DAAM1 and DAAM2 are co-required for myocardial maturation and sarcomere assembly

AbstractWnt ligands regulate heart morphogenesis but the underlying mechanisms remain unclear. Two Formin-related proteins, DAAM1 and 2, were previously found to bind the Wnt effector Disheveled. Here, since DAAM1 and 2 nucleate actin and mediate Wnt-induced cytoskeletal changes, a floxed-allele of Daam1 was used to disrupt its function specifically in the myocardium and investigate Wnt-associated pathways. Homozygous Daam1 conditional knockout (CKO) mice were viable but had misshapen hearts and poor cardiac function. The defects in Daam1 CKO mice were observed by mid-gestation and were associated with a loss of protrusions from cardiomyocytes invading the outflow tract. Further, these mice exhibited noncompaction cardiomyopathy (NCM) and deranged cardiomyocyte polarity. Interestingly, Daam1 CKO mice that were also homozygous for an insertion disrupting Daam2 (DKO) had stronger NCM, severely reduced cardiac function, disrupted sarcomere structure, and increased myocardial proliferation, suggesting that DAAM1 and DAAM2 have redundant functions. While RhoA was unaffected in the hearts of Daam1/2 DKO mice, AKT activity was lower than in controls, raising the issue of whether DAAM1/2 are only mediating Wnt signaling. Daam1-floxed mice were thus bred to Wnt5a null mice to identify genetic interactions. The hearts of Daam1 CKO mice that were also heterozygous for the null allele of Wnt5a had stronger NCM and more severe loss of cardiac function than Daam1 CKO mice, consistent with DAAM1 and Wnt5a acting in a common pathway. However, deleting Daam1 further disrupted Wnt5a homozygous-null hearts, suggesting that DAAM1 also has Wnt5a-independent roles in cardiac development

Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells

Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching

Pofut1 point-mutations that disrupt O-fucosyltransferase activity destabilize the protein and abolish Notch1 signaling during mouse somitogenesis.

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos

Immuno-TEM analysis revealed Notch1 protein accumulation in the ER in <i>Pofut1</i> mutant PSM.

<p>(A-C) TEM analysis of anterior PSM in E8.5 wild-type (A) and <i>Pofut1</i>^Δ<i>/</i>Δ (B and C) embryos. Scale bars = 1 μm (A). (D-L) Immuno-TEM analysis of anterior PSM in E8.5 wild-type (D-F), <i>Pofut1</i>^Δ<i>/</i>Δ (G-I), and <i>Pofut1</i>^{<i>R245A/R245A</i>} (J-K) embryos using anti-Notch1 antibody. Scale bars are 2 μm (D) and 1 μm (E). The boxes in D, G, and J indicate the positions of enlarged images in E, F, H, I, K, and L. Arrows indicate Notch1 signals. Nuclei are indicated as “Nu”. (M) Quantification of subcellular localization of Notch1 in anterior PSM of control (5 embryos, n = 23, 33, 35, 38, and 82 cells), <i>Pofut1</i>^Δ<i>/</i>Δ (3 embryos, n = 46, 37, 51 cells), and <i>Pofut1</i>^{<i>R245A/R245A</i>} (3 embryos, n = 34, 55, 55 cells). Average ratios of each subcellular localization are shown as a bar graph. Control contained wild-type and heterozygous samples of each genotype. Asterisks indicate P<0.01; paired t-test.</p

F0 analysis of <i>Pofut1</i> gene targeted embryos.

<p>F0 analysis of <i>Pofut1</i> gene targeted embryos.</p

<i>O</i>-fucosyltransferase deficient Pofut1 mutant embryos phenocopied Pofut1 null mice.

<p>(A-D) WT, <i>Pofut1</i>^Δ<i>/</i>Δ, <i>Pofut1</i>^{<i>R245A/R245A</i>}, and <i>Pofut1</i>^<i>3G/3G</i> embryos at stage E9.5 are shown. Abnormal somites are shown with arrowheads, and an abnormally enlarged cardiac cavity is shown with an arrow. Scale bar = 500μm (A) (E-P) Immunohistochemistry of anterior PSM in E9.5 WT (E-H), <i>Pofut1</i>^{<i>R245A/R245A</i>} (I-L), and <i>Pofut1</i>^<i>3G/3G</i> (M-P) embryos using anti-Notch1 (green), anti-Pan-cadherin (red: cell surface) antibodies, and Hoechst33324 (blue: Nuclei). The three right panels are magnified images of insets shown in E, I, and M. Scale bars = 10 μm (E), and 5 μm (H).</p

Generation of <i>Pofut1</i>^<i>3G</i> mutant mice.

<p>Generation of <i>Pofut1</i>^<i>3G</i> mutant mice.</p

Pofut1 protein, but not RNA, was reduced in O-fucosyltransferase-deficient Pofut1 mutant embryos.

<p>(A and B) Upper panels show western blot analysis of Pofut1 expression using whole embryos of indicated genotypes at E8.5 (A) and E9.5 (B). Lower panels show the ß-tubulin amount as a loading control. (C) Semi-quantified RT-PCR results using whole embryos of indicated genotypes at E8.5 and E9.5 are shown. <i>Pofut1</i> mRNA amount was normalized with the <i>Hprt</i> mRNA amount. The RNA amount in individual embryos was shown as circles and the average expression levels were shown as a bar graph.</p

Generation of <i>Pofut1</i>^<i>R245A</i> mutant mice.

<p>Generation of <i>Pofut1</i>^<i>R245A</i> mutant mice.</p

Strategy for introducing a point mutation in the <i>Pofut1</i> locus using the CRISPR/Cas9 mediated genome-editing technique.

<p>(A) Schematic of the two independent point mutation sites of the <i>Pofut1</i> gene. The first mutation was introduced in the 5^th exon, and the resulting allele carried the R245A mutation. The second mutation was introduced in the 7^th exon and the resulting allele carried the ERD into GGG (3G) mutation. Each sgRNA coding sequence is capitalized and labeled in green. The protospacer-adjacent motif (PAM) sequence is capitalized and labeled in blue. Two donor oligos and one donor oligo were used for the R245A site and 3G site, respectively. The introduced mutation is indicated in red in the donor oligo sequence, and resulting mutant amino acids are shown below the boxes. Restriction enzyme sites, MscI and StuI, which were created by the mutations, are shown with lines above the donor oligo sequence. PCR primers (R245A Fw, R245A Rv, 3G Fw, and 3G Rv) used for PCR genotyping are shown as arrows. (B) RFLP analysis of F1 embryo samples. Amplified PCR product using primers R245A Fw and R245A Rv, followed by digestion with MscI. The genotypes shown above were genotyping results by direct sequencing. (C) RFLP analysis of F1 embryo samples. Amplified PCR product using primers 3G Fw and 3G Rv, followed by digestion with StuI. The genotypes shown above were genotyping results by direct sequencing. (D,E) Direct sequencing results of PCR products using primers R245A Fw and R245A Rv, and primers 3G Fw and 3G Rv, respectively.</p