Maczkowiak F, Matéos S, Wang E, Roche D, Harland R, Monsoro-Burq AH.

GM42341 NIGMS NIH HHS , R01 GM042341 NIGMS NIH HHS

	Fig. 1. Expression of pax7 and pax3 compared during neurulation in Xenopus laevis. (A) A bootstrap phylogenetic tree illustrates the clear grouping of pax7 and pax3 paralogs in vertebrates while the chordate have a single Pax3/7 gene. Accession numbers are given in Supplemental Fig. 1. (B) RT-PCR analysis on whole embryos shows the early onset of pax3 expression during gastrulation (i.e. at times of neural crest induction), whereas pax7 is detected at mid-neurulation, after muscle actin (MA) is detected. EF1α is used as a baseline control. (C) In situ hybridization on stage-matched sibling embryos confirms the late onset of pax7, in brain first (c) then in paraxial mesoderm, and the lack of expression in the neural crest progenitor area (c, d). In contrast, pax3 and snail2 label neural crest (e–h). Myod marks paraxial mesoderm (i, j).
	Fig. 2. Distinct pax7 and pax3 patterns in central nervous system and paraxial mesoderm at tailbud stages. (A–C) Front views of stage 22 embryos (A) show pax7 expression in the caudal part of the brain (B), whereas pax3 labels both the whole brain and the hatching gland (C). (D–F) Side views (D) illustrate pax7 restriction to mesencephalon, hindbrain and anterior spinal cord whereas pax3 is found along the entire anterior–posterior length of the central nervous system. Pax7 labels the central paraxial mesoderm while pax3 is found in hypaxial cells. (G–L) Dorsal (G–I) and side (J–L) views of tailbud stage 24, with double staining for pax3 or pax7 and krox20, which labels hindbrain rhombomeres r3 and r5 (yellow arrows), confirms the limited extent of pax7 expression along the spinal cord. (M–O) Tailbud stage 24 transverse sections were double-stained with 12–101 myotome marker (brown). This shows pax7 expression in the alar plate of the anterior-most spinal cord and medial myotome (N) and pax3 in the roof plate, alar plates and in the hypaxial myotome (O).
	Fig. 3. pax7 pattern in the brain is positively regulated by FGF, Wnt and retinoid signals. (A–D) FGF8 gain of function expands pax7 expression into forebrain, while FGF8 depletion leads to lack of pax7 at induction stage (stage18, A, B) and in later development (stage 22, C, D). (E–F) Wnt7b over-expression and beta-catenin morpholino injections demonstrate the requirement for Wnt signals in pax7 patterning. (G) Similarly, blocking retinoic acid signaling resulted in defective pax7 expression.
	Fig. 4. Pax3 regulates pax7 expression and alar plate patterning in the brain. (A) Pax3 depletion using an antisense morpholino (Pax3MO) specifically prevents Xenopus laevis pax3 translation in vitro (lane 3). Pax7MO and a control MO have no effect on Xenopus laevis pax3 translation while Mus musculus pax3 is unaffected by Pax3MO. (B) Following Pax3 depletion in vivo (bar 1), pax7 expression is severely reduced, Mus musculus Pax3 mRNA efficiently rescues the decrease in pax7 expression (bar 2). (C) Unilateral depletion of Pax3 results in loss in pax7 expression; (D) a similar effect is observed after Pax3-EnR over-expression. (E) Gain in Pax3 activity increases and expands pax7 expression. (F–I) Transverse sections show that the loss in pax7 (C, D, G, H) is accompanied by reduced alar plates development, while Pax3 increase results in expanded pax7-expressing alar plates (I). Bar = 500 µm.
