Lnx2 ubiquitin ligase is essential for exocrine celldifferentiation in the early zebrafish pancreasMinho Wona,1, Hyunju Rob, and Igor B. Dawida,2

aSection on Developmental Biology, National Institute of Child Health and Human Development, Bethesda, MD 20892-2790, and bDepartment of BiologicalSciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea

Contributed by Igor B. Dawid, August 27, 2015 (sent for review February 25, 2015; reviewed by Didier Y. R. Stainier and Aaron Zorn)

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X(Lnx)2a is expressed in the ventral-anterior pancreatic bud ofzebrafish embryos in addition to its expression in the brain. Knock-down of Lnx2a by using an exon 2/intron 2 splice morpholinoresulted in specific inhibition of the differentiation of ventral budderived exocrine cell types, with little effect on endocrine cell types.A frame shifting null mutation in lnx2a did not mimic this pheno-type, but a mutation that removed the exon 2 splice donor site did.We found that Lnx2b functions in a redundant manner with itsparalog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2skipping and leads to the production of an N-truncated protein thatacts as an interfering molecule. Thus, the phenotype characterizedby inhibition of exocrine cell differentiation requires inactivation ofboth Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb,and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction inthe number of Notch active cells in the pancreas. We suggest thatLnx2a/b function to fine tune the regulation of Notch through Numbin the differentiation of cell types in the early zebrafish pancreas.Further, the complex relationships among genotype, phenotype, andmorpholino effect in this case may be instructive in the ongoing con-sideration of morpholino use.

pancreas | Numb | Notch | morpholino | TALEN

The pancreas is a vertebrate-specific bifunctional organ that iscomposed of exocrine tissue for secretion of digestive en-

zymes and endocrine tissue for production of hormones involvedin regulating glucose homeostasis. Morphogenesis of the devel-oping pancreas has been well characterized in amniotes andother vertebrates (1). The zebrafish has emerged as a useful modelorganism for studying pancreas formation. As in mammals, thezebrafish pancreas develops from two distinct pancreatic anlagenof the endoderm, the dorsal-posterior bud and the ventral-anteriorbud, which subsequently fuse to form the definitive pancreas. Inzebrafish, the dorsal bud gives rise to the primary islet, whereasthe ventral bud gives rise to exocrine cells, the pancreatic duct, andsecondary islets (2, 3).Pancreas development is regulated by a network of tran-

scription factors and signal transduction pathways. The Pdx1homeobox factor is of critical importance to pancreas formationin the mouse (4, 5) and is the earliest marker for cells specifiedas pancreatic precursors in all animals studied including thezebrafish (6). The basic helix-loop-helix factor Ptf1a is essentialfor the development of exocrine precursor cells in the mouse (7)and zebrafish (8, 9) and, furthermore, represents a valuableearly marker for exocrine precursors. Among multiple signalingpathways that have a role in the specification and differentiationof the pancreas, the Notch pathway has received particularattention. Studies in the mouse showed that artificial activationof the Notch pathway prevents precursors from differentiatinginto functional pancreatic cell types (10). Further, manipula-tion of Notch signaling affects the balance between endocrineand exocrine differentiation in the pancreas (11, 12). Severalstudies in zebrafish have focused on the secondary transition inwhich a specific population of ventral bud-derived cells differ-entiates into secondary islets that eventually account for the

majority of endocrine cells in the mature organ (13–15). Thesestudies conclude that cell differentiation in the pancreas de-pends on a reduction or cessation of Notch signaling, whereas,in turn, precursor maintenance requires a Notch signal. Fur-ther, specific levels of Notch signaling can direct precursor cellsto distinct fates, and the Notch pathway affects cell proliferationin this system.In previous work, we have studied the role of the E3 ubiquitin

