Embryonic Arrest at Midgestation and Disruption of Notch Signaling Produced by the Absence of Both Epsin 1 and Epsin 2 in Mice

Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice

Hong Chena,b,1, Genevieve Koa,3, Alessandra Zattic,d,3, Giuseppina Di Giacomoc,d, Lijuan Liua, Elisabetta Raiteric,d, Ezio Peruccoe, Chiara Collesia,c,2, Wang Minb, Caroline Zeissf, Pietro De Camillia,g,h, and Ottavio Cremonac,d,i,4

aHoward Hughes Medical Institute, Departments of Cell Biology and Program in Cellular Neuroscience, Neurodegeneration and Repair, Departments of bPathology, gNeurobiology, and fComparative Medicine, hKavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06536; eDipartimento di Scienze Mediche, Universita del Piemonte Orientale, 28100 Novara, Italy; and cIFOM, Fondazione Istituto FIRC di Oncologia Molecolare, dUniversita`Vita–Salute San Raffaele and iIstituto Nazionale di Neuroscienze, Via Adamello 16, 20139 Milano, Italy

Contributed by Pietro De Camilli, June 24, 2009 (sent for review June 8, 2009) Epsins are endocytic adaptors with putative functions in general that targets ubiquitinated Notch ligands to clathrin-coated pits aspects of clathrin-mediated endocytosis as well as in the inter- for endocytosis (15). nalization of specific membrane proteins. We have now tested the No effect of epsin mutations in flies was observed on synaptic role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by vesicle recycling (20), a process that relies on clathrin-mediated a genetic approach in mice. While either gene is dispensable for endocytosis, although peptide and antibodies microinjection life, their combined inactivation results in embryonic lethality at studies have suggested a role of epsin at the presynapse in lamprey E9.5–E10, i.e., at the beginning of organogenesis. Consistent with giant axons (21). In mammalian cells, knockdown of epsin isoforms studies in Drosophila, where epsin endocytic function was linked was reported to affect the clathrin-dependent internalization of to Notch activation, developmental defects observed in epsin 1/2 specific proteins (22) and of the influenza virus (23). double knockout (DKO) embryos recapitulated those produced by We have now taken a genetic approach in mice to elucidate a global impairment of Notch signaling. Accordingly, expression of epsin function in mammals. Mice express 2 ubiquitous isoforms, Notch primary target genes was severely reduced in DKO embryos. epsin 1 and 2, while a third isoform has been reported to have CELL BIOLOGY However, housekeeping forms of clathrin-mediated endocytosis a highly restricted distribution in repairing epithelia (24). We were not impaired in cells derived from these embryos. These report here the generation and an initial phenotypic character- findings support a role of epsin as a specialized endocytic adaptor, ization of mice that lack epsin 1 and epsin 2. with a critical role in the activation of Notch signaling in mammals. Results cell signaling ͉ endocytosis ͉ gene targeting Tissue Expression of Epsin 1 and Epsin 2. As a premise to the phenotypic characterization of the epsin 1 and 2 knockout (KO) psins are endocytic adaptors conserved from yeast to mam- mice, we conducted a comparative analysis of epsin expression Emals. They comprise a N-terminal ENTH domain that is in adult tissues and embryos (Fig. 1 A and B). In the adult, epsin followed by ubiquitin interacting motif (UIMs) and by predicted 1 and 2 are ubiquitous proteins, with the highest concentration unfolded central and C-terminal regions containing binding sites occurring in the brain (Fig. 1A). Epsin 1 and 2 were already for clathrin, the endocytic clathrin adaptor AP-2, and EH- detectable by quantitative real-time PCR (qRT-PCR) at E7.5 domain-containing proteins, such as the endocytic factors Eps15 (Ct value ϭ 31.2 Ϯ 0.53) and by western blotting of head tissue and intersectin (1). Consistent with these interactions, epsins are at E10 (Fig. 1B), the earliest time points examined. Their levels ubiquitous