	Fig. 5. Pax7 expression in tadpoles. A/ At tailbud stage (st. 25–32) pax7 exhibits a dynamic expression in the myotome, which vanishes at late stage, while brain and spinal cord expression remains restricted to hindbrain and anterior spinal cord (A–B). Pax7 is also found in the lymph heart (B, arrow). This contrasts to pax3 expression in the entire length of the spinal cord, in the hypaxial muscle and trigeminal ganglia (E–F, arrows) or to myod expression in the epaxial muscles (I–J). At swimming tadpole stage (st 41–45), pax7 is expressed in scattered cells on the side of the embryo (yellow arrows) and cells that align at the edge of the myotome cells (see D/), while pax3 and myod mark muscle (G, K). Both pax7 and pax3, but not myod, label melanocytes (D, H, L). B/ During lymph heart development, pax7 is detected earlier than the lymph heart marker prox1 (A, A′, C, C′). By stage 33, both pax7 and prox1 expression overlap in the lymph heart (B, B′, D, D′ and transverse sections b, d, yellow arrows), while prox1 is also expressed adjacent to the cardinal vein (green arrow; Ny et al., 2005). C/ Late tadpoles where photographed before bleaching to position the melanocytes (B) and then pax7 was revealed (A). Most melanocytes were labelled by pax7 (yellow arrows). D/ Some pax7-positive cells aligned at the edge of each myotome in stage 41 tadpoles and may constitute the prospective satellite cell population (arrows).
	Fig. 6. Pax7 morphants are affected in earlier stages than Pax3 morphants, although both display mesoderm and neural crest defects. (A) Pax7MO blocks in vitro translation of pax7 (lane 3) but neither that of a pax7-gfp fusion lacking the MO-binding sequence (lane 4), nor of pax3 (5). (B) Pax7 morphants display early and severe elongation defects shortly after gastrulation (yellow), while a few of them exhibit milder phenotypes allowing us to follow their development further (red). In contrast, Pax3 morphants are rather normal until the end of neurulation except for posterior spina bifida in the more severely affected ones (yellow). (C) Melanocyte development was analyzed in later stage embryos, among the mild phenotype for Pax7 morphants, and in Pax3 morphants. Severe loss refers to the lack of dct positive or pigmented cells, while “mild loss” refers to decreased melanocyte number associated to lack of melanocyte migration. (D) Sibling control embryos (a–e), Pax7 morphants with mild (f–j) or severe (k–o) phenotype, Pax3 morphants with mild (p–t) or severe (u–y) phenotype were analyzed at the end of neurulation (stage 23), tailbud stage 33 or swimming tadpole stage 45, and stained for dct and myod at stage 33. They display prominent head, brain, mesoderm and melanocytes defects. (E)The Pax7MO phenotype is rescued by co-injections with Pax7-myc mRNA, insensitive to the morpholino. See text for details. (F) Both myoD and dct expression are rescued by pax7 mRNA injection into Pax7 morphants (see text for details).
	Fig. 7. Pax7 and Pax3 are essential for hindbrain and neural crest patterning. The early phenotype of Pax7 morphants was analyzed at stage 18. Control siblings show normal expression of krox20 (A) and snail2 (F). Pax7 morphants exhibit either a severe loss of krox20 (B) and snail2 (G), or a posterior shift of rhombomeres r3 and r5 (C, arrows) accompanied by reduced snail2 expression (H). A similar phenotype is observed in Pax7EnR injected embryos (D, I) or Pax3 morphants (E, J). Pax7MO phenotype is rescued by injection of Pax7-myc mRNA), insensitive to the morpholino (Fig. 6): snail2 expression is restored (L shows two morphants and (M) two siblings injected with MO and the rescue construct).
	Fig. 8. Pax7 and Pax3 regulate mid–hindbrain boundary maintenance. Pax7 depletion leads to forebrain–midbrain expansion as seen by otx2 posterior expansion (B, C). This is accompanied by loss or posterior shift of the mid–hindbrain boundary (Gbx2, G, H) and En2 (L, M). A similar phenotype is observed after PaxEnR (D, I, N). Pax3 morphants do not exhibit expanded otx2 domain (E, yellow arrow indicates similar posterior boundaries on each side), but they also display defective or shifted mid–hindbrain boundary (J, O). Red arrows indicate the extent of shift between the posterior boundary on control non-injected (left) side, and the shifted boundary on injected (right) side.
