Cervino AS , Collodel MG , Lopez IA , Roa C , Hochbaum D , Hukriede NA , Cirio MC .

NIH 2R01 DK069403 NIH HHS , R01 DK069403 NIDDK NIH HHS , U54 DK137329 NIDDK NIH HHS

             somite development
             glomus development

                leukemia
                pancreatic cancer
                oligodendroglioma
                esophagus squamous cell carcinoma

Xla WT + ssbp2 MO (Fig. 2e)
Xla WT + ssbp2 MO (Fig. 3 e j m m' n o)
Xla WT + ssbp2 MO (Fig. 4 e f i)
Xla WT + ssbp2 MO (Fig. 5 e, j, o)
Xla WT + ssbp2 MO (Fig. 6. f f' g)
Xla WT + ssbp2 MO (Fig. 6. m o )
Xla WT + ssbp2 MO (Fig S5. e j o p)
Xla WT + ssbp2 MO

	Figure 1. Ssbp2 is expressed in the pronephric anlage and the pronephric kidney during Xenopus development. Ssbp2 expression in Xenopus embryos by whole-mount in situ hybridization (WISH). (a) Early gastrula stage embryo (S10.5). Vegetal view. bp: blastopore. (bd) Early neurula stage embryo (S15). (b) Anterior view, dorsal up. ef: eye field; pop: presumptive olfactory placode; nf: neural folds. (c) Dorsal view, anterior to the left. (d) Lateral view, anterior to the left, dorsal up. (e, f) Late neurula stage embryo (S20). (e) Dorsal view, anterior to the left. nt: neural tube. (f) Lateral view, anterior to the left, dorsal up. (g) Histological preparation (transverse section) of the embryo showed in f (dotted line). sm: somitic mesoderm; nc: notochord; ar: archenteron. (g) Magnification of the dotted square shown in g. im: intermediate mesoderm; lm: lateral plate mesoderm. (h) Late neurula stage embryo (S21), lateral view. ea: eye anlage. (i) Early tailbud stage embryo (S23). Lateral view. op: olfactory placode; e: eye; hb: hindbrain; pa: pronephric anlage. (i) Early tailbud stage embryo (S23), lateral view. (j) Early tailbud stage embryo (S25), lateral view. (k) Early tailbud stage embryo (S26), lateral view. fb: forebrain; ov: otic vesicle. (I, m) Histological preparations (transverse sections) of the embryo shown in k (dotted lines). (l, m) Magnification of the dotted squares shown in l and m, respectively. som: somatic layer of the lateral mesoderm; spl: splanchnic layer of the lateral mesoderm. (n) Late tailbud stage embryo (S32), lateral view. ba: brachial arches; pk: pronephric kidney. (o, p) Histological preparations (transverse sections) of the embryo shown in n (dotted lines). (o, p) Magnification of the dotted squares showed in o and p, respectively. gm: glomus; pt: proximal tubule; dt: distal tubule. (q) Late tailbud stage embryo (S35), lateral view. vbi: ventral blood islands. (r, s) Histological preparations (transverse sections) of the embryo shown in q (dotted lines). (r, s) Magnification of the dotted squares shown in r and s, respectively. da: dorsal aorta. Representative embryos are shown.
	Figure 2. Ssbp2 morpholino knockdown affects pronephric kidney development and function. (a) ssbp2.S/L pseudo-alleles and ssbp2* partial sequences. The initiation codon ATG is underlined. ssbp2-MO target site is highlighted in grey. Note that ssbp2* possesses a 15 nucleotides deletion (crossed out red characters) to prevent ssbp2-MO binding. (b) 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated, fixed at stage 39 and subjected to WISH for the 1-NaK-ATPase to assess formation of the pronephric tubule. The uninjected contralateral side was used as an internal control. The percentage of embryos showing affected pronephros was quantified. Data in the graph is presented as mean. Statistical significance was evaluated using Chi-square test (****p<0.0001). *Represent the comparison to the uninjected group, represent the comparison to the St-MO injected group and # represent the comparison to the ssbp2-MO 15 ng injected group. (ce) 4-cell stage Xenopus embryos were injected into both ventral blastomeres as indicated and edema formation was analyzed at tadpole stage 45. (c) Uninjected embryo (1% with edema; n=88; N=3). (d) St-MO 30 ng injected embryo (11% with edema; n=81; N=3). (e) ssbp2-MO 30 ng injected embryos (38% with edema; n=68; N=3). N: number of independent experiments, n: number of embryos. Representative embryos are shown.
