Horie R , Hazbun A , Chen K , Cao C , Levine M , Horie T .

R01 NS076542 NINDS NIH HHS

	Fig. 1: Lateral plate ectoderm. a, Summary of lateral plate derivatives at early gastrula (110-cell stage). Magenta, progenitors of PSCs (a8.18 and a8.20 lineage) and aATENs (a8.26 lineage) lineages. Green, pATENs and BTNs arising from b8.20 and b8.18 lineages, respectively. Yellow, neural plate. b, Diagram of Ciona tadpole showing the position of PSCs, aATENs, pATENs and BTNs. c, Summary of Ciona lateral plate ectoderm and Xenopus pan-placodal primordium. Magenta, Dmrt.a-expressing blastomeres (a8.20, a8.18 and a8.26 lineage); green, Msxb-expressing blastomeres (b8.20 and b8.18 lineage); purple, prospective Six1/2- and Eya-expressing blastomeres (a8.26 lineage); yellow, Foxc-expressing blastomeres (a8.20 and a8.18 lineage); grey, Pax3/7-expressing blastomeres (a8.26, b8.20 and b8.18 lineage). A, anterior; Ad, adenohypophyseal placode; L, lens placode; OL, olfactory placode; P, posterior; Pr, profundal placode; V, trigeminal placode. d, Left, tailbud embryo injected with Dmrt.a > CFP (green) and Msxb > mCherry (magenta) reporter genes. The arrowhead indicates the boundary that separates the regions in which Dmrt.a and Msxb is expressed. Right, tailbud embryo injected with Six1/2 > CFP and Foxc > mCherry reporter genes. The arrowhead indicates the boundary that separates the regions in which Six1/2 and Foxc is expressed. Anterior is to the left. Scale bars, 100 μm.
	Fig. 2: Functional analysis of the lateral plate ectoderm. a–c, Head regions of larvae that were injected with a Six1/2 > CFP reporter gene. a, Six1/2 expression in the proto-placodal region of control MO injected tadpoles (49 of 49 larvae displayed this expression pattern). The yellow arrowheads identify the normal location of Six1/2 expression. b, Loss of expression in Dmrt.a morphants (49 of 49 larvae). c, Expanded expression of the Six1/2 >  CFP reporter gene (white arrowheads) in Msxb morphants (42 of 50 larvae showed this expansion pattern). d, Head regions of a larva that was injected with Foxc MO and Six1/2  > mCherry reporter gene. There is ectopic expression (white arrowhead) in the palp regions of Foxc morphants (35 of 47 larvae showed this phenotype). e, f, Larvae injected with βγ-crystallin > mCherry reporter gene. Yellow arrowheads indicate the βγ-crystallin expressing PSCs in control MO injected larvae (51 of 51 larvae display this expression pattern). f, There is a loss of these cells in Foxc morphants (108 of 108 larvae showed this phenotype). g, h, Larvae injected with a GnRH > CFP reporter gene. Yellow arrowheads identify the GnRH expressing aATENs in a control larva (59 of 59 larvae displayed expression in aATENs). h, There is ectopic expression in the palp regions of Foxc morphants (white arrowheads) (28 of 40 injected larvae showed this phenotype). i–k, Tail regions of larvae injected with Asic1b > CFP (i) and also injected with βγ-crystallin > mCherry reporter gene (j, k). Yellow arrowheads identify the Asic1b expressing BTNs in a control larva (83 of 83 larvae displayed this phenotype). j, Ectopic expression of the βγ-crystallin > mCherry reporter gene in tail regions (white arrowheads) upon misexpression of Foxc by the Pax3/7 enhancer (26 of 55 larvae showed this phenotype). k, Same as j except that Msxb regulatory sequences were used to misexpress Foxc (31 of 57 larvae showed misexpression of βγ-crystallin > mCherry). Anterior to the left; scale bars, 100 μm (a–h), 20 μm (i–k).
	Fig. 3: Single-cell RNA-seq analysis of BTN transformations. a, The t-SNE projection map of dissociated cells from wild-type and mutant tailbud-stage embryos that misexpress Foxc in tail regions using Pax3/7 regulatory DNAs (Pax3/7 > Foxc transgene). Each dot corresponds to the transcriptome of a single cell, and cells possessing similar transcriptome profiles map near each other. All of the major tissue types in tailbud-stage embryos were identified. CNS, central nervous system; Ep, epidermis; En, endoderm; M/H, heart and muscle; Me, mesenchyme; Noto, notochord; sen, sensory neurons. Identification is based on the expression of known marker genes (Extended Data Fig. 8b, and Supplementary Table 1). Red arrow identifies PSCs. b, Distribution of marker genes expressed in PSCs (Foxg and SP8) and BTNs (Asic1b and synaphin) within t-SNE projections as shown in (a). c, Distribution of cells expressing transgenes, which identifies cells that misexpress the PSC determinant, Foxc. BTNs (dark green dots; n = 21), PSCs (red dots; n = 32), hybrid cells that express both PSC and BTN marker genes (light blue triangles; n = 14), and transformed cells that express PSC markers (blue dots; n = 10). The grey dots (n = 10,103) correspond to all dissociated cells that were sequenced in these experiments. d, Heat map of BTNs, PSCs, transformed cells and hybrid cells showing the relative expression of a select group of genes encoding transcription factors (red), signalling components (green) and cellular effectors (black).

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