ligase Ligand of Numb protein-X (Lnx)2b (16, 17) in embryonicdevelopment (18–20). We proceeded to explore possible func-tions of the other lnx2 paralog in zebrafish, lnx2a. The lnx2a geneis expressed in the pancreas anlage in addition to the nervoussystem, and lnx2a knockdown mediated by a splice morpholino(MO) led to differential inhibition of exocrine cell differentia-tion. Because the role of ubiquitylation and protein stability inpancreas development has received little attention, we pursuedthese observations further. A null mutation in lnx2a did notmimic the MO-induced phenotype because of redundancy of thelnx2a and lnx2b genes. We could show that the splice MO led toexon 2 skipping and the production of an N-truncated Lnx2aprotein that acts as an interfering factor. This effect could bedemonstrated most clearly by studying a mutation that deletesthe exon 2 splice donor site targeted by the MO. The mutantcontained the N-truncated protein also seen in the morphant andfully reproduced the exocrine deficiency phenotype. Further weprovide evidence that Lnx2a and Lnx2b act in pancreas devel-opment by destabilizing Numb, thereby affecting Notch sig-naling. We conclude that regulation of protein stability is animportant mechanism in early pancreas development in zebra-fish. Further, this example shows that nonreplication of anMO phenotype by a null mutation need not indicate off-target

Significance

Pancreas differentiation is of interest as an example of or-ganogenesis and for its medical implications. We report herethat regulation of protein stability is a player in the differen-tiation of certain cell lineages in the early zebrafish pancreas.The related E3 ubiquitin ligases Ligand of Numb protein-X (Lnx)2aand Lnx2b affect differentiation of exocrine cells, apparently bydestabilizing Numb in exocrine progenitor cells. Numb is an in-hibitor of Notch, a key regulator of pancreatic development. Wesuggest that Lnx2a/b destabilize Numb to derepress Notch,allowing precursors to proliferate and support subsequent dif-ferentiation. This study also highlights the fact that detailedanalysis of morpholino versus mutant phenotypes may be re-quired for a full understanding of their relationship.

Author contributions: M.W. and I.B.D. designed research; M.W. and H.R. performed re-search; M.W. and I.B.D. analyzed data; and M.W. and I.B.D. wrote the paper.

Reviewers: D.Y.S., Max Planck Institute of Heart and Lung Research; and A.Z., CincinnatiChildren’s Research Foundation.

The authors declare no conflict of interest.1Present address: Department of Pharmacology, College of Medicine, Chungnam NationalUniversity, Jung-gu, Daejeon 301-747, Korea.

2To whom correspondence should be addressed. Email: idawid@nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1517033112/-/DCSupplemental.

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effects but, in this case, helped reveal a more complex underlyingmechanism.

ResultsEarly Differential Expression of lnx2a in the Ventral Pancreatic Bud. Inthe context of our interest in the function of Lnx family E3ubiquitin ligases in embryonic development (18–20), we exam-ined the expression of lnx2a in the zebrafish embryo. In additionto expression in the CNS, lnx2a is transiently expressed in theventral, but not in the dorsal pancreatic bud from 19 h afterfertilization (hpf; 20–25 somites) to 55 hpf. In addition to anembryo overview, Fig. 1 illustrates the relative expression do-mains of lnx2a and the marker genes pdx1 (pan-pancreas), insulin(β-cells), foxa3 (endoderm), and ptf1a (exocrine cells). SI Ap-pendix, Fig. S1 shows a time course of lnx2a expression and adrawing of zebrafish pancreas development illustrating the lnx2aexpression pattern. Ptf1a, the earliest ventral pancreas-specifictranscription factor, is expressed from 33 hpf on (refs. 8 and 9).Lnx2a seems to be the earliest gene differentially expressed inthe ventral pancreatic bud and, therefore, we tested whetherlnx2a has a role in the specification of different pancreatic do-mains in the zebrafish embryo.

