Gouignard N , Schön T , Holmgren C , Strate I , Taşöz E , Wetzel F , Maccarana M , Pera EM .

             cell-cell adhesion

	Fig 1. Genomic organization, protein structure and probe specificity of Dse/DS-epi1 and Dsel/DS-epi2 in X. laevis embryos. (A) Genomic structures of the Dse and Dsel homeologs. Rectangles with numbers show exons and intervening lines demarcate introns. Filled boxes indicate open reading frames. Accession numbers of the Xenopus laevis genomic DNA / mRNA sequences are: DS-epi1.S, NC_030733 / KU877109; DS-epi1.L, NC_030732 / XM_018263281; DS-epi2.S, NC_030735 / XM_018223616; DS-epi2.L, NC_030734 / KU877110. (B) Overall protein structure of DS-epi1 and DS-epi2, which are encoded by Dse and Dsel, respectively. SP, cleavable signal peptide; TM, transmembrane domain. The arrows represent the antisense RNA probes of Dse and Dsel that were used for the in situ hybridization. (C-F’) Whole-mount in situ hybridization of early gastrula embryos in lateral view. The Dse probe detects injected Dse mRNA (C,C’) but not Dsel mRNA (D,D’). The Dsel probe specifically targets injected Dsel mRNA (E-F’). Arrowheads depict cells that received co-injected nlacZ mRNA as a lineage tracer (red nuclei). Each synthetic mRNA was injected at a dose of 100 pg into the animal pole of a single blastomere at the 4-cell stage. Scale bars are 500 μm (C-F) and 50 μm (C’-F’).
	Fig 2. Dse mRNA is maternally deposited and expressed in the epidermis, neural ectoderm, notochord, somites and dorsal endoderm. Xenopus embryos after whole-mount in situ hybridization in lateral view (A,D,F), dorsal view (C,E) and transversally sectioned through the anterior trunk (G-J). (A) Embryo at the 4-cell stage. Weak expression of Dse mRNA is visible. (B) qPCR analysis of embryonic explants at stage 9. Note Dse transcripts in the animal cap, marginal zone and vegetal region. (C) At stage 13, Dse is expressed in the epidermis and notochord. (D-F) Neurula embryos show ubiquitous Dse expression in the epidermis. The bold lines indicate the levels of sections in G and H. (G) Embryo at stage 15. The arrowhead indicates the border between epidermis and neural ectoderm. Note Dse transcripts in the sensorial layer of the lateral neural plate (arrow), notochord and dorsal endoderm. The bracket in the inset depicts the epidermis with robust Dse expression in the outer layer and lower mRNA levels in the inner layer. (H) Embryo at stage 18. Note Dse signals in the medio-ventral somites. The inset shows Dse expression in the sensorial layer of the ventral neural groove (arrow). The stippled line demarcates the border between the neural plate and the underlying dorsal mesoderm. (I,J) No signals appear in matching sections of sibling embryos that were probed with Dse sense RNA as control. an, animal cap; en, endoderm; epi, epidermis; ma, marginal zone; no, notochord; np, neural plate; so, somite; ve, vegetal region. Scale bars are 500 μm (A,C-F) and 50 μm (G-J).
	Fig 3. Dse is expressed in the pre-migratory neural crest, anterior surface ectoderm, brain, and prechordal mesendoderm. Embryos are shown in anterior view (A-D, G-L) and midsagittally sectioned through the head (E,F). (A-D) Early neurulae. Note Dse expression in the anterior neural ridge and prechordal mesendoderm. Dse transcripts overlap with Twist1 and Snai2 expression in the cranial neural crest (indented arrowheads). The bold line indicates the level of section in E. (E) Embryo at stage 18. Dse is expressed in the superficial layer of the anterior neural ridge and the inner layer of the fore- and midbrain anlage (arrows). The stippled line demarcates the border between the anterior neural plate and the underlying prechordal mesendoderm. (F) Absence of signals in the matching section of a sibling embryo that was probed with Dse sense RNA as control. (G) Late neurula at stage 24. Dse is expressed in the anterior neural ridge and derived sense plate. (H) Tailbud embryo at stage 26. Dse is transcribed in the sense plate. (I) Xag expression demarcates the cement gland and hatching gland. (J-L) Tailbud embryos at stages 27 and 28. Note segregation of anterior Dse expression into the stomodeum-adenohypophysis anlage and posterior domain that partially overlaps with the Pax3+ hatching gland. anr, anterior neural ridge; cg, cement gland; epi, epidermis; ey, eye; fb, forebrain; hg, hatching gland; mb, midbrain; no, notochord; pm, prechordal mesendoderm; se, sense plate; st, stomodeum-adenohypophysis anlage. Scale bars are 500 μm (A-D,G-L) and 50 μm (E,F).