	Figure 3 Ssbp2 is required for normal glomus development. 8-cell stage Xenopus embryos were injected into a single V2 blastomere with 15 ng of St-MO or ssbp2-MO as indicated and the expression of glomus gene markers were analyzed by WISH at stage 35. The uninjected contralateral side was used as an internal control. (ae) WISH for wt1. (a) Uninjected embryo (n=30, N=3). (b, c) St-MO injected embryo (n=27, N=2). (d, e) ssbp2-MO injected embryo (n=54, N=4). (fj) WISH for nphs1. (f) Uninjected embryo (n=38, N=2). (g, h) St-MO injected embryo (n=40, N=2). (i, j) ssbp2-MO injected embryo (n=41, N=2). Representative embryos are shown. Magnification of the glomus region enclosed by the dotted squares. (k, l, m) Histological preparations of nphs1 WISH (transverse sections). nt: neural tube; sm: somites; nc: notochord; gm: glomus. (k, k, l, l, m, m) Magnification of the glomus enclosed by the black squares in k, l and m. *Indicates a reduction in the surface of the developing glomerular filtration barrier. (n,o) Quantification of the area stained by WISH for the different glomus markers (dotted red area in a and f).The ratio between the injected and control side is shown. Data in the graph is presented as mean and standard deviation. Each point represents a single embryo. Statistical significance was evaluated using KruskalWallis test and Dunns multiple comparisons test (****p<0.0001; ***p<0.001; *p<0.1). *Represent the comparison to the uninjected group, represent the comparison to the St-MO injected group. N: number of independent experiments, n: number of embryos.
	Figure 4. Ssbp2 loss-of-function impairs tubule morphogenesis. 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated, fixed at stage 39 and subjected to WISH for 1-NaK-ATPase followed by immunostaining with 12/101 antibody to assess pronephros and somites development, respectively. The uninjected contralateral side was used as the internal control. (a, b) Uninjected embryo. (c, d) St-MO 15 ng injected embryo. (e, f) ssbp2-MO 15 ng injected embryo. (g, h) Rescue experiment. ssbp2-MO 15 ng+ssbp2* mRNA 25 pg coinjected embryo. The white square encloses the tubule convoluted area. dv: dorsalventral; ap: anteriorposterior. Lateral views, anterior to the left. Representative embryos are shown. (k) Quantification of the tubule convoluted area (dv x ap). The ratio between the injected and control side is shown. Data in the graph is presented as mean and standard deviation. Each point represents a single embryo. Statistical significance was evaluated using KruskalWallis test and Dunns multiple comparisons test (****p<0.0001; ***p<0.001). *Represent the comparison to the uninjected group, represent the comparison to the St-MO injected group and # represent the comparison to the ssbp2-MO injected group. N: number of independent experiments, n: number of embryos.
	Figure 5. Ssbp2 depletion reduces the expression domain of proximal and distal tubule markers. 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated. The uninjected contralateral side was used as an internal control. (ae) WISH for pax2 in stage 32 embryos. *Indicate the position of the nephrostomes. (a) Uninjected embryo (8% affected, n=37, N=3). (b, c) St-MO 15 ng injected embryo (23% affected, n=40, N=2). (d, e) ssbp2-MO 15 ng injected embryo (66% affected, n=51, N=3). (fj) WISH for slc5a1 in stage 39 embryos. (f) Uninjected embryo (4% affected, n=47, N=2). (g, h) St-MO 15 ng injected embryo (19% affected, n=39, N=2). (i, j) ssbp2-MO 15 ng injected embryo (50% affected, n=34, N=2). (ko) WISH for clcnkb in stage 39 embryos. (k) Uninjected embryo (3% affected, n=35, N=2). (l, m) St-MO 15 ng injected embryo (23% affected, n=41, N=2). (n, o) ssbp2-MO 15 ng injected embryo (49% affected, n=33, N=2). Magnifications of the pronephric tubules enclosed by the black squares are shown in the left-bottom corner. Statistical significance was evaluated using Chi-square test (****p<0.0001; **p<0.01). *, Represent the comparison to the uninjected and the St-MO injected groups, respectively. Representative embryos are shown.