	Fig. 9. Pax7 is essential for the neural crest inducing activity of the paraxial mesoderm, while Pax3 acts in the ectoderm. (A) We have recombined the stage 9 blastocoel roof ectoderm to stage 10.25 prospective paraxial mesoderm (dorsal–lateral marginal zone), a classical assay for neural crest induction in the ectoderm. Ectoderm and DLMZ were dissected from either control uninjected or Pax3 or Pax7 morphants (see color code). Ectoderm (B) or DLMZ (C) grown alone until stage 18 do not express snail2, while the recombined explants (D) do. When the DLMZ comes from Pax7 morphant (E), snail2 induction is strongly impeded, while Pax3 depleted DLMZ do not perturb snail2 induction (F). RT-PCR analysis (G) further shows that ectoderm alone (lane 3) does not express snail2, myoD or muscle actin (MA), DLMZ alone expresses only myoD and MA (lane 4) while the recombinant expresses both (lane 5). Snail2 induction is impaired if the ectoderm is depleted for Pax3 (lane 6) but not for Pax7 (lane 7). In contrast, snail2 induction is normal if DLMZ comes from Pax3 morphants (lane 8) but is impaired for Pax7 depleted DLMZ (lane 9). In both Pax3- and Pax7-depleted DLMZ, myoD and MA expression are decreased while vent1 is abnormally upregulated.
	Fig. 10. Pax3 and Pax7 do not display redundant functions in neural crest induction and development. Pax3 morphant phenotype (A) is rescued by gain of function for Pax3 (B) but not Pax7 (C). At a later stage, Pax7 does not restore melanocyte development in Pax3 morphants either (G). Conversely, Pax3 is not sufficient to rescue Pax7 depletion (D, F), in contrast to Pax7 gain of function (E). These data indicate that the two proteins do not display similar roles in neural crest induction and development. Injected side is on the right (yellow arrow).
	prox1 (prospero homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
	prox1 (prospero homeobox 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32/33, lateral view, anterior left, dorsal up.
	pax3 (paired box 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, anterior view, dorsal up.
	pax3 (paired box 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
	pax3 (paired box 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior right, dorsal up.
	pax3 (paired box 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	myod1(myogenic differentiation 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, lateral view, anterior right, dorsal up.
	myod1 (myogenic differentiation 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	dct (dopachrome tautomerase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	Supp. Fig 3. Pharmacological inhibition of FGF signaling at mid-neurulation does not affect pax7 patterning. Control embryos were grown in DMSO (carrier) from stage 12 to stages 18–22, they exhibit normal sox2, hoxb9 and pax7 expression (100% of the embryos). In contrast, sibling embryos grown in SU5402 between stages 12 and 18–22 have severe defects in neural patterning (hoxb9) while sox2 is normal. In this condition, pax7 expression remains largely comparable to controls (60% of the embryos).
	Supp. Fig. 4 . Pax3 morphants display strongly affected myotome segmentation. Although Pax3 morphants display more prominent MyoD expression than Pax7 morphants, myotome segmentation is perturbed, ranging from mild to severe defects.
	Supp Fig. 5. Pax7 mismatch MO phenotype. A 5-mismatched MO corresponding to the translation start site (same target site as Pax7MO, see Supplemental Fig. 2) was injected. Embryo development, early gene expression (myod and snail2) as well as later gene expression (myod, dct) were normal compared to sibling or between the injected and control sides for unilateral injections.
	Sup. Fig 6. Pax7 splice MO phenotype. A splice-blocking morpholino was designed (see Supplemental Fig. 2). Injections in whole embryos were monitored for pax7 expression by semi-quantitative RT-PCR followed by phospho-imager analysis. This shows that the Pax7 splice MO decreases pax7 expression by 60% at the dose used here, at tailbud stage 22 (A). The morphology of the embryos (whole embryo injections) was similar to the one observed for Pax7MO (translation blocking MO) (B) with a slightly milder percent of severe embryos. Marker genes analyzed during neurulation or later on displayed similar defects as those observed with the Pax7MO (see Fig. 6).
	By stage 33, both pax7 and prox1 expression overlap in the lymph heart ( transverse sections, yellow arrows),
	Some pax7-positive cells aligned at the edge of each myotome in stage 41 tadpoles and may constitute the prospective satellite cell population (arrows)

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