Fig 2: XEN cells contribute to the parietal endoderm of chimeric embryos.(a–c) Pre-XEN cell line X-ICM-4 contributes to the PE of E6.5 and E7.5 embryos. GFP+ cells are derived from the injected pre-XEN cells. (a) Wholemount of E7.5 embryo. (b,c) Sections of the decidua of E6.5 embryos, showing the merged image of immunofluorescence for PDGFRa (red) and E-cadherin (blue), and fluorescence of DAPI (white) and GFP (green). (d–k) Post-XEN cell lines (X-E5.5-9, X-E6.5-Z0617-5, and X-E6.5-Z0617-2) contribute to the PE of E6.5 or E7.5 embryos. (d–f) Wholemount. GFP+ cells derived from X-E5.5-9 contribute to an E7.5 embryo. (g) Wholemount. GFP+ cells derived from X-E6.5-Z0617-5 contribute to an E7.5 embryo. (h,i) Wholemount. GFP+ cells derived from X-E6.5-Z0617-2 contribute to an E6.5 embryo. (j,k) Sections of the decidua of E7.5 embryos containing GFP+ cells derived from X-E6.5-Z0617-5, with immunofluorescence for PDGFRa (red) and E-cadherin (blue), fluorescence of DAPI (white) and GFP (green), and merge. The single GFP+ cell in (k) apposes but does not belong to the VE, as it is not immunoreactive for E-cadherin. (a–g) and (j,k) were imaged using a Zeiss LSM 710 confocal microscope, and (h–i) were imaged using a Nikon SMZ25 stereofluorescence microscope. In (a) and (e–g) the red counterstain is propidium iodide.

Fig 3: Roles of FgNTH and FgATH in modulating sexual reproduction and their subcellular localization in Fusarium graminearum. (a) The colocalization of FgNTH and FgATH with cell nucleus. (b) The colocalization of FgNTH and FgATH with vacuoles. The fusion strains FgNTH-GFP and FgATH-GFP were cultured in YEPD liquid medium for 1 day at 25 °C to harvest mycelia and in CMC medium for 3 days at 25 °C to produce conidia. The mycelia and conidia were stained with 4,6-diamidino-2-phenylindole (DAPI) or 7-amino-4-chloro-methylcoumarin (CMAC) to observe fluorescence using a Leica TCS SP5 confocal microscope. GFP fusion protein: green fluorescence; DAPI and CAMC: blue fluorescence. Bar = 10 µm. (c) The localization of FgNTH in the mutant ?FgATH. (d) The localization of FgATH in the mutant ?FgNTH. The mycelia and conidia of strain ?FgATH FgNTH-GFP were harvested and stained with CMAC and DAPI, respectively, as described above. Bar = 10 µm. (e) Conidial morphology. Conidia were produced in CMC medium and stained with calcofluor white (CFW) to observe the septa using an Olympus IX-71 inverted fluorescence microscope. Left image, bright field; right image, ultravioletlight excitation. Bar = 10 µm. (f) Conidial germination. Conidia were cultured in YEPD liquid medium for 8 hr and stained with CFW to observe the germination using an Olympus IX-71 inverted fluorescence microscope. Top image, bright field; bottom image, ultraviolet light excitation. Bar = 20 µm. (g) Sexual reproduction. All strains were incubated on carrot medium for 14 days to produce perithecia. The perithecia and ascospores were observed using a Nikon SMZ25 fluorescence stereomicroscope and an Olympus IX-71 inverted fluorescence microscope, respectively. Top image, perithecia, bar = 1 mm; bottom image, asci and ascospores, bar = 50 µm

Fig 4: Cell Growth, NanoString Gene Expression Analysis, and Chimeric Embryos(A) Heatmap NanoString analysis of one PDGFRA-GFP heterozygous ESC line (ESC-18), two PDGFRA-deficient ESC lines (ESC-23, ESC-111), five PDGFRA-GFP heterozygous XEN cell lines (X-E6.5-82053-1, X-E6.5-79642-1, X-E6.5-79642-4, X-E6.5-79637-1, and cXEN-18), and four PDGFRA-deficient XEN cell lines (X-E6.5-79642-8, X-E6.5-79637-6, cXEN-23, and cXEN-111). The PDGFRA-deficient cell lines are indicated in red. Heatmap colors correspond to log10 values of normalized counts as indicated in the color key, from dark blue (low) to dark orange (high).(B) Cell proliferation: a.u. (arbitrary units), difference between days 4 and 1. n.s., not significant (t test). Line at mean, error bars at SD. Six cell lines were used for each of the two genotypes, representing six biological replicates per genotype. Three technical replicates per cell line were seeded and measured on a daily basis for 4 days.(C–E) PDGFRA-deficient post-XEN cell line X-E6.5-79642-8. A whole mount of an E7.5 chimeric embryo was imaged in bright field and fluorescence (C) and in fluorescence (D), using a Nikon SMZ25 stereofluorescence microscope. The same embryo was imaged using a Zeiss LSM 710 confocal microscope (E).(F–H) PDGFRA-deficient pre-XEN cell line X-ICM-97025-4. A whole mount of an E7.5 chimeric embryo was imaged in bright field and fluorescence (F) using a Nikon SMZ25, and in fluorescence alone (G), with DAPI (blue) and GFP (green), using a Zeiss LSM 710. A section of the decidua of another E7.5 embryo, showing the merged image of fluorescence from DAPI, GFP, and F-actin (red), was imaged using a Zeiss LSM 710 (H).

Fig 5: Epifluorescence whole-mount images of bulbs of transgenic mice expressing rat OR I7 from a mouse MOR23 promoter and gap-YFP with and without Nrp1. Images of medial and lateral views of bulbs were taken with a Nikon SMZ25 stereomicroscope. Signal represents the intrinsic fluorescence of YFP. Dorsal is up, ventral is down. A, Views on the medial and lateral aspects of a left bulb (left two images) and a right bulb (right two images) of two I7-Cre-YFP Tg mice at PD17. Axons coalesce into a single glomerulus. B, Views on the medial and lateral aspects of the right bulbs of six I7-Cre-YFP Tg × n5247-flox littermates at PD14. Images are pairwise for an individual mouse. Glomeruli are indicated with yellow arrows. Axons coalesce into multiple glomeruli, in particular in the lateral aspect.