Roche DD, Liu KJ, Harland RM, Monsoro-Burq AH.

GM42341 NIGMS NIH HHS , Biotechnology and Biological Sciences Research Council , Wellcome Trust , BB/E013872/1 Biotechnology and Biological Sciences Research Council , R01 GM042341 NIGMS NIH HHS , BB_BB/E013872/1 Biotechnology and Biological Sciences Research Council

	Fig. 1. Temporal and spatial expression of dazap2 mRNA. (A) RT-PCR using dazap2 primers shows that dazap2 is expressed during blastula to neurula stages. (B-I) Dazap2 spatial expression pattern was analyzed by whole-mount ISH, using either antisense (B, C, E-G and H, I) or sense (D and G′) full-length xl-dazap2 probe. Embryos are seen in dorsal view with anterior to top (B and D, E), posterior view with dorsal on top (C), lateral view with head to the left (F, G, G′ and H) or ventral view (I). The blue arrow indicates the blastopore. Embryos hybridized with the sense probe in the same ISH conditions show no staining at all stages tested (D and G′). (B) At mid-neurula stage 16 (B) and late neural stage 18 (C, posterior view), dazap2 is present in the head folds (red arrowhead) and throughout the neural plate (black arrow). The staining is enhanced when neurulae are cleared (stage 19, E and F). By stage 32 (G, G′), tailbud embryos express dazap2 in trunk and head region, including the brain (red arrowhead) and spinal cord, the eye, the branchial arches (arrowhead) and the pronephros (green arrow). A faint staining is found in the dorsal fin (arrow) and the proctodeum (blue arrow). Cleared stage 44 tadpoles (H, I) express dazap2 in the brain (red arrowhead), the pronephros (green arrows) and caecum (blue arrow).
	Fig. 2. Dazap2 silencing results in a loss of hoxb9 with concomitant posterior shift of anterior neural markers. Embryos co-injected with Dazap2 SP-MO and lacZ mRNA were analyzed at stage 18 (shown in dorsal views, with anterior at the top). (A, B) Sox2 expression domain was only marginally affected. (C, D) Otx2 domain was expanded and shifted posteriorly within the neural plate (blue and red lines indicate normal and shifted posterior limits respectively). (E, F) Krox20 expression (rhombomeres 3 and 5) also shows a posterior shift in SP-MO injected embryos (blue and red lines indicate normal and posteriorized rhombomeres 3 and 5 respectively). Expression of hoxb9 is decreased/lost in injected cells (G, H) and can be rescued when dazap2 mRNA (250 pg) is co-injected with SP-MO (I). The line indicates the sagittal midline
	Fig. 3. FGF8a posteriorizing activity requires Dazap2. Embryos were co-injected with lacZ mRNA and either dazap2 SP-MO (20 ng, B), fgf8a (10 pg, C) or a combination of SP-MO and fgf8a (20 ng and 10 pg, respectively, D) into one cell at the 4-cell stage. Stage 18 embryos are shown in dorsal views, with anterior to the top (the line indicates the sagittal plane). Compared to control embryos (A), the SP-MO blocks hoxb9 expression on the injected side (B, arrow) while Fgf8a induces ectopic hoxb9 expression (C, arrowhead). When fgf8a and SP-MO are combined, hoxb9 expression is blocked and no ectopic expression is observed (D, arrow). (E) RT-PCR was performed on whole embryos injected with SP-MO (either 40 ng/cell or 20 ng/cell) into two cells at the 2-cell stage, then aged until stage 20 (Ctl, control sibling controls; -RT, no reverse transcriptase control). Hoxb9 expression is strongly decreased (lanes 3, 4), especially at the highest concentration of SP-MO, while cdx4 and sox2 expression remain unaffected. (F) Animal caps were dissected at stage 9 from embryos injected with either dazap2 mRNA (250 pg/embryo, lane 4), SP-MO (40 ng/embryo, lane 5), fgf8a (200 pg/embryo, lane 6), a combination of SP-MO and fgf8a (40 ng/embryo + 200 pg/embryo, lane 7) or a combination of fgf8a, SP-MO and dazap2 mRNA (40 ng + 200 pg + 250 pg/embryo, lane 8) at the 2-cell stage (Ni: non-injected caps). At stage 18, hoxb9 is induced by fgf8a (lane 6) but this induction is blocked in the presence of SP-MO (lane 7). This loss is rescued by addition of dazap2 mRNA (lane 8). In all combinations with Fgf8a, cdx4 is robustly induced.