	Fig 4. Dse is expressed in the eye, migratory neural crest and the hypochord. Embryos are shown in lateral view (A,D-E,H-J’), and transversally sectioned through the trunk (B-C) and head (F,G). (A) Embryo at stage 25. The bold line indicates the level of the section in B. (B,B’) Dse is expressed in the epidermis, notochord and hypochord. The arrowhead shows Dse transcripts in trunk neural crest cells adjacent to the spinal cord. (C) Matching section of a sibling embryo showing Twist1 expression in trunk neural crest cells (arrowhead). (D-I) Tailbud embryos. The bold lines indicate the level of sections in F and G. Dse is expressed in the forebrain, proximal eye and Twist1+ cranial neural crest (indented arrowheads). The numbers indicate the mandibular (1), hyoid (2) and branchial neural crest (3,4). (J,J’) Embryo at stage 31 after removal of the epidermis. Dse is expressed in the spinal nerves (arrow) and migrating trunk neural crest cells (arrowhead). cg, cement gland; epi, epidermis; ey, eye; fb, forebrain; hc, hypochord; no, notochord; sp, spinal cord; so, somite. Scale bars are 500 μm (A,D-J) and 100 μm (B-C,J’).
	Fig 5. Dse is expressed in the adenohypophysis, melanophores, dermomyotome and sclerotome. Embryos are shown in lateral view (A,A’,F), dorsal view (G), transversal sections (B-D’,H,I) and horizontal section (E). (A,A’) Embryo at stage 33. The bold lines indicate the level of sections in B-E. (B-E) Vibratome sections showing Dse expression in the adenohypophysis, forebrain, ventral midbrain, epidermis, notochord, hypochord, dermomyotome, and sclerotome. Note also Dse transcripts in cranial neural crest cells (indented arrowheads) and melanophores (filled arrowhead). (F-I) Embryo at stage 40. The bold lines indicate the level of sections in H and I. Dse is expressed in post-migratory cranial neural crest cells (indented arrowheads) and the notochord. ad, adenohypophysis; dm, dermomyotome; epi, epidermis; ey, eye; fb, forebrain; hc, hypochord; he, heart; mb, midbrain; no, notochord; sc, sclerotome; so, somite; sp, spinal cord. Scale bars are 1 mm (A,A’,F-H) and 100 μm (B-E,I).
	Fig 6. Dsel is expressed maternally and in the eye, brain and cranial neural crest. Embryos after whole-mount in situ hybridization are shown in animal view (A), lateral view (B,E,E’,I,I’), hemi-sectioned (C) and transversally sectioned (F-H). (A) 4-cell stage embryo. (B,C) Blastula embryos. The bold line indicates the level of section in C. (D) qPCR analysis at stage 9. Note ubiquitous expression of Dsel in the animal cap, marginal zone and vegetal region. (E-I’) Tailbud embryos. The bold lines indicate the level of sections in F-H. Note Dsel expression in the eye, interneurons of the mid- and hindbrain (arrows), cranial neural crest (indented arrowheads) and bilateral trigeminal ganglia. an, animal cap; ey, eye; hb, hindbrain; ma, marginal zone; mb, midbrain; V, trigeminal ganglion, vg, vegetal region. Scale bars are 500 μm.
	Fig 7. Dsel is expressed in differentiated neurons, cranial sensory ganglia and the spinal cord. Embryos are shown in lateral view (A-B’,K,K’) and transversal vibratome sections (C-J). (A,A’) Embryo at stage 33. The bold lines indicate the level of sections in C-J. Magnification in A’ depicts Dsel mRNA in cranial sensory ganglia: V, trigeminal ganglion; VII, geniculate ganglion; IX, petrosal ganglion; X, nodose ganglion. (B,B’) Sibling embryo depicting N-tubulin expression in the central nervous system (stars) and cranial ganglia. (C-J) Dsel is expressed in the adenohypophysis and distinct neurons (arrows) of the forebrain, midbrain, hindbrain and spinal cord. The indented arrowhead labels signals in migrating cranial neural crest cells. (K,K’) Embryo at stage 34. The indented arrowhead labels robust Dsel transcripts in post-migratory cranial neural crest cells. ad, adenohypophysis; df, dorsal fin; ea, ear; ey, eye; fb, forebrain; hb, hindbrain; ist, isthmus; IX, petrosal ganglion; mb, midbrain; no, notochord; pm, sp, spinal cord; V, trigeminal ganglion; VII, geniculate ganglion; IX, petrosal ganglion; X, nodose ganglion. Scale bars are 1 mm (A,B,K,K’) and 200 μm (A’,B’,C-J).
