, and sum intensity of the channel being modeled was used to threshold. Threshold cutoff values for spot models were set at 1500 for all figures except Figure 6. In Figure 6 the thresholds values (a.u.) for the sum intensity are: low (blue), 1,000–6,000; low-medium (green), 6,000–11,000; medium (yellow), 11,000–16,000; medium-high (red), 16,000–21,000; high (white), 21,000 or greater. ‘Heat maps’ in Figures 4 and ​and77 were generated within the Imaris software using a linear color scale; the scale in Figure 4 ranged from intensity values of 7000 (blue) to 20,000 (red), the scale in Figure 7 ranged from 1000 (blue) to 15,000 (red).