An obligatory role of mind bomb-1 in notch signaling of mammalian development

Abstract

Background: The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood.

Methodology/principal findings: Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2(-/-) mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants.

Conclusions/significance: Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable.

Figure 1. Generation of Neur1 and Neur2 double knockout mice and their dispensable role in mammalian cerebral development.

(A) Targeted disruption of the murine neuralized-2 (Neur2) locus. Schematic drawings of the wild-type (wt) and recombinant (mt) loci and the targeting vector (tv) are shown. The homologous recombination event deletes exons 2-3 and places the IRES-LacZ gene within the exon 2. H, HindIII. (B) Schematic drawing of the Neur1 and Neur2 proteins with the deleted region (green). (C) Southern blot analysis showing the recombination event. The RT-PCR analysis shows the loss of Neur1 and Neur2 transcripts in each mutant indicated. β-actin was used for normalization. wt, wild-type band; mt, Neur2 mutant band. (D) Heterozygote intercrosses of Neur1^+/−;Neur2^+/− mice. (E) H&E sections of the neocortex (left panel) and hippocampus (right panel) of wild-type and Neur1&2^DKO mice. Note that there is no difference between the wild-type and Neur1&2^DKO mice.

Figure 2. Generation of Mib2^−/− mice and its dispensable role for mammalian development.

(A) Gene targeting of the murine Mib2 locus. The homologous recombination event deletes exons 5–15 and places the IRES-LacZ gene within exon 5. Schematic structures of the wild-type (wt) and recombinant loci (mt) and the targeting vector (tv) are shown. E, EcoRI; B, BamHI; K, KpnI; spe, SpeI. (B) Southern blot analysis of tail DNA after EcoRI digestion with the flanking probe shown in (A). The positions of the wild-type (23kb) and the targeted (6.3kb) allele are indicated. (C) Northern blot analysis of Mib2 gene expression in adult brain mRNA from wild-type (+/+) and Mib2^−/− (−/−) mice. Loading and integrity of the RNA were assessed by ethidium bromide staining of the 28S RNA in the gel prior to membrane transfer. (D) RT-PCR analysis of adult brain using Mib2-specific primers. No PCR product was detected from Mib2^−/− cDNA. β-actin was used for the normalization. (E) H&E sections of the hippocampus of wild-type (left) and Mib2^−/− (right) mice. Note that there is no difference between the wild-type and Mib2^−/− mice. (F) Whole-mount images of E9.5 embryos. Mib1^+/−;Mib2^−/− (i), Mib1^−/−;Mib2^+/− (ii), and Mib1^−/−;Mib2^−/− (iii).

Figure 3. Conditional inactivation of Mib1 in endothelial cells generates a Dll4 mutant phenotype.

(A, B) External view of embryonic day 9.5 (E9.5) yolk sacs (A) and embryos (B). The Tie2-cre;Mib1^f/f yolk sac (A, right) has failed to remodel the primary vascular plexus to form large vitelline blood vessels. The Tie2-cre;Mib1^f/f embryo (B, right) exhibits growth retardation and pericardial effusion. (C–F) Whole-mount PECAM staining of E9.5 wild-type (C, E) and Tie2-cre;Mib1^f/f (D, F) embryos. The large cranial vessels appear truncated and degenerated in the mutants (D, arrows). Intersomitic vessels are present, but the angiogenic sprouts are disorganized in the mutants (F). (G, H) Sections of PECAM-1 stained embryos. The dorsal aortas of Tie2-cre;Mib1^f/f embryos are reduced in diameter (H). ACV, anterior cardinal vein; DA, dorsal aorta. Scale bar, 200 µm. (I–L) Sections of ephrinB2 (I,J) and CD44 (K,L) stained embryos. The dorsal aortas of Tie2-cre;Mib1^f/f (J,L) embryos are negative for the arterial markers, ephrinB2 and CD44. (M–P) Sections of sm22 (M,N) and αSMA (O,P) stained embryos. The smooth muscle cell markers, sm22 and αSMA, are lost in the dorsal aortas of Tie2-cre;Mib1^f/f embryos (N,P). (Q) Semiquantitative RT-PCR in the yolk sac (ys) and para-aortic splenchnopleura (P-Sp) of wild-type (wt) and Tie2-cre;Mib1^f/f (mt). Hey1 is down-regulated in the mt yolk sac (left panel). Dll4 expression in the mt P-Sp is similar to that of wt (right panel). β-actin was used for normalization.

