Montequin A and LaBonne C (2026), Dynamic and non-uniform expression of key trans...

XB-ART-61743

Development 2026 Feb 15;1534:. doi: 10.1242/dev.205254.

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Dynamic and non-uniform expression of key transcription factors provides insights into the emergence of neural crest cells at the neural plate border.

Montequin A, LaBonne C.

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The neural crest is a vertebrate stem cell population with broad developmental potential. While a gene regulatory network describing establishment of these cells has been generated, much remains to be learned about the dynamics of this process. Here, we use fluorescent in situ hybridization chain reaction to quantify the spatiotemporal dynamics of neural crest formation in Xenopus. We find that the initial onset of neural crest genes is broad and partially overlapping, with distinct anterior-posterior and medio-lateral biases. A shared neural crest domain emerges, but some genes retain relative expression differences that persist into migratory stages, producing stream-specific gene expression patterns. These differences correlate with dynamic expression of the neural plate border factors pax3 and zic1. Correlating relative intensities of pax3 and zic1 with the presence or absence of nascent neural crest transcripts predicts that these factors can differentially regulate snai2 and sox8, which we confirm experimentally. Strikingly, later stages display an inverse correlation between neural crest and neural plate border factors, suggesting that pax3 and zic1 initially promote neural crest gene activation but are downregulated as neural crest identity emerges.

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Species referenced: Xenopus laevis
Genes referenced: foxd3 msx1 pax3 snai2 sox10 sox8 sox9 tfap2a zic1
GO keywords: neural plate development [+]