	Fig. 4. Cdx4 induction of hoxb9 does not require dazap2. Embryos were injected with either SP-MO (20 ng, B), cdx4 (250 pg, C), or a combination of SP-MO and cdx4 (20 ng and 250 pg, respectively, D) into one cell of 4-cell stages (all were co-injected with lacZ mRNA). Stage 18 embryos are shown in dorsal views, with anterior at the top. As previously stated, SP-MO blocks the endogenous expression of hoxb9 (B, arrow), but has no effect on cdx4-induced ectopic expression of hoxb9 (C and D, arrowheads). (E) Ectodermal explants show that cdx4 induces hoxb9 in Noggin-neuralized caps (lane 7) and this is not affected by the silencing of dazap2 (lane 8). WE: whole embryo, -RT: no reverse transcriptase, Ni: not injected
	Fig. 5. Dazap2 gain-of-function expands the neural plate and induces a posterior identity at the expense of anterior fates. Injections of dazap2 mRNA were targeted either dorsally (A′-H′, red arrow) or ventrally (A′-E′, green arrow). Embryos were analyzed at stages 16-18 (shown in dorsal views with anterior at the top (A-E′), or frontal views (F-H′)). The injected side is on the right, injected cells are marked by the red nuclear stain. General neural markers nrp1 (A-A′), sox2 (B-B′) and sox11 (C-C′) are expanded when Dazap2 is expressed in or around the neural plate but not when dazap2 is injected on the ventral side of the embryo. MyoD (D-D′) expression is not affected by either dorsal or ventral injections. Hoxb9 (E-E′) is strongly ectopically expressed in dorsal but not ventral targeted injections. The expression of anterior neural markers cpl1 (F, F′) and pax6 (G, G′) is decreased when dazap2 is targeted to the anterior neural plate (arrows, bar indicates the midline).
	Fig. 6. Dazap2 has potent posteriorizing activities in neuralized tissues. (A) Both cells of 2-cell stage embryos were injected with the following mRNAs: NI, non-injected; dazap2 (DZ2, 250 pg/embryo); fgf8a (F8a, 100 pg/embryo); noggin (12.5 pg/embryo); or combination as indicated above lanes. RT-PCR analysis shows expression of a complete range of anterior and posterior markers in whole embryo (WE, line 1) but none in the NI caps (lane 3). Dazap2 and fgf8a both induce posterior genes hoxd1 and hoxb9, but Dazap2 also activates otx2 and en2 while fgf8 activates cdx1, 2 and 4 (lanes 4, 5). Upon neuralization by noggin (indicated by sox2 expression), animal caps express anterior markers only (otx2, lane 6). Dazap2 and noggin co-injections (lane 7) induce a complete range of anterior and posterior markers (but not cdx1, 2 or 4), while Fgf8 and Noggin co-injections only partially posteriorize the explants (en2 induction, lane 8). (B) A range of dazap2 mRNA (125 pg/cell, 50 pg/cell, 10 pg/cell or 2 pg/cell) alone or in combination with noggin (12.5 pg/cell) was injected into both cells of 2-cell stage embryos. Explants were dissected at stage 9 from these embryos and aged until stage 18. Decreasing levels of dazap2, alone or in combination, show a decrease level of induction of en2, hoxd1 or hoxb9. This is the opposite effect for otx2, which shows a decrease of induction alone but an increase induction in combination with noggin. Additionally, sox2 induction requires noggin. For in vivo analysis, embryos were injected at 4-cell stage with lacZ (100 pg/cell, C�E), dazap2 (250 pg) and lacZ (G�I), noggin (25 pg) and lacZ (K�M) or a combination of dazap2, noggin and lacZ (O�Q). Dazap2 injected embryos show a reduction of otx2 and krox20 expression (arrow) and ectopic expression of hoxb9 (arrowhead) in the anterior neural plate at stages 16�18 (G�I). Noggin-injected embryos show an increase in both otx2 and krox20 (arrow), and a decrease in hoxb9 expression (arrowhead) (K�M). The combination of dazap2 and noggin results in rescued otx2 and krox20 expression in injected cells (arrows) and hoxb9 expression is activated ectopically in the anterior neural plate (arrowhead) (O�Q).