components of endocytic clathrin-coated pits (1–3). then steadily increased throughout gestation. Collectively, these They are recruited at early stages of endocytosis via interactions findings indicate that epsin 1 and 2 are housekeeping proteins with other coat components and via the binding of the ENTH broadly expressed in all tissues from early stages of development. domain to PI(4,5)P2, a phosphoinositide concentrated at the plasma membrane (4). The ENTH was also shown to have No Obvious Phenotypic Defects in Epsin 1 and Epsin 2 Single KO Mice. membrane bending properties, suggesting a contribution of epsin The genes encoding epsin 1 and 2 (Epn1 and Epn2) were disrupted to the generation of membrane curvature at the growing pit. by insertion of a neomycin cassette to delete the 3Ј-end of the first Based on these and other data, epsins have been considered coding exon and the beginning of the adjacent downstream intron housekeeping factors in clathrin-mediated endocytosis with an (Fig. S1A). KO mice for either genes were born with Mendelian additional role in the internalization of ubiquitinated cargo (1, 2, distribution, did not exhibit any obvious phenotypic defect, grew 5–7). Consistent with this idea, mutations of yeast epsins impair normally and were fertile. As revealed by western blot analysis of endocytosis and also actin function (8, 9), in agreement with the strong link between actin and endocytosis in this organism (10). However, studies in other species have produced conflicting Author contributions: H.C., G.K., A.Z., P.D.C., and O.C. designed research; H.C., G.K., A.Z., G.d.G., L.L., E.R., E..P., C.C., W.M., C.J.Z., and O.C. performed research; P.D.C. and O.C. results concerning an essential role of epsin in clathrin-mediated contributed new reagents/analytic tools; P.D.C. and O.C. analyzed data; and H.C., A.Z., endocytosis and have suggested a role as a cargo-specific adap- P.D.C., and O.C. wrote the paper. tor. In Dictyostelium, epsin is dispensable for clathrin-mediated The authors declare no conflict of interest. endocytosis (11). In Drosophila, the only epsin ortholog, liquid 1Present address: Cardiovascular Biology Research Program, Oklahoma Medical Research facets, was shown to have a critical role in the Notch signaling Foundation, 825 N.E. 13th Street, Oklahoma City, Oklahoma 73104. pathway most likely via a specific endocytic function in Notch 2Present address: Molecular Medicine Laboratory, International Centre for Genetic Engi- ligands expressing cells (12–15). Activation of Notch critically neering and Biotechnology, Trieste, Italy. requires its proteolytic cleavage, which is triggered by the 3G.K. and A.Z. contributed equally to this work. transendocytosis of Notch receptors by Notch ligands (16–18), 4To whom correspondence should be addressed. E-mail: [email protected]. an action that depends on ubiquitination of these latter proteins This article contains supporting information online at www.pnas.org/cgi/content/full/ (19). Hence, it was proposed that epsin functions as an adaptor 0907008106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907008106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 less frequently and prominently (Fig. S2 A and B), thus indicating that three-allele mutant mice suffer of a gene dosage effect of the epsin loci. These alterations were not further investigated in this study. A genotypic analysis on staged embryos derived from crossing of three-allele mutants (mostly Epn 1ϩ/Ϫ/2Ϫ/Ϫ mice) was performed. Matings were tightly controlled with a window-time of 4 h. Embryos were genotyped from the yolk sacs and staged by somite counting and by crown-rump length measurement of wild-type littermates. As shown in Table 1, Mendelian distribution was preserved until E9.5. However, DKO E9.5 embryos stopped growing. No DKOs were detected after E11.5, while at E10.5 their frequency was dramatically reduced. Taken together, these results suggest a date of embryonic lethality for the DKOs at around E10.