Figure 4. Conditional inactivation of Mib1 in skin epithelial cells generates a Jag1 mutant phenotype.

(A) External view of postnatal day 28 (P28) mice. The Msx2-cre;Mib1^f/f (lower) mouse shows loss of hairs in the dorsal midline (asterisk) and fused digits (arrow). (B, C) H&E sections of P28 wild-type (B) and Msx2-cre;Mib1^f/f (C) skin. The Msx2-cre;Mib1^f/f skin shows hyperplasia of skin epithelial cells (bracket) with lots of cysts (asterisks). Scale bar, 100 µm. (D–I) Sections of wild-type (D, F, H) and Msx2-cre;Mib1^f/f (E, G, I) skin stained with K14/PCNA (D, E), K10/PCNA (F, G) and Loricrin (H, I). The Msx2-cre;Mib1^f/f skin shows basal cell proliferation. K14, basal cell marker; K10, spinous cell marker; loricrin, granular cell marker; PCNA, proliferating cell marker. Scale bar, 100 µm.

Figure 5. Conditional inactivation of Mib1 in cerebellum generates a Jag1 mutant phenotype.

(A, B) Western blot analysis of P15 vermis (A) and whole-mount images of P9 vermis (B). wt, wild-type; mt, hGFAP-cre;Mib1^f/f. β-actin was used for normalization. (C–F) Nissle stained sections of P15 (C, D) and P17 (E, F) vermis from wild-type (C, E) and hGFAP-cre;Mib1^f/f (D, F) mice. hGFAP-cre;Mib1^f/f sections show the accumulation of granule cells (D, F, bracket). (G–J) DNA staining by Hoechst (G, H) and Ki67 immunostaining (I, J) of wild-type (G, I) and hGFAP-cre;Mib1^f/f (H, J) sections of P4 mice. Note that the hGFAP-cre;Mib1^f/f EGL has a similar level of proliferation as compared to the wild-type EGL. (K–N) DNA staining by Hoechst (K, L) and NeuN immunostaining (M, N) of wild-type (K, M) and hGFAP-cre;Mib1^f/f (L, N) sections of P15 mice. Note that the hGFAP-cre;Mib1^f/f mouse shows delayed migration of postmitotic granule cells in EGL (arrow) and ML (arrowhead). (O–R) BLBP (O, P) and GFAP (Q, R) immunostaining of wild-type (O, Q) and hGFAP-cre;Mib1^f/f (P, R) sections of P15 mice. The hGFAP-cre;Mib1^f/f mouse shows truncated Bergmann glial fibers that fail to extend to the pial surface (P, R, arrowhead). EGL, external germinal layer; ML, molecular layer; IGL, internal granule cell layer; PC, Purkinje cell layer. Scale bars, 50 µm.

Figure 6. Conditional inactivation of Mib1 in apical ectodermal ridge generates a Jag2 mutant phenotype.

(A) Whole-mount in situ hybridization of Mib1 in E10.5 apical ectodermal ridge (AER). The image on the left is an enlarged view of the boxed area in the right image. (B–G) Hind limbs of E13.5 (B, C), E15.5 (D, E), and P21 (F, G) of wild-type (B, D, F) and Msx2-cre;Mib1^f/f (C, E, G) mice. Note that the mutants show the fusion of digits 2 and 3 (C, E, G, asterisk). Numbers indicate the digit identity. (H, I) Stained skeletal preparations of neonatal hind limbs of wild-type (H) and Msx2-cre;Mib1^f/f (I) mice. Numbers indicate the digit identity. (J–O) Whole-mount in situ hybridization of Fgf8 (J, K, E11.5), Bmp2 (L, M, E13.5), and Jag2 (N, O, E11.5) in wild-type (J, L, N) and Msx2-cre;Mib1^f/f (K, M, O) embryonic hind limbs. Msx2-cre;Mib1^f/f embryos have broad Fgf8 expression (K) and show the loss of interdigital Bmp2 expression (M, arrow).