	Fig. 7. Loss of FGF receptor signaling in the neural plate is rescued by dazap2. Uninjected control embryos (A-D) or embryos injected with dazap2 mRNA (250 pg, one cell at 4-cell stage) (co-injected with lacZ mRNA, A′-D′) were treated with either DMSO (1%, carrier control) or SU5402 (80 μM) at stage 12. Stage 18 embryos are shown in dorsal views, with anterior at the top. Uninjected embryos show no change in sox2 expression after SU5402 treatment (B), in contrast, hoxb9 expression was strongly reduced (D) in treated embryos, when compared to sibling controls (A and C, respectively). Dazap2 mRNA injected embryos show both an expansion (arrow) of sox2 expression (A′ and B′) and ectopic (arrowhead) expression hoxb9 (C′ and D′) when treated with either DMSO or SU5402.
	Fig. 8. Dazap2 activity is Cdx4-independent. mRNAs for dazap2 (250 pg, B), cdx-EnR (250 pg, C) or a combination of dazap2 and cdx-EnR (250 pg each, D) were injected into one cell of 4-cell stage embryos (all were co-injected with lacZ mRNA). Stage 18 embryos are shown in dorsal views, with anterior at the top. Dazap2 injections induce ectopic hoxb9 expression (B, arrowhead) while cdx4-EnR overexpression blocks hoxb9 induction (C, arrow). Combined Dazap2 and cdx-EnR injection results in robust hoxb9 induction (D, arrowhead). Parallel experiments in explants (E) show that Dazap2 induces hoxb9 either alone (lane 4) or in the presence of Cdx-EnR (lane 5).
	Fig. 9. Dazap2 activity does not depend on canonical WNT signaling. (A) RT-PCR was performed using whole embryos injected with fgf8a (100 pg/cell) either alone or in combination with β-catenin MO (either 40 ng/cell or 60 ng/cell) into two cells at the 2-cell stage, animal caps were dissected and then aged until stage 19 (WE, whole embryo sibling controls; -RT, no reverse transcriptase control; Ni, non-injected explant control). Hoxb9 expression is lost at both concentrations of β-catenin MO (lanes 5 and 6) when compared to Fgf8a alone (lane 4). (B) When the experiment is repeated using Dazap2 (125 pg/cell), β-catenin MO is unable to block Dazap2 induction of HoxB9 expression (compare lane 4, with co-injection lanes 5 and 6)
	Supplemental Figure 1. Dazap2 is conserved in the deuterostome clade. (A) Dazap2 encodes a 168 amino acid protein that contains several conserved SH2 (Yxxφ, indicated by light grey boxes) and SH3 (PxxP, indicated by dark blue boxes) consensus target sequences, as well as a polyproline rich region (red box). (B) The alignment of fourteen Dazap2 proteins found throughout the deuterostome clade shows a high conservation (Metazome.net). The conserved domains are highlighted in red, with the annotation based on the Xenopus laevis protein sequence. (Hsa, Homo sapiens; Cfa, Canis familiaris; Rno, Rattus norvegicus; Mmu, Mus musculus; Xtr, Xenopus tropicalis; Xla, Xenopus laevis; Dre, Danio rerio; Gac, Gasterosteus aculeatus; Tru, Takifugu rubripes; Ola, Oryzias latipes; Csa, Ciona savignyi; Cin, Ciona intestinalis; Bfl, Branchiostoma floridae; Spu, Strongylocentrotus purpuratus.) (C) A phylogenic tree was generated to demonstrate the relative distances of proteins using a UPGMA best tree method (MacVector).
	dazap1 (DAZ associated protein 1) gene expression in Xenopus laevis embryos, NF stage 19, as assayed by in situ hybridization, dorsal view, anterior up.
	dazap1 ( DAZ associated protein 1) gene expression in Xenopus laevis embryos, NF stage 19, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	dazap1 (DAZ associated protein 1) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	dazap1 (DAZ associated protein 1) gene expression in Xenopus laevis embryos, NF stage 44, assayed by in situ hybridization, ventral view, anterior left.
	dazap1 ( DAZ associated protein 1) gene expression in Xenopus laevis embryos, NF stage 44, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.

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