We aimed to compare the differentiation pattern of HD isogenic lines (Fig. 1A) in our micropattern-based 2D gastruloid assay. Consistent with our earlier observations, after 48 h of BMP4 treatment, wild-type RUES2 colonies differentiated into 2D gastruloids containing spatially ordered specification domains with SOX2+ ectoderm, brachyury+ (BRA) mesoderm and CDX2+ extraembryonic tissue from center to edge (Figs 1B, S8). Under the same conditions, all HD isogenic hESC lines also induced the three embryonic germ layers, demonstrating that their differentiation potential was not affected. However, although the germ layers were induced similarly to the parental line, the radii associated with each germ layer ring were altered in the concentric circles (Fig. 1B). The analysis of several replicates per cell line revealed that the central ectodermal SOX2+ domain decreased in size proportionally to the increase in CAG length (Fig. 1C-E). Surprisingly, and in contrast to the CAG-expanded lines, HTT−/− did not display a SOX2 reduction (Figs 1F,G, S8). To confirm the relationship between HTT-CAG expansion and reduced SOX2 area in an independent cell source, we used CAG-expanded induced pluripotent stem cells (iPSCs) generated from patient-derived fibroblasts. BMP4-induced 2D gastruloids of HD iPSCs displayed a reduction in their SOX2+ domain compared with the non-HD iPSC (Fig. S1C), similar to our hESC data. Furthermore, CRISPR/Cas9-correction of the HD mutation (Fig. S1A,B) rescued the SOX2 area to close to wild-type levels, demonstrating that the defect in ectodermal patterning is indeed caused by the CAG expansion (Fig. S1D-F). In order to track the dynamics of ectodermal fate acquisition, we generated SOX2 live-reporter lines using CRISPR/Cas9 gene-editing technology to fuse mCitrine to the C-terminus of SOX2 in 20CAG, 56CAG and 72CAG genetic background hESCs (Fig. 1H). As expected, the transcription factor SOX2 was homogeneously expressed in pluripotency, before BMP4 was added to the colonies (Fig. 1I). After stimulation with BMP4, the SOX2 signal remained high throughout the whole colony for 20 h, and then rapidly decreased at the colony edge (Fig. 1I,J). This is consistent with our previous demonstration that self-organization of 2D gastruloids follows a wave of differentiation that begins at the periphery and moves inwards (Warmflash et al., 2014; Etoc et al., 2016; Martyn et al., 2018). After 24 h, the SOX2 signal was solely localized at the colony center and gradually decreased in intensity. When comparing the parental 20CAG line with the CAG-expanded lines, no difference was observed during the first wave of differentiation. However, HD 2D gastruloids displayed a different dynamic of SOX2 expression: while the SOX2+ area of wild-type 2D gastruloids slowly retreated to the center of the colonies (Fig. 1I,J), HD 2D gastruloids displayed a much more abrupt reduction (Fig. 1J). This demonstrates that HTT mutation does not modify the early cell-intrinsic response to BMP4, but rather perturbs a process downstream of BMP4 signaling, which occurs 24 h post-stimulation.