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First published online 6 July 2005
doi: 10.1242/dev.01922

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Mind bomb 1 is essential for generating functional Notch ligands to activate Notch

Bon-Kyoung Koo^1,*, Hyoung-Soo Lim^1,*, Ran Song¹, Mi-Jeong Yoon¹, Ki-Jun Yoon¹, Jin-Sook Moon¹, Young-Woong Kim¹, Min-chul Kwon¹, Kyeong-Won Yoo³, Myung-Phil Kong¹, Jinie Lee¹, Ajay B. Chitnis², Cheol-Hee Kim³ and Young-Yun Kong^1,

¹ Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, South Korea
² Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
³ Department of Biology, Chungnam National University, Taejeon 305-764, South Korea

View larger version (78K): [in a new window]   Fig. 1. Pleiotropic Notch defects in Mib1–/– embryos. (A,B) Wild-type (A) and Mib1–/– (B) embryos at E9.0. The wild-type embryo has the first and second branchial arches (A, arrows), while the Mib1–/– embryo only has the first branchial arch (B, arrow) with a distended pericardial sac (asterisk) and small, irregular somites (arrowheads). (C-F) Vascular defects in the yolk sac of E9.5 Mib1–/– embryos (D,F), as compared with the wild type (C,E). The blood vessels are indicated (asterisks). (G,H) Transverse sections of wild-type (G) and Mib1–/– (H) embryos at E9.5. The Mib1–/– embryo has a smaller dorsal aorta (da; inset i), a thinner neural tube (nt), loss of mesenchymal cells (asterisk), an enlarged pericardial cavity (#) and a fused notochord (inset ii). (I,J) Transverse section of wild-type (I) and coronal section of Mib1–/– (J) embryos at E9.5. A kinked neural tube (asterisk) and irregular somites (arrows) are evident in the Mib1–/– embryo (K) Myogenin expression (bracket) in E9.5 wild-type (left) and Mib1–/– (right) embryos. (L-P) Expression of Uncx4.1 (L), Dll1 (M), Hes7 (N), lunatic fringe (Lfng) (O) and Heyl (P) in E8.5 wild-type (left) and Mib1–/– (right) embryos. The Mib1–/– embryos lack the characteristic expression of Uncx4.1 (L, arrows), Dll1 (M, arrows and inset), Hes7 (M, arrow and bracket), Lfng (O, arrow and bracket) and Heyl (P, arrow and inset).

View larger version (99K): [in a new window]   Fig. 2. Premature neurogenesis in Mib1–/– embryos. (A,B) Sagittal sections of wild-type (A) and Mib1–/– (B) embryos at E9.5. The Mib1–/– embryo has a smaller head, with picnotic cells in the brain and inside the ventricles. Enlarged views of the forebrain are shown in insets. (C-H) Sagittal and transverse sections of E9.0-E9.5 brain regions from wild-type (C,E,G) and Mib1–/– (D,F,H) embryos. Sections were stained for nestin (C,D; in red) and huC/D (E-H; in red). BrdU incorporation (E,F; in green) and TUNEL staining (C,D,G,H; in green) were used for the detection of proliferative and apoptotic cells, respectively. Nuclear DNA was stained with Hoechst (in blue). (I) Impaired neurosphere formation in Mib1–/– forebrains. Neural stem cells are absent in E9.5 (n=3) and E9.75 (n=5) Mib1–/– forebrains. (J) Expression of neurogenin 1 (Ngn1), Dll1, Hes5 and Lfng in the neural tubes of E9.5 wild-type and Mib1–/– embryos. (K,L) Neurogenin 1 (Ngn1) (K) and Neurod1 (L) expression in E9.5 wild-type (left) and Mib1–/– (right) embryos. Note that the expression of both Ngn1 and Neurod1 in the trigeminal ganglia is increased in the Mib1–/– embryos (arrows), and Ngn1 expression is also increased in the neural tube of the Mib1–/– embryos (asterisk).