lnx2a Knockdown Using a Splice MO Inhibits Exocrine PancreasDifferentiation. To explore the pancreatic role of Lnx2a, weused a splice MO (hereafter named lnx2a-MO) that effectivelyblocks splicing between the first coding exon, exon 2, and exon 3(Fig. 2A and SI Appendix, Figs. S2 and S3). Whole-mount in situhybridization (WISH) using markers for ventral/exocrine (trypsin,ptf1a) and dorsal/endocrine cells (insulin, glucagon) revealed thatinjection of lnx2a-MO causes a severe deficit in ventral but notdorsal markers (Fig. 2 B–E). Reduction in ventral pancreasformation was confirmed by using WISH with additional markersat different stages of development (SI Appendix, Fig. S2 C andD). Using the transgenic strain Tg(ptf1a:EGFP), cell type-specificantibodies, and confocal laser scanning microscopy, we illustratethe reduction in ventral pancreatic markers at the protein levelas well (SI Appendix, Fig. S2E). In contrast to the strong re-duction of ventral pancreatic cells, other organ precursors in theendoderm formed normally, suggesting that Lnx2a is involved

in the development of the ventral pancreas but not the initialspecification of the endoderm (SI Appendix, Fig. S2 C and D).

A lnx2a Null Mutant Does Not Recapitulate the Morphant Phenotype.The validity of MO-induced phenotypes has been questionedrecently (21), yet the high regional precision of the lnx2a-MO–

induced defects seems to argue against a nonspecific effect. Toinvestigate this issue, we generated lnx2a null mutants by usingTALEN-mediated gene targeting (22). The lnx2aΔ70 mutationhas a frame shift early in the protein coding region, leading toearly termination of translation (Fig. 3 A and B), and contains nodetectable Lnx2a protein (Fig. 3C and SI Appendix, Fig. S3A).Thus, lnx2aΔ70 is almost certainly a null mutation, yet the mutantembryos displayed no pancreatic phenotype (Fig. 3 D1, D3, andE). Further, homozygous mutant fish are morphologically nor-mal, and are viable and fertile. Thus, we investigated the possiblefunctional redundancy of lnx2a with lnx2b, a paralog encoding asimilar protein (SI Appendix, Fig. S3B); lnx2b is expressed in theendoderm including the pancreas in zebrafish embryos (SI Ap-pendix, Fig. S3C). To test for possible redundancy, we injected avalidated (18) MO against lnx2b (SI Appendix, Fig. S3 D and E)into WT and lnx2aΔ70 mutant embryos, and found that the MOgenerated a ventral pancreas deficiency phenotype in mutant butnot in WT embryos (Fig. 3 D2, D4, and E). Thus, redundantlnx2b function may account for the fact that lnx2a is dispensablefor pancreatic development.Because a block to exon 2/3 splicing disconnects the standard

initiation site in exon 2 from downstream coding, the lnx2amorphant cannot produce full-length Lnx2a protein (Fig. 2A andSI Appendix, Fig. S2 A and B). Nevertheless, RT-PCR and an-tibody blotting showed that morphant embryos produce exon 2skipped RNA (Fig. 3 F and H) and, importantly, abundant levels

Fig. 1. lnx2a is expressed in the ventral pancreas during early pancreas spec-ification. Lateral views (A and I), dorsal views (B–E), and ventral views (F–H).(A) lnx2a transcripts (blue) are detected in regions of the endoderm (arrow),forebrain (arrowhead), and the spinal cord at 24 hpf. Expression of pdx1 (B, D,and G), ins (C, E, and I) and foxa3 (F) are in red. lnx2a expression is observed inthe antero-ventral part of pdx1-positive pancreas precursors at 21 (B) and 26(D) hpf, but excluded from β-cells (ins+) in the dorsal pancreas at 21 (C), 26 (E),and 48 (I) hpf. At 48 hpf, lnx2a expression is detected in the ventral pancreasbut not in the intestine, swim bladder, or liver (F–H). SB, swim bladder; In, in-testine; Li, liver; Pa, pancreas; VP, ventral pancreas. (Scale bars: 50 μm.)