Housekeeping Forms of Endocytosis Occur in the Absence of Both Epsin 1 and Epsin 2. The survival and development of epsin DKO embryos up to E9.5–E10 indicate that the collective epsin 1/2 function is dispensable for cell viability. This result speaks against an essential role of epsin 1 and 2 in clathrin-dependent endocytosis, since this process appears to be indispensable from the earliest stages of development in mammalian organisms (25). The dynamics of housekeeping forms of clathrin-mediated endocytosis in the complete absence of epsin 1 and 2 were assessed in mouse embryonic fibroblasts (MEFs) and in middle Fig. 1. Expression of epsin 1 and 2, and of other endocytic and control T antigen-immortalized cells (26, 27) derived from either wild- proteins, in wild-type and mutant mice. (A) Tissue distribution of epsin 1 and type or DKO embryos. DKO cells did not reveal proliferation 2. Total homogenates from wild-type, epsin 1Ϫ/Ϫ, or epsin 2Ϫ/Ϫ mice tissues defects relative to cells derived from wild-type embryos. Levels were analyzed by western blotting with epsin isoforms specific antibodies. (B) of clathrin were the same in these mutant cells as in control cells Pattern of epsin 1 and 2 expression during development as revealed by (Fig. S3). Likewise, immunofluorescence analysis did not show western blotting of total homogenates of embryo heads (E) and from post- any obvious difference in the punctate distribution (known to natal brains (P) of wild-type mice. (C) Total brain homogenates from wild-type (WT) and mutant mice, were analyzed by western blotting with antibodies represent clathrin-coated pits) of clathrin heavy chain (Fig. 2 specific for epsin 1 or epsin 2. (D) Total brain homogenates from wild-type, A–F) between control and DKO cells. Furthermore, uptake of epsin 1ϩ/ϩ/2Ϫ/Ϫ, epsin 1ϩ/Ϫ/2Ϫ/Ϫ, epsin 1Ϫ/Ϫ/2ϩ/ϩ, and epsin 1Ϫ/Ϫ/2ϩ/Ϫ mice were transferrin (an assay of constitutive clathrin-mediated endocy- analyzed by western blotting with antibodies specific for epsin interacting tosis) (Fig. 2 G–I) and of EGF (1.5 ng/mL) (an assay of proteins and control proteins as indicated. ligand-triggered clathrin-mediated endocytosis) (Fig. 2 J) were indistinguishable in the two genotypes. This is in contrast to the reported partial impairment of EGF internalization in epsin 1 various tissues of single KO mice, corresponding epsin protein knockdown cells (22). Even the internalization of EGF at higher expression was missing (Fig. 1 A and C). Notably, in brain, no concentration (20 ng/mL), which is thought to occur mainly by changes in the expression levels of major epsin interactors, including a clathrin-independent pathway, was unaffected by the simulta- clathrin, AP-2, Eps15, and intersectin, or of other major synaptic neous lack of epsin 1 and epsin 2 (Fig. S4). proteins were observed in single homozygous and three-allele (see below) mutant mice (Fig. 1D). Major Developmental Defects in DKO Embryos. Inspection of DKO embryos at E9.5 revealed major defects both in the embryos Embryonic Lethality of Epsin 1/2 Double KO Mice. The intercrossing proper and in the extraembryonic structures relative to epsin of double heterozygous epsin 1 and 2 mice yielded three-allele 1ϩ/Ϫ/2Ϫ/Ϫ or epsin 1ϩ/ϩ/2Ϫ/Ϫ embryos, hence defined as ‘‘con- Ϫ Ϫ ϩ Ϫ ϩ Ϫ Ϫ Ϫ mutant mice, i.e., Epsin 1 / /2 / mice and Epsin 1 / /2 / trols.’’ DKO embryos were smaller, showed failure of axis mice, but no double KO (DKO) mice indicating embryonic rotation, and an open neural tube (Fig. 3 A–D). Next, the lethality of this genotype. Epn 1Ϫ/Ϫ/2ϩ/Ϫ mutants showed re- extraembryonic vasculature was examined, since its malfunction duced fertility, delayed growth (Fig. S2A), shortened life span, is a primary cause of developmental arrest at this stage. Whole and increased embryonic and perinatal mortality (Fig. S2B). mount preparations of the yolk sac failed to show large vitelline Similar defects were also observed in Epn 1ϩ/Ϫ/2Ϫ/Ϫ mutants, but arteries (Fig. 3 E–H), and histological analysis revealed that

Table 1. Genotyping results from the intercrossing of Epn1؉/؊/Epn2؊/؊ mice Gestation Days E1 ϩ/ϩ E1 ϩ/Ϫ E1 Ϫ/Ϫ Total

Day 7.5 5 (4) 8 3 (4) 16 Day 8.5 8 (11) 23 10 (11) 41 Day 9.5 33 (32) 64 28 (32) 125 Day 10.5 17 (17) 34 9*(17) 60 Day 11.5 9 (8) 17 0 (8) 26

Total number of embryos isolated for each genotype at the indicated gestational age. Asterisk indicates that some of these DKO embryos already showed sign of reabsorption at the time of isolation. Values in brackets indicate the number of embryos for an expected Mendelian distribution.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907008106 Chen et al. Downloaded by guest on September 24, 2021 A D A B C D