View larger version (86K): [in a new window]   Fig. 3. Vascular defects in Mib1–/– embryos. (A-D) Whole-mount Flk1 antibody staining of E9.0 wild-type (A,C) and Mib1–/– (B,D) embryos. Mib1–/– embryos have relatively thin and disorganized blood vessels. (A,B) Lateral view of the head; (C,D) Dorsal view of the trunk. (E-N) Transverse sections of E9.0 wild-type (E,G,I,K,M) and Mib1–/– (F,H,J,L,N) embryos, stained with an anti-PECAM antibody (E,F) or labeled by in situ hybridization with specific probes for ephrin B2 (G,H), Sm22 (I,J), Dll4 (K,L) and Hey1 (M,N). The PECAM-stained sections revealed the marked reduction or loss of the dorsal aorta (da) in the Mib1–/– embryos. The lack of vascular ephrin B2 expression (H; inset), smooth muscle cell recruitment (J; inset), and Hey1 expression (N; inset) in the Mib1–/– embryos is evident, whereas the Dll4 expression is normal (L; inset). (O,P) Expression of vascular Notch target genes (Hey1 and Hey2) and genes for vasculogenesis (ephrin B2, EphB4, Vegf and Shh). Total RNA from E9.0 wild-type and Mib1–/–yolk sacs was analyzed by semi-quantitative RT-PCR (O). The expression of Hey1 and Hey2 was analyzed by real-time quantitative RT-PCR (P). The numbers on each bar indicate the mean fold of induction, and the error bars indicate the standard deviation. ß-actin was used for normalization. The results are representative of three independent experiments. ***P<0.0001, *P<0.01.

View larger version (65K): [in a new window]   Fig. 4. Notch signaling defects in Mib1–/– embryos. (A) Lateral view of E8.759.0 wild-type (wt; a-h) and Mib1–/– (mt; a'-h') embryos probed for Dll1 (a,a'), Jag1 (b,b'), Notch1 (c,c'), Notch2 (d,d') Lfng (e,e'), Hes1 (f,f'), Hes5 (g,g') and Hey1 (h,h') expression. There is ectopic overexpression of Dll1 in the neural tube (a'; asterisk) and the downregulation of Dll1 in the somite and PSM (a; arrows, a'; arrowhead); loss of Jag1 expression in the head, branchial arches and presomitic region (b; arrows, b'; arrowhead); loss of Notch1 and Notch2 expression in somites (c,d; arrows, c',d'; arrowheads); and ectopic overexpression of Notch1 in the neural tube and trigeminal ganglia (c'; asterisk); the loss of Lfng expression in the trigeminal ganglia, newly forming somites and PSM (e'; arrowheads); and the loss of Hes1, Hes5 and Hey1 expression in the first branchial arches and PSM (Hes1, f'; arrowheads), the forebrain and neural tube (Hes5, g'; arrowheads), and the first branchial arches and newly forming somites (Hey1, h'; arrowheads) of the Mib1–/– embryos. (B) Expression of Notch target genes (Hes1, Hes5, Hes7, Hey1, Hey2, Heyl) and Notch pathway genes [Notch1, Notch2, Neur, RBP-j, Pres1 (presenilin 1), Pres2 (presenilin 2), Maml1 (mastermind-like1), Dll1, Jag1 and Mib1]. Total RNA from E8.5 wild-type (wt) and Mib1–/– (mt) embryos was analyzed by RT-PCR. ß-actin was used for normalization. The results are representative of three independent experiments. (C) Real-time quantitative RT-PCR for Notch1, Dll1, Jag1, Hes5, Hey1 and Hey2, using RNA from E8.5 wild-type (white bars) and Mib1–/– (black bars) embryos. Numbers in each bar indicate the mean fold of induction, and error bars indicate the standard deviation. ***P<0.0001, **P<0.001.