Fig. 2. lnx2a knockdown causes defects in the exocrine pancreas. (A) Sche-matic drawing of the lnx2a locus containing 11 exons, and the Lnx2a protein(737 aa) containing a RING-finger domain (RF), the Numb binding NPAF motif(F), and four PDZ domains. The translation initiation site is located in exon 2,which encodes the RING-finger domain. The lnx2a-MO targets the splice donorsite of exon 2. Primers in exon 1 (F1) and exon 11 (R1) are shown, as is theepitope for the Lnx2a antibody in the third PDZ domain. (B and C) The lnx2a-MO(5 ng) leads to inhibition of exocrine markers (try, ptf1a), but not endocrinemarkers (ins, glu), as seen by two-color WISH at 60 hpf. (D and E) Quantificationof marker expression; effects were classified as strong, partial, and unaffected.MO, lnx2a-MO; UN, uninjected. (Scale bar: 100 μm.)

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of Lnx2a immunoreactive protein with a molecular size smallerthan the wild type (Fig. 3G). Using mass spectrometry, we showedthat the short form of Lnx2a represents an N-terminally truncatedform that is initiated from a cryptic start site in exon 3 (Fig. 3Hand SI Appendix, Fig. S4). Truncated Lnx2a has lost the RINGdomain that has been shown previously to be required for E3ubiquitin ligase activity of Lnx proteins (16–18), as confirmed in SIAppendix, Fig. S5 A and B. The N-truncated protein retains theNPAF motif and the PDZ domains that mediate protein–proteininteractions (Figs. 2A and 3H), suggesting that it could act as anegative interfering form. This suggestion is borne out by theobservation that injection of the truncated mRNA into lnx2aΔ70

mutant embryos re-created the loss of exocrine marker phenotype(Fig. 3 I and J). A weak phenotype was seen after truncatedmRNA injection into WT embryos, most likely because thetruncated protein has only a moderately strong interfering effect.We conclude that lnx2a and lnx2b have redundant function inpancreas development so that loss of expression of one paraloghas no phenotype but sensitizes the organism to further inter-ference with Lnx2 protein function.

The lnx2aΔ329 Mutant Recapitulates the Morphant Phenotype. Usingthe same TALEN pair described above, we isolated a largerdeletion, lnx2aΔ329 (Fig. 4 A and B). In this mutation, the exon 2splice donor site has been lost, providing a definitive geneticmodel for the putative effect of the lnx2a-MO. As predicted,lnx2aΔ329 mutant embryos express the same truncated form ofLnx2a protein as the morphant (Fig. 4C and SI Appendix, Fig.S4). Most critically, lnx2aΔ329 mutants shows the same pan-creatic phenotype as the morphant, with specific inhibition ofexocrine marker expression, whereas endocrine markers are es-sentially unaffected (Fig. 4 D and E). This fact is true for bothzygotic and maternal/zygotic mutants (Fig. 4E). It should be notedthat homozygous lnx2aΔ329 zebrafish show high embryonic le-thality, but some mutant fish survive, allowing the cross illustratedin bars 2 and 3 of Fig. 4E. The behavior of the lnx2aΔ329 mutantprovides support for the conclusion that the phenotype is dueto loss of functional Lnx2a combined with interference withthe function of Lnx2b, and that the lnx2a morphant phenotype isbased on the same mechanism. These results legitimize the useand interpretation of the lnx2a-MO.