	Figure 6. Ssbp2 depletion affects pronephros terminal differentiation. 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated. The uninjected contralateral side was used as an internal control. (af) 3G8 whole-mount immunostaining was carried out at stage 37/38. (a, b) Uninjected embryo. (c, d) St-MO 15 ng injected embryo. (e, f) ssbp2-MO 15 ng injected embryo. (af) Magnifications (bottom) and schemes (top) of the proximal tubules enclosed by the white squares in a-f. Numbers indicate the tubule components. (g) The pronephric index (PNI) was scored as the difference between the number of proximal tubules components on both sides of the same embryo (PNI=0 indicates two identical proximal tubules). The percentage of embryos exhibiting different PNI values is shown. Data in the graph is presented as mean. (ho) 4A6 whole-mount immunostaining were carried out at stage 42. Magnifications of the distal tubule enclosed by the white square are shown for each side of the embryo. (h, i) Uninjected embryo (8% affected; n=40; N=2). (j, k) St-MO 15 ng injected embryo (28% affected; n=34; N=2). (l, m) ssbp2-MO-MO 15 ng injected embryo (59% affected; n=61; N=3). Statistical significance was evaluated using Chi-square test (****p<0.0001; **p<0.01). *, Represent the comparison to the uninjected and the St-MO injected groups, respectively. N: number of independent experiments, n: number of embryos. (n, o) Histological preparations (transverse sections) of the embryo shown in l and m. pt: proximal tubule; sm: somites; dt: distal tubule. Representative embryos are shown.
	ssbp2 (single-stranded DNA binding protein 2) gene expression in X. laevis embryo, NF stage 15, assayed via in situ hybridization, anterior view, dorsal up.
	ssbp2 (single-stranded DNA binding protein 2) gene expression in X. laevis embryo, NF stage 20 assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	ssbp2 (single-stranded DNA binding protein 2) gene expression in X. laevis embryo, assayed via in situ hybridization at NF stage 20, mid-trunk transverse section, dorsal up.
	ssbp2 (single-stranded DNA binding protein 2) gene expression in X. laevis embryo, NF stage 21 assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	ssbp2 (single-stranded DNA binding protein 2) gene expression in X. laevis embryo, NF stage 25 assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	ssbp2 (single-stranded DNA binding protein 2) gene expression in X. laevis embryo, NF stage 32 assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	atp1b1 (ATPase Na+/K+ transporting subunit beta 1) gene expression in X. laevis embryo, NF stage 39, assayed via in situ hybridization. trunk region, lateral view, anterior left, dorsal up.
	clcnkb (chloride voltage-gated channel Kb) gene expression in X. laevis embryo, NF stage 39, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	Supplementary Figure S2. Validation of the ssbp2-MO specificity. (a) Scheme of the ssbp2-eGFP mRNA reporter showing the ssbp2-MO target site. (b-e) 2-cell stage Xenopus embryos were injected into both blastomeres with 1 ng of ssbp2-eGFP mRNA in the presence or absence of the standard control morpholino (St-MO) or the ssbp2-MO (15 ng). Green fluorescence was analyzed in early gastrula stage embryos (S10.5) to allow ssbp2-eGFP mRNA translation. Vegetal views. (b) Uninjected embryos (0/40 with fluorescence). (c) ssbp2-eGFP injected embryos (39/40 with fluorescence). (d) ssbp2-eGFP + St-MO co-injected embryos (43/43 with fluorescence). (e) ssbp2-eGFP + ssbp2-MO co-injected embryos (2/52 with fluorescence). Note ssbp2-MO specifically reduced ssbp2-eGFP translation. Two independent experiments were performed. Representative embryos are shown.
	Supplementary Figure S3. Ssbp2 is not essential for establishment of the pronephric field. 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated. The uninjected contralateral side was used as internal control. (a-c) WISH for pax8 in late neurula stage embryos (S20). Dorsal views, anterior up. (a) Uninjected embryo (3% affected; n = 107; N = 6). (b) St-MO 15 ng injected embryo (11% affected; n = 36; N = 2). (c) ssbp2-MO 15 ng injected embryo (18% affected; n = 89; N = 5). (d-e) WISH for ors2 in mid-neurula stage embryos (S17). Transverse hemi sections, dorsal up. (d) Uninjected embryo (7% affected; n = 29; N = 2). (e) St-MO 15 ng injected embryo (7% affected; n = 43; N = 3). (f) ssbp2-MO 15 ng injected embryo (12% affected; n = 60; N = 3). Statistical significance was evaluated using Chi-square test. No significant differences were found between groups. N: number of independent experiments, n: number of embryos. Representative embryos are shown.