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G H K L CELL BIOLOGY I J Fig. 3. Major developmental defects in epsin DKO embryos. (A–D) Whole- mount (A and C) and parasagittal sections (B and D) of E9.0 embryos (WT and DKO as indicated). The DKO embryo is smaller than its control counterpart, has an open neural tube (arrows) and did not undergo axis rotation in the sagittal plane. (E–H) Whole-mount (E and G) and H&E stained cross sections (F and H) of yolk sac of control and DKO embryos at E 9.5; staining of whole-mount yolk sacs with the pan endothelial marker PECAM-1 is shown in insets of fields (F) and (H). The H&E-stained cross-sections reveal presence of large vascular sinusoids in the DKO yolk sac wall instead of the network of large and small Fig. 2. Occurrence of clathrin-mediated endocytosis in epsin DKO cells. MEFs vessels visible in the control sample. Large vitelline vessels are absent from derived from wild-type (A and B) and DKO (D and E) E9.5 embryos were stained DKO yolk sacs [insets of (F) and (H)]. (I–L) Whole-mount E9.5 WT and DKO by immunofluorescence for clathrin light chain (green) and epsin 1 (red)as embryos immunostained for the pan-endothelial cell marker PECAM-1. The indicated. Boxed areas are shown as higher magnification merged images in telencephalic (I and J) and intersomitic (K and L) regions are shown. (C) and (F). (G–I) Uptake of Alexa 488-transferrin (1.5 ␮g/mL) in WT and DKO large T antigen immortalized cells as revealed by epifluorescence after 30-min incubation (G–H) and by FACS analysis (i). (J) Uptake of I125-labeled EGF (1.5 ng/mL) in immortalized MEFs (Scale bar, 3 ␮m). was also dramatically impaired. Somites were fewer and of irregular size and shape (compare Figs. 3 K and L).

sinusoids were much larger and dilated in DKO embryos than in Impaired Notch Signaling in Double KO Embryos. Collectively, the controls, thus indicating a normal development of a primary defects observed in DKO embryos are highly reminiscent of vascular plexus but failure in its remodeling (Fig. 3 F and H). defects observed in mutants of Notch genes [Notch1/Notch4 Sections of mutant placentas demonstrated absence of the DKO mice (28)] or in genes essential for the activation of the labyrinthine layer and no intermingling between maternal and embryonic vessels (Fig. S5 A–C). Vascular defects were also present in the DKO embryo proper at E9.5. Although their heart in situ was beating, an atrophic ventricular wall was observed in H&E stained sections (Fig. 4). Further abnormalities were seen in DKO E9.0 embryos stained for PECAM-1, an early endothelial marker (Fig. 3 I–L). In the telencephalic region of DKOs, vessels did not form finely branched trees composed of large and small vessels as in controls (Fig. 3I), but rather a coarse network (Fig. 3J) where vessels of large uniform diameter prevailed. In the intersomitic region of mutant embryos, vessels were prominent only in the area where somites developed (see below). These vessels were quite disor- ganized relative to the vessels of control embryos (compare Figs. 3 K and L). Histological observations of the neural tube revealed picnotic cells and increased labeling for caspase 3 (active subunit), thus Fig. 4. Heart abnormalities in epsin DKO mice. H&E staining of heart sections indicating activation of the apoptotic pathway. Somitogenesis including the ventricles of E9.5 WT and DKO embryos.

