DeLay BD , Baldwin TA , Miller RK .

K01 DK092320 NIDDK NIH HHS , R03 DK118771 NIDDK NIH HHS

Xla wt + dnmbp CRSPR (Fig. 5 EFG)
Xla wt + dnmbp CRSPR (Fig. 6 A r2, B)
Xla wt + dnmbp CRSPR (Fig. 9)
Xla Wt + dnmbp MO (Fig. 10)
Xla Wt + dnmbp MO (Fig. 10)
Xla Wt + dnmbp MO (Fig 2 C r3, r4)
Xla Wt + dnmbp MO (Fig. 3C r2, 3D)
Xla Wt + dnmbp MO (Fig. 4 43, r4)
Xla Wt + dnmbp MO2 (Fig S2 r3, r4)

	FIGURE 1. Dnmbp is present throughout Xenopus pronephric development. (A) Western blot showing that Dnmbp is present in embryos ranging from stage one to stage 38. ∗ indicates Dnmbp. (B) In situ hybridization using probes against dnmbp.S and dnmbp.L showing dnmbp expression in the developing pronephros from stage 26 through stage 37/38. White bar is 500 μm.
	FIGURE 2. Knockdown of Dnmbp results in reduced kidney tubulogenesis. (A) Western blot showing the efficiency of two different MOs targeting Dnmbp. Single cell embryos were injected with 40ng of Dnmbp MO 1, Dnmbp MO 2 or Standard MO. One embryo equivalent per lane was loaded on to the SDS-PAGE gel. (B) Schematic of the Xenopus pronephros showing the proximal, distal and connecting tubule regions. (C) Unilateral injection of 20ng Dnmbp MO 1 into blastomere V2 at the 8-cell stage leads to defects in kidney tubulogenesis in comparison to embryos injected with Standard MO. Antibody 3G8 used to label the lumen of the proximal tubule, antibody 4A6 used to label the distal and connecting tubules. memRFP used as an injection tracer. White bar indicates 200 μm.∗ indicates injected side of embryo. (D) Knockdown of Dnmbp leads to reduced expression of differentiated kidney tubule markers in comparison to embryos injected with Standard MO. n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 3. Dnmbp knockdown is specific and does not cause somite development defects. (A) Unilateral injection of 20 ng Dnmbp MO 1 into blastomere V2 at the 8-cell stage does not cause somite defects in comparison to embryos injected with Standard MO. 12/101 antibody labels somites, lectin labels the proximal tubule lumen, rhodamine used as a tracer. Images are stitched from 6 tiles. White bar is 200 μm. (B) Knockdown of Dnmbp does not lead to reduced somite development compared to embryos injected with Standard MO. (C) Representative embryos showing that co-injection of β-galactosidase RNA with Dnmbp MO 1 does leads to kidney defects in comparison to control embryos injected with Standard MO and β-galactosidase RNA. Co-injection of human DNMBP mRNA with Dnmbp MO 1 rescues the knockdown phenotype. Stage 40 embryos stained with antibody 3G8 to label the proximal tubule and antibody 4A6 to label the distal and connecting tubules. White bar is 200 μm. (D) Quantitation of the rescue phenotype. n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different, p < 0.05.
	FIGURE 4. Dnmbp knockdown alters ciliogenesis. (A) Unilateral injection of 20ng Dnmbp MO 1 into blastomere V2 at the 8-cell stage causes altered primary ciliogenesis in comparison to embryos injected with Standard MO. Longer primary cilia are visible in the proximal tubules of the Dnmbp knockdown embryos (“more prominent cilia” phenotype). Acetylated tubulin antibody used to label cilia and nerves and lectin used to label the proximal tubule lumen. Images are stitched from 6 tiles. White bar is 50 μm, yellow bar is 25 μm. White arrowhead indicates nephrostome. White box indicates region represented in the enlarged merge panel. (B) Knockdown of Dnmbp leads to altered primary cilia development compared to embryos injected with Standard MO. n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 5. sgRNA targeting dnmbp efficiently edits Xenopus embryo DNA. Stage 40 embryos injected with dnmbp sgRNA and Cas9 protein at the 1-cell stage. (A) Chromatogram showing CRISPR editing of dnmbp.L in a single embryo. The underlined sequence corresponds to the dnmbp sgRNA binding sequence, and the arrow indicates sequence degradation due to CRISPR. (B) Chromatogram showing CRISPR editing of dnmbp.S in a single embryo. The underlined sequence corresponds to the dnmbp sgRNA binding sequence, and the arrow indicates sequence degradation due to CRISPR. (C) TIDE analysis of dnmbp.L sequence trace degradation after the expected Cas9 cut site. ∗p < 0.001. Percentage of dnmbp.L DNA sequence containing insertions and deletions. Bars indicate the mean of the percent of insertion/deletion sequences from four embryos, with the error bars representing the Standard error of the mean. Results are the mean of sequencing data from four embryos. (D) TIDE analysis of dnmbp.S sequence trace degradation after the expected Cas9 cut site. ∗p < 0.001. Percentage of dnmbp.S DNA sequence containing insertions and deletions. Bars indicate the mean of the percent of insertion/deletion sequences from four embryos, with the error bars representing the Standard error of the mean. Results are the mean of sequencing data from four embryos. (E) Representative stage 40 embryos showing that 8-cell targeted knockout of dnmbp leads to disrupted kidney tubulogenesis in comparison to slc45a2 knockout controls. Antibody 3G8 labels the proximal tubule lumen and antibody 4A6 labels cell membranes of the distal and connecting tubules. White bar is 200 μm. (F) Knockout of dnmbp