	Supplementary Figure S4. Ssbp2 is necessary for pronephric field morphogenesis. 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated. Pax8 expression domain was analyzed by WISH in early tailbud stage embryos (S26) and the uninjected contralateral side was used as internal control. (a,b) Uninjected embryo (0% affected, n = 36, N = 3). (c,d) St-MO 15 ng injected embryo (14% affected, n = 29, N = 2). (e-h) ssbp2-MO 15 ng injected embryos (55% affected, n = 40, N = 3). Representative embryos are shown. N: number of independent experiments, n: number of embryos. Representative embryos are shown.
	Supplementary Figure S5. Ssbp2 depletion affects proximal and distal tubule development. 8-cell stage Xenopus embryos were injected into a single V2 blastomere as indicated. The uninjected contralateral side was used as an internal control. (a-e) WISH for lhx1 in 32 stage embryos. (a) Uninjected embryo (2% affected, n = 54, N = 3). (b,c) St-MO 15 ng injected embryo (5% affected, n = 56, N = 3). (d,e) ssbp2-MO 15 ng injected embryo (43% affected, n = 70, N = 3). (f-j) WISH for pax8 in 32 stage embryos. (f) Uninjected embryo (5% affected, n = 40, N = 2). (g,h) St-MO 15 ng injected embryo (10% affected, n = 41, N = 2). (i,j) ssbp2-MO 15 ng injected embryo (44% affected, n = 54, N = 3). (k-o) WISH for hoxb7 in stage 32 embryos. (k) Uninjected embryo (10% affected, n = 40, N = 2). (l,m) St-MO 15 ng injected embryo (31% affected, n = 35, N = 2). (n,o) ssbp2-MO 15 ng injected embryo (68% affected, n = 38, N = 2). Magnifications of the pronephric tubules enclosed by the black squares are shown in the left-bottom corner. Statistical significance was evaluated using Chi-square test (****p < 0.0001; ***p < 0.001). Representative embryos are shown. (p) Quantification of the tubule length in the most anterior hoxb7 expression domain revealed by WISH (dotted red lines in f-j). The ratio between the injected and the control side is shown. Data in graph is presented as mean and standard deviation. Each point represents a single embryo. Statistical significance was evaluated using KruskalWallis test and Dunns multiple comparisons test (**** p < 0.0001; ***p < 0.001). * represent the comparison to the uninjected group and represents the comparison to the St-MO injected group. N: number of independent experiments, n: number of embryos.
	osr2 (odd-skipped related transcription factor 2) gene expression in X. laevis embryo assayed via in situ hybridization, NF stage 17, in transverse section, dorsal up.
	Figure 3. Ssbp2 is required for normal glomus development. 8-cell stage Xenopus embryos were injected into a single V2 blastomere with 15 ng of St-MO or ssbp2-MO as indicated and the expression of glomus gene markers were analyzed by WISH at stage 35. The uninjected contralateral side was used as an internal control. (a–e) WISH for wt1. (a) Uninjected embryo (n = 30, N = 3). (b, c) St-MO injected embryo (n = 27, N = 2). (d, e) ssbp2-MO injected embryo (n = 54, N = 4). (f–j) WISH for nphs1. (f) Uninjected embryo (n = 38, N = 2). (g, h) St-MO injected embryo (n = 40, N = 2). (i, j) ssbp2-MO injected embryo (n = 41, N = 2). Representative embryos are shown. Magnification of the glomus region enclosed by the dotted squares. (k, l, m) Histological preparations of nphs1 WISH (transverse sections). nt: neural tube; sm: somites; nc: notochord; gm: glomus. (k’, k’’, l’, l’’, m’, m’’) Magnification of the glomus enclosed by the black squares in k, l and m. *Indicates a reduction in the surface of the developing glomerular filtration barrier. (n,o) Quantification of the area stained by WISH for the different glomus markers (dotted red area in a and f).The ratio between the injected and control side is shown. Data in the graph is presented as mean and standard deviation. Each point represents a single embryo. Statistical significance was evaluated using Kruskal–Wallis test and Dunn’s multiple comparisons test (****p < 0.0001; ***p < 0.001; *p < 0.1). *Represent the comparison to the uninjected group, †represent the comparison to the St-MO injected group. N: number of independent experiments, n: number of embryos.

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