The Ventral Pancreas Defects in lnx2aΔ329 Mutants and lnx2a MorphantsAre Rescued by Knockdown of Numb. Lnx is known to target Numbfor ubiquitylation and degradation (16), and Numb is an inhibitorof Notch signaling (23–26). Because Notch signaling has a majorrole in the specification and differentiation of various pancreaticcell types (10, 13–15) we asked whether the Lnx2a morphant andmutant phenotype is due to a change in the activity of Numb.Numb is expressed in the embryonic endoderm including thepancreas, but whereas numb RNA could be detected in the entirepancreas, the protein was detected only in the dorsal bud-derivedprimary islet (SI Appendix, Fig. S5 C and D). Because the knownubiquitylation and degradation of Numb by Lnx proteins (16, 18)was confirmed for zebrafish Lnx2a (SI Appendix, Fig. S5 A and B),it seemed possible that Lnx2a reduces the level of Numb proteinin the ventral pancreas where Lnx2a is expressed (Fig. 1). Thelnx2amutant/morphant phenotype might then be due to abnormalstabilization of Numb; we illustrate in the next section that thereis, in fact, an increase of Numb-positive cells in lnx2a morphants.Therefore, we tested whether reduction of Numb expression couldrescue the lnx2a-deficient phenotype. This prediction provedcorrect: Both lnx2aΔ329 mutant and lnx2a morphant phenotypeswere substantially rescued by injection of numb-MO (Fig. 5 and SIAppendix, Fig. S5E). The absence of a gross morphological effectof the numb-MO itself is consistent with the work of Brescianiet al. (27). This agreement, and the fact that we observe a specificrescue phenotype rather than developmental malformation, shouldhelp alleviate concerns regarding the validity of the numb-MO inthis context. These results support the view that Lnx2a and Lnx2b

Fig. 3. Generation of lnx2aΔ70 mutant using TALENs, and the functional re-dundancy of lnx2 genes. (A) Schematic representation of the lnx2a locus and theTALEN target site in the first protein coding exon (exon 2). TALEN targets (left,orange and right, green), and the lnx2aΔ70 mutation are shown below thedrawing. Next, the protein sequences of WT and the lnx2aΔ70 frameshift alleleare shown. Genotype of lnx2aΔ70 mutant was analyzed by the genomic PCR (B),and immunoblotting of endogenous Lnx2a protein (C). For validation of theanti-Lnx2a antibody, see SI Appendix, Fig. S2F. (D) Marker gene (try and glu)expression shows little effect in lnx2aΔ70 null mutants, and in lnx2b-MO–injectedembryos at 60 hpf. However, lnx2b-MO injection into lnx2aΔ70 mutant embryosshows suppression of exocrine markers. (E) Quantification of pancreatic defectsby analysis of trypsin expression, classified as in Fig. 2D. See SI Appendix, Fig. S3Dand E for effectiveness of lnx2b-MO. (F–H) lnx2a-MO leads to production ofN-truncated Lnx2a protein. (F) RT-PCR using primers F1 and R1 (defined in Fig.2A) and 48 hpf embryo RNA, followed by sequencing, shows that lnx2a-MOinjection results in exon 2 skipping and stabilization of the resulting transcript.lnx2b expression was not changed by lnx2a-MO injection. (G) Endogenous Lnx2ashowed the expected size in controls, but a smaller protein was seen in lnx2a-MO–injected embryos, which was identified by mass spectrometry (SI Appendix,Fig. S4) as an N-truncated protein (587 aa) arising from an alternate start site inexon 3. (H) Schematic drawing of exon 2 skipping, alternate translation start site,and N-truncated protein in lnx2a-MO–injected embryos. (I) N-truncated Lnx2a(exon 2-skipped, as shown in F–H) has interfering effect. Tr-mRNA was injectedinto the WT and lnx2aΔ70 mutant embryos and analyzed at 48 hpf. (J) Quanti-fication of pancreatic defects in I by analysis of ptf1a expression. Injection ofTr-mRNA into WT embryos has little effect, but leads to the exocrine pancreasphenotype in lnx2aΔ70 mutants. (Scale bars: D, 100 μm; I, 50 μm.)

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function in early pancreas development to limit the extent andarea of Numb protein accumulation and activity.