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	Fig. 1. Neural crest gene expression is dynamic and non-uniform during gastrulation and neurulation. (A) Time-course collection protocol for wild-type Xenopus laevis embryos at 19°C. Images adapted from Zahn et al. (2022). Xenopus illustrations © Natalya Zahn (2022). Xenbase (www.xenbase.org RRID:SCR_003280). (B) Maximum intensity z-projections of neural crest genes snai2 and foxd3. (C) Maximum intensity z-projections of neural crest genes snai2 and sox8. All images in B,C are views of the dorsal midline, with the anterior pole facing upwards. Images are representative of a minimum of 4 samples per stage and probe combination. Scale bars: 1 mm (A); 250 μm (B,C).
	Fig. 2. Gene expression patterns in the ectoderm differ between cell layers. (A) Overview of the computational surface-mapping protocol. The program detects the surface of the embryo within 3D z-stacks from confocal microscopy images, fits and shifts the surface to an appropriate depth, and creates a ‘flattened’ z-stack following the shape of the embryo surface. (B) Maximum intensity z-projections from the full, unmapped image (top row), the superficial ectodermal cell layer (middle row) and the deep ectodermal cell layer (bottom row). The stage 15 embryo was double-labeled with probes targeting pax3 and snai2. Scale bar: 250 μm (B, top row, unmapped images). Surface-mapped images are displayed without a scale bar because the mapping process induces minor length distortions that vary across the image. Images are representative of 15 samples.
	Fig. 3. Non-uniform spatial onset of neural crest gene expression resolves during neurulation. (A,B) Maximum intensity z-projection of NPB marker tfap2a. Dashed lines mark the dorsal midline, annotated to show the anterior and posterior directions. Insets mark the regions shown at higher magnification in A′ and B′. (A′,B′) Maximum intensity z-projections of snai2 and sox8 from the fields of view denoted in A and B. Dashed lines denote approximate boundaries of detected expression. Scale bars: 250 μm (A-B′). Images are representative of 4 samples. (C,D) Normalized line intensity profile measurements for double-labeled snai2/sox8 images at stage 13 (n=16) (C) and at stage 15 (n=13) (D). Grayscale images on the left are representative mean-intensity projections of slices taken only from the deep cell layer after mapping, and the dashed lines denote the regions used for line profile measurements. Shaded areas represent s.d. for normalized intensity measurements at each pixel location. L, lateral; M, medial.
	Fig. 4. Relative expression differences within the neural crest persist through neural crest migration. (A) Gene expression patterns of snai2 and sox8 within the deep ectodermal cell layer at stages 15, 17 and 20. The box in stage 15 images indicates a region of high snai2/low foxd3 in the top half of the box, and low snai2/high foxd3 in the bottom half of the box. (B) Gene expression patterns of snai2 and foxd3 within the deep ectodermal cell layer at stages 15, 17 and 20. Labels in the bottom right panels of A,B indicate the mandibular (M), hyoid (H) and branchial (B) migratory streams, as well as the direction of the anterior-posterior (A-P) axis. Images are representative of a minimum of 3 samples per stage and probe combination. Surface-mapped images are presented without a scale bar due to minor length distortions induced by the mapping.
	Fig. 5. Expression patterns of pax3 and zic1 are dynamic during gastrulation and neurulation. (A) Maximum intensity z-projections of NPB genes pax3 and zic1 at stages 12-15. Scale bars: 250 μm. Images are representative of a minimum of 4 samples per stage. (B) Z-scored voxel intensities of pax3 and zic1 from 10× surface-mapped images selecting the deep cell layer of the dorsal ectoderm.
	Fig. 6. Distinct probabilities of early snai2 and sox8 transcription based on local pax3 and zic1 expression levels in the NPB. (A) Maximum intensity z-projection of DAPI and snai2 intronic probes at stage 12.5. (B) Maximum intensity z-projection of DAPI and sox8 intronic probes at stage 12.5. Insets in the left panels of A,B denote the digital zoom displayed in the following three panels. Arrows indicate examples of intronic probe detection and localization with nuclei/DAPI. Scale bars: 100 μm. (A,B) Images are representative of a minimum of 13 samples per stage and probe combination. (C) Overview of the image analysis pipeline used to estimate the levels of snai2 and sox8 transcription given local pax3 and zic1 intensities. (D) Heat map depicting t-statistics from two-sided t-tests between bootstrapped sample and null distributions estimating the percentage of neural crest-positive voxels within each bin of pax3/zic1 intensity. Data collected from n=15 images for the snai2 intronic labeling, and n=13 images for the sox8 intronic labeling.
	Fig. 7. Pax3 and Zic1 drive differential expression of snai2 and sox8 in animal caps. (A-D) HCR-FISH images of neural crest genes snai2 and sox8 in stage 15 animal caps for uninjected (A), Pax3-GR (B), Zic1-GR (C) and Pax3-GR+Zic1-GR (D) conditions. Images are representative of a minimum of 16 samples per condition. Scale bars: 100 μm.
	Fig. 8. Expression of pax3 and zic1 is differentially reduced in the anterior and posterior neural crest. (A) Normalized line intensity profiles for double-labeled snai2/pax3 images at stage 15 (n=12) in the deep ectodermal cell layer. Deep cell layers images from Fig. 2B (left panels) are annotated to indicate the regions analyzed. (B) Normalized line intensity profiles for double-labeled snai2/zic1 images at stage 15 (n=13) in the deep ectodermal cell layer. Grayscale images on the left of A,B are representative mean-intensity projections of slices taken only from the deep cell layer after mapping, and the dashed lines denote the regions used for line profile measurements. Shaded areas represent s.d. for normalized intensity measurements at each pixel location. L, lateral; M, medial. (C,D) Surface-mapped images of zic1/snai2 expression (C) and foxd3/snai2 expression (D; from Fig. 4A) used to indicate (dashed line) the region used to make the xy projections shown in C′ and D′, respectively. A, anterior; P, posterior. Images are representative of a minimum of 9 samples per probe combination. Scale bars: 100 μm (C,D); 100 μm (C′,D′).
