Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain

Vesicoureteral Reflux and Other Urinary Tract Malformations in Mice Compound Heterozygous for Pax2 and Emx2

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. This disease group includes a spectrum of urinary tract defects including vesicoureteral reflux, duplex kidneys and other developmental defects that can be found alone or in combination. To identify new regulators of CAKUT, we tested the genetic cooperativity between several key regulators of urogenital system development in mice. We found a high incidence of urinary tract anomalies in Pax2;Emx2 compound heterozygous mice that are not found in single heterozygous mice. Pax2+/−;Emx2+/− mice harbor duplex systems associated with urinary tract obstruction, bifid ureter and a high penetrance of vesicoureteral reflux. Remarkably, most compound heterozygous mice refluxed at low intravesical pressure. Early analysis of Pax2+/−;Emx2+/− embryos point to ureter budding defects as the primary cause of urinary tract anomalies. We additionally establish Pax2 as a direct regulator of Emx2 expression in the Wolffian duct. Together, these results identify a haploinsufficient genetic combination resulting in CAKUT-like phenotype, including a high sensitivity to vesicoureteral reflux. As both genes are located on human chromosome 10q, which is lost in a proportion of VUR patients, these findings may help understand VUR and CAKUT in humans

A Point Mutation in p190A RhoGAP Affects Ciliogenesis and Leads to Glomerulocystic Kidney Defects.

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease

Crosses between <i>Arhgap35</i>^<i>D34</i> and an independent <i>Arhgap35</i> mutant allele demonstrate non-complementation, resulting in hypoplastic and glomerulocystic kidneys.

<p><i>(A-F)</i> Whole urogenital systems dissected at E17.5. In contrast to normal sized kidneys in control animals (panels A,B,D), <i>Arhgap35</i>^{<i>D34/D34</i>}(C), <i>Arhgap35</i>^<i>-/-</i>(E), and <i>Arhgap35</i>^<i>-/D34</i>(F) kidneys exhibit hypoplasia (arrows) or agenesis (arrowheads). <i>(G-L)</i> Hematoxylin and Eosin stained sections (20X) show large glomerular cysts (asterisks), accompanied by occasional proximal tubule [PT] dilation in <i>Arhgap35</i>-deficient animals (I,K,L). <i>(G’-L’)</i> Higher magnification (63X) images show that glomerular architecture is largely preserved in cystic glomeruli. Scale bars, 20μm <i>(M)</i> Kidneys from the <i>Arhgap35</i>^<i>D34/-</i> allelic series show a similar proportion of cystic glomeruli, counted from H&E stained sections. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (one-way ANOVA)</p

Mapping of ENU-generated mutation identifies a point mutation in <i>Arhgap35</i> (p190A RhoGAP) that results in kidney hypodysplasia.

<p><i>(A)</i> The ENU-mutant line D34 shows exencephaly and spina bifida (open arrowheads), concomitant with kidney hypodysplasia (brackets) at E17.5. <i>(B)</i> Single nucleotide polymorphism (SNP)-array screening of 76 embryos identifies a single significant region on chromosome 7 associated with kidney hypodysplasia using algorithms EM [black], Haley-Knott [blue] and Multiple Imputation [red]. Significance threshold is indicated by horizontal line for each algorithm. <i>(C)</i> Restriction fragment length polymorphism analysis between the wild type C3H/HeNCrl and mutated C57BL/6J strains using marker rs31200925 (N = 346 embryos) confirms the association with kidney hypodysplasia. <i>(D)</i> Sanger sequencing of the <i>Arhgap35</i> A to T sequence variant identified by exome sequencing reveals a strong association with kidney hypodysplasia (N = 59 embryos).</p

Mouse embryonic fibroblasts (MEFs) derived from <i>Arhgap35</i>^{<i>D34/D34</i>} embryos exhibit defects in cilia elongation, associated with a failure of p190A_L1396Q to be recruited to the base of the cilium.

<p><i>(A)</i> Immunofluorescent staining of MEFs stimulated to form cilia by serum withdrawal reveals a defect in cilia length (acetylated α-tubulin, open arrowhead) independent of the basal body (γ-tubulin, closed arrowhead). <i>(B)</i> Quantification of number of ciliated cells based on basal body (γ-tubulin) and axoneme (acetylated α-tubulin). <i>(C)</i> The positioning of the basal body with respect to the cell surface is similar between control and <i>Arhgap35</i>^{<i>D34/D34</i>} MEFs. <i>(D-E)</i> The defects in cilia number (panel D) and length (panel E) in <i>Arhgap35</i>^{<i>D34/D34</i>} MEFs can be rescued by introduction of full-length wild-type p190A, but not the p190A_L1396Q mutant protein. <i>(F)</i> Full-length GFP-tagged p190A_WT and p190A_GAP constructs are enriched at the basal body (marked by γ-tubulin; arrowheads) while p190A_L1396Q, P190A_R1284A, p190A_GBD, and p190A_{GBD_FF} constructs fail to specifically localize in wild type MEFs. The percentage of cells with ciliary localization is indicated (N>200 cells per construct), which suggests a transient recruitment to the basal body. Scale bars, 2.5μm. *p<0.05, **p<0.01, ***p<0.005 (one-way ANOVA)</p

Glomerulocystic phenotype in <i>Arhgap35</i>^{<i>D34/D34</i>} is associated with a ciliogenesis defect.

<p><i>(A)</i> Immunofluorescent staining of control and mutant glomeruli sections at E17.5 show that podocytes (marked by nephrin expression) form ZO-1⁺ tight junctions, reflective of an intact slit diaphragm (closed arrowheads). Scale bars, 10μm <i>(B-D)</i> Immunofluorescent staining for acetylated α-tubulin and Arl13b in proximal tubules (marked by Lotus Tetragonolobus Lectin; LTL)(panel B) shows a significant decrease in the number (panel C) and length (panel D) of ciliated cells in <i>Arhgap35</i>^{<i>D34/D34</i>} animals compared to wild type (open arrowheads). Scale bars, 5μm. *p<0.05, **p<0.01, ***p<0.005 (unpaired, two-tailed Student’s <i>t</i>-test)</p

Ciliogenesis defects in <i>Arhgap35</i>^{<i>D34/D34</i>} MEFs can be rescued by inhibition of Rho GTPases and depolymerization of F-actin.

<p><i>(A-C)</i> Wild type and <i>Arhgap35</i>^{<i>D34/D34</i>} MEFs were stimulated to form cilia by serum withdrawal and treated with inhibitors of ROCK1/2 (GSK 429286A), Rac1 (NSC 23766) or the F-actin polymerization inhibitor Cytocholasin D. <i>(A)</i> Ciliary defects of <i>Arhgap35</i>^{<i>D34/D34</i>} MEFs are fully rescued by ROCK1/2 and F-actin inhibition and partially rescued by Rac1 inhibition as shown by immunofluorescence staining against γ-tubulin (basal body) and acetylated α-tubulin (axoneme)(arrowheads). Scale bars, 2.5μm <i>(B-C)</i> Quantification of cilia number (B) and length (C) in wild type and mutant MEFs treated with ROCK, Rac and F-actin polymerization inhibitors from (A). *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (one-way ANOVA)</p