View larger version (41K): [in a new window]   Fig. 5. Defective Notch activation in Mib1–/– embryos. (A) Notch1 intracellular domain (N1icd) generation and Hes5 expression. Lysates from E9.0 wild-type (wt) and Mib1–/– (mt) embryos were immunoblotted with rabbit anti-N1icd, mouse anti-Notch1, rabbit anti-mouse Hes5 and rabbit anti-Mib1 antibodies. The results are representative of three independent experiments. (B-D) S3 cleavage of Notch1 by -secretase. Embryonic fibroblasts (EFs) isolated from E9.5 wild-type (wt) and Mib1–/– (mt) embryos were infected with the MSCV vector driving the expression of Myc-tagged extracellular domain-deleted Notch1 (EN1). (B) Western blot analysis of Nicd generation. Cell lysates were immunoblotted with anti-Notch1, anti-N1icd and anti-Mib1 antibodies. HEK293A cells (HEK) were used as a positive control for infection and N1icd generation. There is intact N1icd generation in Mib1–/– EFs. (C) Nuclear transport of Nicd. Infected cells were fixed and immunostained with an anti-Myc antibody, followed by anti-mouse Alexa-488 (in green). Nuclear DNA was stained with Hoechst (in blue). N1icd are transported intra-nuclearly in Mib1–/– EFs. (D) CBF-luciferase (Luc) activation by Nicd. The 8x wild-type and mutant CBF-Luc vectors were transfected to measure the activity of N1icd in MSCV_EN1-infected wild-type and Mib1–/– EFs. The 8x mutant CBF-Luc lacks the CBF-binding sites and was used as a control. The relative Luc activities of wild-type and Mib1–/– EFs are comparable. (E) Internalization of Delta by murine Mib1. COS7 cells were transfected with either mock GFP or HA and Myc-tagged Xenopus Delta (HA-XD-Myc) (black) or Mib1-GFP and HA-XD-Myc (green). Twenty-four hours after transfection, the cells were stained with anti-HA Ab followed by PE-conjugated anti-mouse Ab and analyzed by flow cytometry. (F) Defective Notch signaling in the Mib1–/– EFs. The wild-type (white bar) and Mib1–/– (black bar) EFs infected with MSCV vector driving the expression of Myc-tagged Xenopus Delta (XD), Myc-tagged mouse Delta1 (Dll1) or Myc-tagged mouse jagged 1 (Jag1) were co-cultured with C2C12-Notch1 cells transfected with the 8x wild-type and mutant CBF-Luc vectors. Twenty-four hours after co-culture, luciferase activity was measured. The 8x mutant CBF Luc was used as a control for Notch activation.

View larger version (68K): [in a new window]   Fig. 6. Interactions between Mib1 and all known Notch ligands. (A) Co-immunoprecipitation (Co-IP) of murine Mib1 with murine Notch ligands (Dll1, Dll3, Dll4, Jag1 and Jag2). HA-tagged Mib1 (HA-Mib1) or control vectors were co-expressed with Myc-tagged Notch ligands in HEK293A cells. The top panels show IP of Notch ligands by HA-Mib1, and the middle and bottom panels show the expression of HA-Mib1 and Notch ligand-Myc, respectively, in total cell lysates. (B) Subcellular localization of Mib1 (in green) and Notch ligands (Dll1, Jag1 and XD; in red). Mib1-GFP and/or Myc-tagged Notch ligand constructs were co-expressed in COS7 cells. Myc epitopes were detected with an anti-Myc antibody followed by a TRITC-labeled antibody. Nuclear DNA was stained with Hoechst (in blue). (a-c) Expression of Mib1-GFP (a), Dll1-Myc (b) and Jag1-Myc (c). (d-f) Co-transfection of Mib1-GFP with either Dll1-Myc (d), Jag1-Myc (e) or XD-Myc (f). Overlapping expression is yellow. (C) Transverse sections of E9.0 wild-type (wt) and Mib1–/– (mt) embryos stained with anti-Dll1 antibody. Dll1 is localized in the cytoplasm in wild-type neural tube, and accumulates in the plasma membrane in Mib1–/– neural tube (in red). Nuclear DNA was stained with Hoechst (in blue). The overall images are shown in insets. (D) Notch signal transduction. The pan-Notch phenotype in Mib1–/– embryos and the molecular interactions between Mib1 and multiple Notch ligands suggest a new core-Notch component that regulates the endocytosis of Dll and Jag ligands. The endocytosis of the Notch ligands by Mib1 stimulates the S2 and S3 cleavages of Notch receptors and the released Nicd translocates to the nucleus to express the Notch target genes, such as Hes5, Hey1 and Heyl.