Loss of Lnx2 Activity Causes a Reduction of Notch-Responsive Cells inthe Pancreas. Notch signaling is a key regulator of pancreas for-mation, affecting specification of different cell types and control-ling their quiescence or proliferation (10, 13–15, 28). We askedwhether Lnx2a, through its modulation of Numb protein stability,can affect Notch activity in the embryonic pancreas. For thispurpose, we used the transgenic strain Tg(Tp1:H2BmCherry);

Tg(Tp1:VenusPest) in which a Notch-responsive element drivesexpression of stable nuclear mCherry and unstable Venus (13). Inthis strain, red nuclei signify cells in which Notch signaling waspreviously active but has now been turned off, whereas green anddouble-positive cells are active for Notch signaling when exam-ined. We find that lnx2a-MO increased Numb-positive cells andreduced Notch-active cells in the pancreas (Fig. 6 A, 2 and B, 1and 2). Furthermore, coinjection of numb-MO rescues the effectof lnx2a-MO on the abundance of Tg(Tp1:VenusPest) expressingNotch-ON cells (Fig. 6 A, 4 and B). We propose that numb istranscribed in the entire pancreatic anlage, but the protein isdestabilized by Lnx2 in the ventral bud. Loss of Lnx2 allows Numbaccumulation in the ventral bud, interfering with Notch regulationand cell differentiation.To test how Lnx2 affects ventral bud precursor populations,

we tested Notch activity and Numb accumulation at earlierstages. In Lnx2a morphants at 36 hpf, the number of Tg(Tp1:VenusPest)-positive cells is reduced but not as strongly as at 48hpf, and the number of Numb+ cells is increased (Fig. 6 and SIAppendix, Fig. S6). At 24 hpf, before the ventral bud markerptf1a is expressed, no difference between wild type and morphantembryos was seen (SI Appendix, Fig. S6).

Reduced Cell Proliferation in Lnx2a-Deficient Pancreas. Notch sig-naling affects the cell cycle in the early pancreas (13, 14). Weused Tg(ptf1a:EGFP) embryos to visualize ventral pancreaticcells and BrdU to mark proliferating cells. Injection of lnx2a-MOresulted in a substantial reduction in EGFP+ cells and an increasein Numb+ cells, in agreement with the phenotype described earlier(Fig. 7 A, 1 and 3 and B). Further, the number of BrdU+/EGFP+

cells decreased in concert with the reduction of total EGFP+ cells,consistent with the view that proliferation of Ptf1a-expresssingcells is reduced in the Lnx2-deficient pancreas (Fig. 7).Are fewer ventral bud precursors specified in mutants/

morphants, or does the initial population fail to proliferate nor-mally? Early ventral bud markers, ptf1a and mnr2a, are alreadyreduced in the morphants at the earliest time when they can bedetected, but the degree of reduction appears to increase as de-velopment proceeds (SI Appendix, Fig. S7). This fact suggests thatLnx2a regulates both the entry of cells into the ventral pancreasprecursor pool and the expansion of this population duringdevelopment.

Fig. 4. The lnx2aΔ329 mutant recapitulates the morphant phenotype. (A) Se-quence of the lnx2aΔ329 mutation (Fig. 3A). The lowercase sequence in themutant indicates that it is in intron 2, with lnx2aΔ329 having lost the exon 2splice donor site. (B and C) Genotyping of lnx2aΔ329 by genomic PCR (Δ329),RT-PCR (Δ510 equaling exon 2) (B), and byWestern blotting of embryo extractsshowing the presence of N-truncated protein (C). (D) Phenotypic analysis oflnx2aΔ329 mutants at 48 hpf. (Scale bar: 50 μm.) (E) The quantification ofpancreatic defects by analysis of ptf1a expression, classified as in Fig. 2D. De-velopment of the exocrine pancreas is specifically suppressed in lnx2aΔ329.

Fig. 5. Ventral pancreas defects in lnx2aΔ329 mutant and lnx2a-MO–injected embryos can be rescued by knockdown of Numb. (A) The translation blockingMO for Numb (5 ng) was injected into WT, lnx2aΔ329-, and lnx2a-MO–injected embryos, and pancreatic phenotype was examined at 60 hpf. (Scale bar: 50 μm.)(B) Quantification of pancreatic defects by analysis of ptf1a expression. Exocrine pancreas defects were substantially rescued by knockdown of Numb; numb-MO hadlittle effect in WT embryos. (C) Immunoblotting of embryo extracts with Numb antibody shows the efficiency of the numb-MO.