Chen et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 24, 2021 redundantly by both epsin 1 and epsin 2, i.e., the two predom- inant and ubiquitous isoforms of this endocytic protein family. These findings do not rule out specific and unique roles of either epsin 1 or epsin 2, but imply that isoform-specific functions are not essential for life. Our data also implies that the third endocytic epsin present in mammalian organisms, epsin 3, cannot compensate for the lack of the other two epsins during embryonic development, consistent with its restricted pattern of expression (24). Importantly, as we have shown by studies in cells derived from DKO embryos, absence of both epsin 1 and 2 does not globally impair clathrin-dependent [e.g., transferrin uptake and EGF uptake at low concentration (39)] and clathrin-independent endocytosis [e.g., EGF uptake at high concentration (39)]. Accordingly, DKO embryos can complete gastrulation and begin organogenesis, while embryos lacking a functional clathrin adaptor AP-2, the major endocytic clathrin adaptor, die before E3.5 (25). Thus, if the embryonic lethality of DKO embryos is due to an endocytic defect, such defect must affect the internalization of selected cargos. A role in EGF-receptor internalization revealed by epsin 1 knockdown studies (22) was not reproduced in our DKO cells, possibly reflecting compensatory mechanisms that develop under these conditions. Based on the finding that Fig. 5. Impaired Notch signaling in DKO embryos. (A) Lysates from E9.0 the ENTH domain of epsin has membrane curvature generating embryo pools, MEFs and T-antigen immortalized cells were analyzed by properties, it was proposed that one function of epsin is to bend western blotting with antibodies directed against NICD. (B) Total RNA from the membrane at nascent endocytic clathrin-coated buds (2). control and epsin 1/2 DKO embryos at E9.0–E9.5 were analyzed by qRT-PCR Our findings do not rule out this function but speak against an using primers to speciﬁed genes. Gene expression of DKO embryos has been essential role of epsin 1 and 2 in this process. normalized on expression levels of wild-type (wt) littermates and then plotted in a logarithmic scale. (C) In situ hybridization for Hes5 of whole-mount E9.0 Epsin DKO embryos die at E9.5–E10, i.e., at the beginning of WT and DKO embryos counterstained. organogenesis, when lethality if often due to vasculature defects, including inability to establish an adequate yolk-sac circulation, heart tube formation defects, and cardiac failure (40, 41). Notch signaling pathway, including the Hey1/2 DKO (29), the Accordingly, in DKO embryos, placenta, and yolk-sac display RBP-Jk KO (30), the presenilin1/2 DKO (31), and POFUT1 KO massive angiogenesis defects. mice (32). In the embryo proper, we observed a major subversion of the In view of the critical role of epsin as an enabler of Notch three main developmental programs active at E9.5–E10, i.e., signaling in Drosophila, levels of the Notch intracellular domain cardiovascular development, somitogenesis, and neural tube (NICD), a marker of Notch activation, were examined by differentiation. The sum of these defects is highly reminiscent of western blotting. A significant reduction of NICD was observed the embryonic phenotype produced by impairment of the Notch in DKO E9.0 embryos relative to controls (Fig. 5A). Since signaling, a pathway involved in cell fate determination of all 3 alterations of Notch processing in DKO embryos could be an germ layers (16, 42). This similarity is of special interest given the artifact due to the severe hypoxic conditions experienced by established and critical role in Notch signaling, via an action in those mutants, we analyzed NICD levels in MEFs and middle T the signal sending cell, of the only epsin gene present in antigen-immortalized cells derived from wild-type and DKO Drosophila (12–15). Indeed, a massive down-regulation of Hes embryos. As observed in embryos, reduction of Notch processing and Hey family genes in DKO E9.0 embryos was observed in our strictly correlated with epsin absence (Fig. 5A). DKO mutants. These genes are the mammalian counterparts of Next, qRT-PCR was used to investigate the expression in whole the Hairy and Enhancer-of-split Drosophila genes and represent embryos of well-characterized primary target genes of the Notch the best-characterized primary targets of Delta-Notch signaling. pathway, i.e., the Hes and Hesr/Hey families of basic helix-loop- Their misregulation is associated with developmental defects helix transcriptional repressors (Fig. 5B) (33). Expression of these that closely resemble those observed in DKO epsin mutants (29, factors was lower in KO embryos, as expected if signaling by Notch 43, 44). Moreover, the vasculogenic phenotype observed in epsin ligands was reduced. Conversely, expression of the Notch ligand Delta-like 4 (Dll4) was higher, possibly reflecting a compensatory DKOs embryos is consistent with inactivation of both Notch1 response of the signal-sending cell to lack of engagement of the and Notch4 (28), while the somitogenic phenotype highly re- Notch receptor. Expression of other genes used as controls, for sembles that observed in Notch1 (45), Notch1/Notch2 double example Pitx2 [a homeobox transcription factor of the TGF-␤ (46), and in Notch1/Notch4 double (28) KOs embryos. Finally, signaling pathway (34)], gli1 [a Sonic Hedgehog pathway responsive neural defects of epsin DKOs were also observed in other Notch gene (35)], and c-Jun [a tyrosine kinase involved in several signaling mutants; an open neural tube was seen in Notch1 KO embryos pathways, including the MAPK and Wnt pathways (36, 37)] was (28) and increased apoptosis in the neural tissue of Notch2 KOs normal (Fig. 5B). embryos (47). Thus, the phenotype of epsin DKO mice is more Finally, the impact of epsin absence on the activation of Notch severe than that observed for any single or double Notch KO(s), signaling was supported by whole-mount in situ hybridization and can be explained by the functional impairment of multiple analysis of a specific Notch primary target gene—Hes5, an Notch genes. RBP-J binding gene (38). Hes5 expression was dramatically A similar phenotype is observed in embryos that lack genes of reduced, although not absent in E9.0 DKO embryos (Fig. 5C). the core activation machinery of the Notch signaling pathway, including (i) Mindbomb 1 mutant embryos, where Notch ligands Discussion fail to be ubiquitinated and thus activated (48); (ii) embryos with This study demonstrates an essential role of the epsin genes in mutations of O-fucosyltransferase 1 (Pofut1), where lack of mouse organogenesis. It shows that this function is contributed Notch fucosylation reduces binding of Notch ligands (32); (iii)

