Marquez J , Bhattacharya D , Lusk CP , Khokha MK .

R01 HL124402 NHLBI NIH HHS , T32 GM007205 NIGMS NIH HHS , T32 GM007223 NIGMS NIH HHS , UL1 TR001863 NCATS NIH HHS

             nuclear pore inner ring
             nuclear pore complex assembly

                congenital heart disease

Xtr wt + nup188 MO + nup205 MO (Fig. 3 B r2c2, r2c3)
Xtr wt+ nup205 MO (Fig. 1 C)
Xtr wt+ nup205 MO (Fig.1 D)
Xtr wt+ nup205 MO (Fig. 1 E)
Xtr wt+ nup205 MO (Fig. 2 A)
Xtr wt+ nup205 MO (Fig. 2 B)
Xtr wt+ nup205 MO (Fig. 3 C r1c1)
Xtr wt+ nup205 MO (Fig. 4 A)
Xtr wt + nup93 MO (Fig.3 B r1c2)
Xtr wt + nup93 MO (Fig. 4 E)

	Fig. 1. Knockdown of nup205 Disrupts LR Patterning. (A) Schematic of one-cell injections and regions analyzed for LR patterning defects. (B) Representative blot of Nup205 protein levels in whole stage 45 Xenopus protein extracts. (C) Representative images of normal and abnormal cardiac OFT directionality (ventral views with anterior to the top) along with quantitation of frequency of abnormal morphology in control and nup205 morphant embryos. (D) Representative images of normal and abnormal pitx2c expression patterns (lateral views, dorsal to the top) along with quantitation of frequency of abnormal patterns in control and nup205 morphant embryos. (E) Representative images of normal and abnormal dand5 expression patterns (ventral views, anterior to the top) along with quantitation of frequency of abnormal patterns in control and nup205 morphant embryos. For all graphs, Ns indicate number of individual embryos assessed across a minimum of 3 replicates. Graph colors correspond to example border colors. Bar graph color coding depicts proportion of total MCCs assessed, while error bar is based on frequency of combined abnormal phenotypes. p < 0.005 and p < 0.0005 for ** and *** respectively by two-tailed T-Test. A – anterior, P – posterior, R – right, L-left. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 2. Knockdown of nup205 Disrupts Cilia in Multiple Tissues. (A) Representative images and quantitation of cilia number for control and nup205 morphant embryo LROs (ventral view with anterior to the top). Ns indicate number of distinct LROs analyzed. (B) Schematic of two-cell injections and representative images of control and nup205 morphant sides of an embryo (lateral view with dorsal to the top, top panel left side, bottom panel right side) and severity of ciliation defects in individual MCCs along with quantitation of remaining multiciliated structures and severity of MCC defect frequency. Ns indicate number of distinct embryos for which both sides were analyzed (dot plot) and numer of MCCs analyzed on either control MO or nup205 MO injected sides of embryos with no more than 10 MCCs assessed per a single side of an embryo (bar graph). Graph colors correspond to example border colors. Bar graph color coding depicts proportion of total MCCs assessed, while error bar is based on frequency of combined severe and abnormal phenotypes. (C) Representative localization of overexpressed GFP-NUP205 at the nuclear periphery and cilium in the Xenopus LRO and MCCs (yellow dotted line designates magnified area). p < 0.0001 for **** by two-tailed T-Test for dot plots or Chi Squared Test for bar graph. Graphed values were obtained across three replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. Knockdown of Inner-Ring NUPs Disrupts Cilia. (A) Examples of cilia components. (B) Representative images of cilia within MCCs of control and inner-ring NUP knockdown morphant embryo epidermis. Black arrowheads indicate properly positioned ciliary components, red arrowheads indicate mispositioned ciliary components beneath the cell surface. Note the apparent inclusion body with multiple ciliary components in the cases of nup188 + nup205 knockdown. (C) Percentages of visualized cells with abnormally positioned basal bodies at cell surface for each condition (basal bodies within 100 nm of surface were considered to be normally localized). Ns indicate number of cells assessed from a single embryo per condition. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 4. Inner-Ring Nucleoporin Roles Overlap. (A–D) Knockdown of nup205 loss of MCC cilia can be rescued with GFP-NUP188 or GFP-NUP205 respectively. Similarly, knockdown of nup188 loss of MCC cilia can be rescued with GFP-NUP205 (E–H) Knockdown of nup93 and loss of MCC cilia can be rescued with GFP-NUP93, but knockdown of nup205 and loss of MCC cilia cannot be rescued not be rescued with GFP-NUP93 nor can knockdown of nup93 and loss of MCC cilia be rescued with GFP-NUP205 (I–J) Knockdown of nup205 and loss of MCC cilia can be rescued with GFP-NUP205 (3131C > T) but not GFP-NUP205 (4829C > G). (K) Quantitation of rescue efficiencies amongst morphant and reintroduced NUPs/NUP variants with pairwise comparisons assessed via two-tailed T-test with * designating p < 0.05. Ns indicate number of MCCs assessed with 3 MCCs assessed per embryo across 3 replicates per condition. A: Apical B: Basal.
	Fig. S1. F0 CRISPR Knockout of nup205 Disrupts LR Patterning. (A) Quantitation of abnormal cardiac OFT morphology frequency in control and nup205 morphant embryos. (B) Fragment analysis of one control embryo and two crispant embryos shows the various edits achieved in F0 embryo tissue (expected size: 365). Ns indicate number of individual embryos assessed across 3 replicates. p<0.0001 for **** by two-tailed T-Test.
	Fig. S2. Pronephric Development and Ciliation is Dysregulated by Knockdown of nup205. (A) WISH for expression of atp1a1 as a marker of pronephric development in control and nup205 morphant sides of embryos. (B) Proximal pronephros cilia in control and nup205 morphant sides of embryos along with quantitation. Indicate number of embryos in which proximal pronephric cross-sections were assessed on both control MO injected and nup205 MO injected sides. p<0.0001 for **** by paired T-Test.
	Fig. S3. MCC Cell fate and Wnt signaling are Unaffected by Knockdown of nup205. WISH for expression of dnah9 and foxj1 as markers of multiciliated cell fate and in the case of foxj1 of Wnt signaling in the Xenopus epidermis appears unchanged in nup205 morphant sides of embryos as compared to control sides. Lateral views with dorsal to the top. Top panel – left side. Bottom panel – right side.
	Fig. S4. Knockdown of nup205 Disrupts Basal Body Distribution within MCCs. Representative images and quantitation of basal body number for control and nup205 morphant embryo MCCs. Ns indicate number of individual MCCs assessed across a minimum of 3 replicates with one MCC assessed per individual embryo. n.s. – not significant by two-tailed T-Test.
	Fig. S5. NUP205 Variant May Weaken Protein-Protein Interactions. The published structure of the NPC was used to determine the location of amino acid residue changes resulting from variants in an individual with heterotaxy. Left: The 3D structure of NUP93 and NUP205 (pdb: 5IJO): NUP93 (green) and NUP205 (red). Inset on right: The 3D structure of an interface region between NUP93 and NUP205.
	Fig. S6. Automated Counting of LRO Cilia and MCC Basal Bodies Workflow. Examples of counting workflow used in this study
	dnah9 (dynein, axonemal, heavy chain 9) gene expression in X.tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	foxj1 (forkhead box J1) gene expression in X.tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.

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