	Fig 8. Dsel is expressed in the brainstem in tadpole embryos. Specimen after whole-mount in situ hybridization are shown in dorsal view (A,A’), lateral view (B,D) and ventral view (C,E). (A,A’) Embryo at stage 36. Arrows label distinct Dsel expression domains in the midbrain and hindbrain. The arrowhead labels the position of the midbrain-hindbrain boundary. (B-E) Isolated central nervous system from stage 43 embryos after removing of surrounding tissues. Dsel mRNA accumulates in bilateral domains in the brainstem (arrows). Expression of Foxg1 demarcates the forebrain and En2 the midbrain-hindbrain isthmus (arrowhead). hb, hindbrain; mb, midbrain; sp, spinal cord. Scale bars are 1 mm (A-E) and 100 μm (A’).
	Fig 9. Comparison of Dse and Dsel expression in the head. Embryos at stage 32 are shown in lateral views (A,E) and transversal sections (B-D’, F-H’). (A) Dse expression. The bold lines indicate the level of sections in B-D. (B-D’) Dse expression is found in the cranial neural crest (indented arrowheads). Note ventral expression domains in the forebrain, posterior midbrain, hindbrain, and notochord. Weak expression is visible in the trigeminal ganglia. (E) Dsel expression. The bold lines indicate the level of sections in F-H. (F-H’) Dsel expression appears in the cranial neural crest (indented arrowheads) and trigeminal ganglia. Note expression domain in the prechordal mesendoderm. Apparent are low-level transcripts in the entire ventral brain and robust bilateral expression domains in the ventral marginal zone of the mid- and hindbrain (arrows). fb, forebrain; hb, hindbrain; he, heart; mb, midbrain; no, notochord; pm, prechordal mesendoderm; V, trigeminal ganglion. Scale bars are 1 mm (A-H) and 200 μm (B’-H’).
	Fig 10. Comparison of Dse and Dsel expression in the trunk. Embryos at stage 32 are shown in lateral views (B,C) and transversal sections (D-G’) after whole-mount in situ hybridization. (A) qPCR analysis of embryonic explants at stage 32. The numbers on the y-axis indicate fold expression of (Dse / eEF1A1) x 1000 (top) and (Dsel / eEF1A1) x 1000 (bottom). Note equal distribution of Dse transcripts in the head, dorsal trunk and ventral trunk. Dsel mRNA is most abundant in the head and is expressed at comparable levels in the dorsal and ventral trunk. (B) Dse expression. The bold lines indicate the level of sections in D and E. (D-E’) Dse expression is detected in the notochord, dermatome, sclerotome and hypochord (inset). (C) Dsel expression. The bold lines indicate the level of sections in F and G. (F-G’) Dsel expression appears in the ventral spinal cord, migrating trunk neural crest cells (arrowhead), myotome, sclerotome, hypochord (inset) and posterior dorsal hindgut endoderm. dm, dermatome; en, endoderm; hc, hypochord; my, myotome; no, notochord; sc, sclerotome; sp, spinal cord. Scale bars are 1 mm (B,C) and 200 μm (D-G’).
	Table 1. Summary of gene expression for Dse and Dsel in Xenopus laevis embryo.
	dse (dermatan sulfate epimerase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior up.
	dse (dermatan sulfate epimerase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
	dse (dermatan sulfate epimerase) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, ateral view, anterior left, dorsal up.
	dse (dermatan sulfate epimerase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage16, anterior view, dorsal up.
	dse (dermatan sulfate epimerase) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, midsagittally sectioned through the head, dorsal up.
	dse (dermatan sulfate epimerase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, anterior view, dorsal up.
	dse (dermatan sulfate epimerase) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, head and anterior trunk region, lateral view, anterior left, dorsal up.
	dse (dermatan sulfate epimerase) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, head and anteror trunk region, ateral view, anterior left, dorsal up.
	dse (dermatan sulfate epimerase) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, F lateral view, anterior right, dorsal up, G dorsal view and H, transverse section as indicated in F , through hindbrain and pharyngeal region.
	dsel (dermatan sulfate epimerase like) gene expression in Xenopus laevis embryo, hemi-sectioned , assayed via in situ hybridization, NF stage 9, anterior up.
	dsel (dermatan sulfate epimerase like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	dsel (dermatan sulfate epimerase like) gene expression in Xenopus laevis tadpole brain, assayed via in situ hybridization, NF stage 43, B: lateral view, anterior left, dorsal up; C dorsal view, anterior left.

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