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907008106 Chen et al. Downloaded by guest on September 24, 2021 embryos lacking presenilin 1 and 2, where no Notch intracellular for reverse transcription (First-strand synthesis kit; Invitrogen) with random domain can be released upon ligand binding (31); and (iv)KO primers, and qRT-PCRs were performed with TaqMan chemistry (MasterMix; embryos for the Notch transcription activator RBP-Jk, where Applied Biosystems), using an ABI Prism 7900 sequence detector (TaqMan PCR; activation of Notch-dependent transcriptional activity is im- Applied Biosystems). Samples were run in triplicate, and at least 3 indepen- paired (30). Hence, our results support a crucial role of epsin in dent embryos for each genotype and for each gestational age were analyzed. enabling Notch signaling. Standard curves were established by measuring 6 replicates per data point. A critical open question is a mechanistic understanding of how The relative expression of the various mRNAs was normalized to the amount of glyceraldehyde 3-phosphate dehydrogenase or 18s RNA. epsin achieves its effect on Notch signaling. A plausible scenario is that epsin functions as an endocytic adaptor for ubiquitinated Cell Culture. MEFs derived from E9.0 wild-type and DKO embryos were im- Notch ligands, thus mediating their recruitment to clathrin mortalized by culturing them for at least 19 passages in vitro (PIV19) at 3% O2 coated pits. While we and others have demonstrated a UIM- as described in (51). Embryonic cells were also immortalized by Polyoma mediated interaction of epsin with ubiquitinated proteins (6, 7, middle-T retrovirus infection [kindly provided by Elisabetta Dejana (IFOM, 22), the precise effect of epsin on Notch ligands traffic, as well Milano, Italy)] as described (27). These latter cells were propagated on 0.1% as the mechanisms through epsin-dependent traffic of such gelatin-coated dishes in DMEM with 20% FCS (Life Technologies), supple- ligands may affect Notch activation in the signal receiving cell mented with endothelial cell growth supplement (50 ␮g/mL; Life Technolo- remain to be elucidated. Actions of epsin independent of clath- gies) and heparin (100 ␮g/mL; Sigma). rin, and mediated by its ENTH domain only (11, 49), also need to be understood. In conclusion, this study demonstrates that Endocytosis Assays. For transferrin internalization assays, cells were serum- epsin 1 and 2 are dispensable for basic aspects of clathrin- starved for 4 h, loaded with with 1.5 ␮g/mL Alexa-488 conjugated transferrin mediated endocytosis and support a cargo-specific function of (Molecular Probes) at 4 °C for 40 min, and then incubated for 10 or 30 min at these endocytic factors. Some general function of epsin 1 and 2 37 °C. At the end of each time point, cells were subjected to acid wash (0.5 M in clathrin-mediated endocytosis cannot be ruled out, but such NaCl, 0.5 M acetic acid, pH 4.5) for 5 min at 4 °C, trypsinized, and then fixed action(s) would have to be redundant with that of other endo- with 1% formaldehyde in PBS. Specific uptake of transferrin above a set threshold was measured as percentage of fluorescent cells by flow cytometric cytic proteins. analysis using a 4-color FACSCalibur (Becton Dickinson). For each sample, Methods 10,000 events were collected. Diva FACS software was used for the analyses. For EGF internalization assays, cells were serum-starved for 3 h before the Inactivation of the Epsin1 and 2 Loci. The epsin 1 and 2 genes were disrupted addition of 1.5 or 20 ng/mL 125I-EGF (Amersham Biosciences) in binding