	Fig. 9. Differential onset and resolution of neural crest gene expression in the NPB. Our data suggest that neural crest gene expression is differentially induced broadly throughout the ectoderm and resolves to a non-uniform domain during neurulation. Gene name colors correspond to shaded areas overlaid on the drawings, with cross-hatched areas indicating regions of shared gene expression. NC, neural crest. Adapted from Nieuwkoop and Faber (1994) (www.xenbase.org/xenbase/anatomy/alldev.do).
	Fig. S1. Neural crest gene expression is dynamic and non-uniform during gastrulation and neurulation (a) 4x maximum intensity z-projections of neural crest genes sox9 and sox10. All images are views of the dorsal midline, with the anterior pole facing upwards. Scale bars represent 250 μm. (b) Schematic depicting the approximate temporal onset of gene expression within the presumptive neural crest domain for imaged neural crest genes. (c) Schematic depicting approximate spatial boundaries of gene expression for sox8, foxd3 and snai2 at gastrula and neurula stages. Hatched pattern indicates areas of shared expression.
	Fig. S2. HCR-FISH images provide linear signal intensities with low noise for relative transcript quantification (a) Schematic of the redundant detection control experiment. Pax3 transcripts were labeled with orthogonal probe sets and detected in the red and far-red channels. (b) Scaled voxel intensities for pax3-B1-647 (far red channel) and pax3-B3-546 (red channel) probes in the deep ectodermal cell layer. (c) Intrinsic noise measurements made within bins of equal voxel counts along the diagonal line in (b). Solid line for each replicate represents the mean noise, and error bars represent the 2.5th and 97.5th percentiles of the mean noise estimated using bootstrapping.
	Fig. S3. Non-uniform spatial onset of neural crest gene expression (a) 20x maximum intensity z-projection of neural crest gene snai2. Dashed line marks the dorsal midline, annotated to show the anterior and posterior directions. (b-c) Cropped regions denoted by the insets in (a) showing foxd3 and sox9 expression. Scale bars in represent 250 μm.
	Fig. S4. Relative expression differences within the neural crest persist through neural crest migration [[ Panels (a) and (b) ]] (a) Maximum intensity z-projections of gene expression patterns of snai2 and sox8 within dorsal ectoderm at stages 15, 17 and 21. (b) Maximum intensity z-projections of gene expression patterns of snai2 and foxd3 within the dorsal and anterior ectoderm at stages 15, 17 and 20. Scale bars represent 250 μm.
	Fig. S4. Relative expression differences within the neural crest persist through neural crest migration [[ Panels (c) and (d) ]] (c) Maximum intensity z-projections of snai2 and sox8 at Stage 23. (d) Maximum intensity z-projections of snai2 and foxd3 at Stage 23. Scale bars represent 250 μm.
	Fig. S5. Expression patterns of neural plate border genes are dynamic during gastrulation and neurulation (a-e) 4x maximum intensity z-projections of neural plate border genes tfap2a and msx1 at stages 12-15. Scale bars represent 250 μm.
	Fig. S6. 6. Diﬀerential association of sox8 transcription with pax3 and zic1 expression predicts sox8 expression in the anterior neural plate border (a-b) Sample and null distributions generated through bootstrapping to estimate the fraction of neural crest-positive voxels within ranges of pax3 and zic1 intensities. (c) 10x images of wild-type Stage 12.5 embryos labeled with zic1 and pax3 HCR-FISH probes. Images are surface mapped, and only the deep cell layer is displayed. (d-e) Binning of pax3 and zic1 intensities and color-coding based on the heatmap displayed in Fig. 6d. Red box indicates a region of interest within the anterior neural plate border. (f) 10x image of a Stage 12.5 wild-type embryo displaying co-localization of zic1 and sox8 within the anterior neural plate border. Images are mapped and only the deep cell layer is displayed. Dashed line indicates the location of the dorsal midline, and the box represents the cropped region displayed on the bottom row.
	Fig. S7. Animal cap quantification (a-b) Cumulative distribution functions for snai2 (a) and sox8 (b), voxel intensities within animal cap images. (c-d) Box plots displaying the 99th percentile voxel intensity for each animal cap image. P-values from two-sided t-tests comparing the intensities Pax3-GR and Zic1-GR to uninjected caps are listed in (c) and (d). (e-f) Cumulative distribution functions for pax3 (e) and zic1 (f) voxel intensities within all animal cap images. (i-l) HCR-FISH images of neural plate border genes pax3 and zic1 in Stage 15 animal caps for uninjected (i), Pax3-GR (J), Zic1-GR(k), and Pax3-GR + Zic1-GR (L) conditions. Scale bars represent 100 μm.
	Fig. S8. Snai2 is essential for neurula-stage patterning of pax3 and zic1 (a-b) HCR-FISH images in embryos injected with a morpholino blocking Snai2 translation. GFP-Myc indicates the injected side. (c-d) Chromogenic in situ hybridization images in embryos injected with a morpholino blocking Snai2 translation. Asterisk indicates the injected side. (e) Western blot demonstrating eﬃcacy of Snai2 MO. Translation of Snai2-Myc constructs is blocked in the presence of the morpholino when its target sequence in the 5' UTR is intact.