reduces proximal tubule development. (G) Knockout of dnmbp reduces distal and intermediate tubule development. (F,G) n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 6. Human DNMBP mRNA rescues kidney defects caused by CRISPR dnmbp knockout in Xenopus embryos. (A) Representative embryos showing that co-injection of β-galactosidase RNA with a sgRNA against dnmbp does leads to kidney defects in comparison to control embryos injected with a sgRNA against slc45a2 and β-galactosidase RNA. Co-injection of human DNMBP mRNA with a sgRNA against dnmbp rescues the knockdown phenotype. Stage 40 embryos stained with antibody 3G8 to label the proximal tubule and antibody 4A6 to label the distal and connecting tubules. White bar is 200 μm. (B) Quantitation of the rescue phenotype. n = number of embryos across 2 replications. Error bars represent Standard error. ∗Significantly different, p < 0.05.
	FIGURE 7. Overexpression of human DNMBP results in kidney tubule defects. (A) Western blot showing expression of both endogenous (Xenopus) Dnmbp (lower band) and exogenous (human) HA::DNMBP (upper band). Embryos injected at the one-cell stage and assayed at stage 10–12. One embryo equivalent loaded per lane. (B) Representative stage 40 embryos injected with either β-galactosidase control mRNA or human DNMBP mRNA. Overexpression of hDNMBP leads to disrupted kidney tubulogenesis in comparison to controls. Embryos injected at the 8-cell stage (blastomere V2) to target the kidney and stained with 3G8 antibodies to label the proximal tubule and 4A6 antibodies to label the distal and connecting tubules. White bar indicates 200 μm. (C) Quantitation of phenotypes showing that overexpression of hDNMBP leads to a reduction in proximal, distal and connecting tubule staining. n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 8. Dnmbp knockdown does not alter expression of early markers of kidney development, as assessed by in situ analysis. Embryos injected unilaterally at the 8-cell stage (blastomere V2) to target the kidney and reared to stage 29–30 for lhx1 expression assessment, stage 32–33 for hnf1β expression assessment and stage 33–34 for pax2 expression assessment. Representative embryo phenotypes shown. Scale bar indicates 500 μm. n = number of embryos across 2 (pax2) or 3 replications (lhx1, hnf1β). Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 9. dnmbp CRISPR knockout does not alter expression of early markers of kidney development, however late marker expression is altered by in situ analysis. Embryos injected unilaterally at the 8-cell stage (blastomere V2) to target the kidney and reared to stage 29–30 for lhx1 expression assessment and stage 40–41 for atp1a1 expression assessment. Representative embryo phenotypes shown. Scale bar indicates 500 μm. n = number of embryos across 2 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 10. Dnmbp knockdown alters expression of late markers of kidney development, as assessed by in situ analysis. Embryos injected unilaterally at the 8-cell stage (blastomere V2) to target the kidney and reared to stage 40–41 for slc5a1, clcnkb, and atp1a1 expression assessment or stage 36–37 for nphs1 expression assessment. Representative embryo phenotypes shown. Scale bar indicates 500 μm. n = number of embryos across 2 (slc5a1, clcnkb, nphs1) or 3 replications (atp1a1). Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	FIGURE 11. dnmbp CRISPR knockout results in edema formation. (A) Bilateral injection of dnmbp sgRNA and Cas9 protein into both ventral blastomeres at the 4-cell stage leads to edema formation in comparison to embryos injected with slc45a2 sgRNA and Cas9 protein. Representative stage 45–46 embryos shown. White arrowhead indicates area of fluid accumulation. Scale bar indicates 500 μm. (B) Percent of embryos exhibiting edema formation. n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	dnmbp (dynamin binding protein ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	In situ hybridization of both homeologs of dnmbp. Antisense probes (AS) labeling dnmbp expression in the pronephros, head structures and somites. Sense (S) probes shown as a control for non-specific probe binding were processed in parallel with the AS probes. Scale bar indicates 500 μm.
	Knockdown of Dnmbp results in reduced kidney tubulogenesis. (A) Unilateral injection of 20 ng Dnmbp MO 2 into blastomere V2 at the 8-cell stage leads to defects in kidney tubulogenesis in comparison to embryos injected with Standard MO. Antibody 3G8 used to label the lumen of the proximal tubule, antibody 4A6 used to label the distal and connecting tubules. memRFP used as an injection tracer. White bar indicates 200 μm. ∗Indicates injected side of embryo. (B) Knockdown of Dnmbp leads to reduced expression of differentiated kidney tubule markers in comparison to embryos injected with Standard MO. n = number of embryos across 3 replications. Error bars represent Standard error. ∗Significantly different from control, p < 0.05.
	dnmbp (dynamin binding protein ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
	dnmbp (dynamin binding protein ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 29 & 30, lateral view, anterior left, dorsal up.

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