by deletion of the 3Ј-end of the first coding exon and the 5Ј-end of the CELL BIOLOGY medium (DMEM with 20 mM HEPES, pH 7.4, and 0.1% BSA) for 2, 4, 6, and 8 following intron (Fig. S1A). 129Sv/J ES cells were electroporated with the min at 37 °C. At the end of each time point, cells were subjected to acid wash linearized construct, selected, and then screened by PCR (Fig. S1B). Recombi- (0.5 M NaCl, 2 M acetic acid, pH 2.8) for 5 min at 4 °C; surface-bound radio- nant clones were microinjected into C57BL/6 blastocysts (50). Genotyping was activity (i.e., the radioactivity present in the acid-wash) and internalized carried out by PCR on tail DNA (newborn animals) or on yolk sac DNA radioactivity (residual cell radioactivity) were measured by liquid scintillation. (embryos). Single epsin KO mice were backcrossed for at least 5 generations in 125 the C57BL/6 strain. Staged embryos were obtained by 4-h matings. Data are expressed as the ratio between internalized and surface I-ligand for each time point. Nonspecific binding was measured at each time point in the presence of a 100-fold molar excess of cold EGF and was always less than Antibodies and Reagents. Polyclonal rabbit antibodies were obtained from the following sources: Anti-epsin 1, anti-epsin 2, and anti-OCRL, from our labo- 5% of the total counts. For the 20 ng/mL EGF uptake assays, a mixture of ratory as previously described (1, 5, 51, 52); anti-dynamin 1, from Santa Cruz radioactive and cold EGF was used at a molar ratio of 1:4, i.e., with a large Biotechnology; anti-clathrin light chain and anti-intersectin, kind gifts from P. excess of cold EGF. McPherson (McGill University, Montreal, Canada). The polyclonal goat anti- body anti-PECAM-1 was from Santa Cruz Biotechnology. Mouse monoclonal Other Miscellaneous Procedures. Embryos were fixed in either 4% paraformal- antibodies were obtained from the following sources: Anti-tubulin, from dehyde (for H&E staining) or in MetOH:DMSO ϭ 4:1 (for whole mount inspec- Sigma; anti-HA from Covance; anti-clathrin heavy chain and anti-AP-2, from tion and immunohistochemistry) (54). Affinity Reagents; anti-synaptotagmin 1 from Synaptic Systems; anti-NICD Western blotting analysis, immunofluorescence, GST fusion protein pro- (Val 1744) from Cell Signaling Technology; anti-Eps15, a kind gift from PP Di duction, and PCR were performed as described (1). Fiore [IFOM, Milan, Italy]. Alexa 488-transferrin was from Invitrogen, and 125I-EGF was from Amer- ACKNOWLEDGMENTS. We thank Yun He and Simona Ferron for critical help; sham Biosciences. Simona Polo, Pier Paolo Di Fiore, Elisabetta Dejana, and Giorgio Scita for discussion and gifts of reagents; Loris Bernard and colleagues from COGEN- In Situ Hybridization. Sense and antisense riboprobes for Hes5 were cloned by TECH for qRT-PCR analyses; and Alberto Gobbi and Manuela Capillo for PCR from an embryonic mouse cDNA library (Invitrogen); the riboprobes assistance in animal husbandry. This work was supported in part by a Human spanned a region of about 1 kb of the transcript, including its 3Ј-UTR se- Frontiers Science Program grant, grants from the National Institutes of Health quence. Whole-mount in situ hybridization was performed on littermate (NS36251, CA46128 and DK45735), and a gift from the G. Harold and Leila Y. Mathers Charitable Foundation to P.D.C., by a pilot grant from Diabetes and embryos using digoxygenin-labeled antisense RNA probes as previously de- Endocrinology Research Center (DERC) at Yale University and a national scribed (53). Scientist Developmental Grant from American Heart Association to H.C. and by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Quantitative PCR Analysis of Gene Expression in Embryos. Total RNA was Association for International Cancer Research-U.K. (AICR-U.K.), Telethon, extracted from single E9.0–E9.5 embryos using standard techniques and Fondo per gli Investimenti della Ricerca di Base (FIRB), Interlink, and Progetti quantified by measuring of OD260 absorbance. RNA aliquots (2 mg) were used di Ricerca di Interesse Nazionale (PRIN) to O.C.

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