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DiscussionFunctional Redundancy Between Lnx2a and Lnx2b. The mammalianLNX family is composed of five members, but only LNX1 andLNX2 share an N-terminal RING-finger domain, a NPAY/F motiffor NUMB binding, and four PDZ domains (29, 30). The in vivofunctions of LNX1/2 remain incompletely known. MammalianLNX1 was shown to act as an E3 ubiquitin ligase that mediatesubiquitylation and degradation of Numb in cultured cells (16, 17).Lnx1 and Lnx2 are expressed in the nervous system and may have arole in neuronal cell proliferation and differentiation (31, 32).Further, interactions of Lnx1/2 have been analyzed with individualproteins or at a global proteomic level (33–35). However, to ourknowledge, no genetic test of Lnx1/2 function has been reported.In zebrafish, three LNX1/2 homologs (lnx1, lnx2a, and lnx2b)

have been identified (18), and Lnx2b affects dorso-ventral pat-terning by modulation of Dharma (Bozozok, Nieuwkoid) sta-bility (18, 19). In eutherian mammals, LNX-2b has been lost bypseudogenization, contributing several exons to the noncodingXist RNA (36, 37). We show here that zebrafish Lnx2a, likehuman LNX1, can mediate Numb ubiquitylation and degradation.In studying the zebrafish pancreas, we show that Lnx2a is expressedin the ventral bud at an early stage of embryogenesis. Further,Lnx2a and Lnx2b function redundantly in zebrafish pancreasdevelopment through the destabilization of Numb. It remains tobe tested whether the single Lnx2 gene in mammals carries outan analogous function.

Control of Protein Stability as a Regulatory Mechanism in PancreasFormation. Our results support the view that Lnx2a and Lnx2bmediate the specification of exocrine pancreatic cells by limiting

Numb stability. Numb has an inhibitory effect on Notch signaling(23–26, 38), and the Notch pathway has a major role in pancreasdevelopment. In the mouse, interfering with Notch activity in theearly pancreas led to accelerated differentiation of endocrine celltypes while inhibiting formation of exocrine cells (11), whereasoverexpression of the constitutively active Notch intercellulardomain led to inhibition of differentiation of all pancreatic celltypes (10, 28). These observations led to the view that Notchsignaling maintains precursor populations, whereas down-regu-lation of the pathway allows differentiation to proceed. In theformation of secondary islets in zebrafish, Notch activation inhibitsexocrine differentiation (39), and specification of particular celltypes depends on different Notch ligands (12). Secondary isletformation requires cells that are initially Notch active but un-dergo Notch down-regulation before differentiation (13, 14).Further, specific levels of Notch activity control quiescence orproliferation of precursor cells and their eventual entry into theendocrine differentiation pathway (13).In our context, the above conclusions may be summarized as

(i) Notch signaling is required in pancreatic cell differentiation;(ii) high Notch activity maintains precursor pools, lower activityallows differentiation, in certain cases preceded by proliferation;and (iii) cells that down-regulate Notch cannot maintain theirprecursor status. Our observations suggest that Lnx2a, togetherwith Lnx2b, destabilizes Numb in ventral bud-derived cells, allowingNotch activity, which may affect specification and expansion of

Fig. 6. lnx2a-MO injection causes a reduction of Notch-responsive cells inthe pancreas. (A) Tg(Tp1:H2BmCherry); Tg(Tp1:VenusPest) embryos wereinjected with lnx2a-MO (A2), numb-MO (A3), or both (A4). At 48 hpf, em-bryos were stained with anti-Numb antibody and analyzed by confocal mi-croscopy; Z projections are shown. (Scale bar: 10 μm.) (B) Total number ofNotch-OFF (Tp1:H2BmCherry+) and Notch-ON (Tp1:H2BmCherry+ and Tp1:VenusPest+) cells, as well as Numb-positive cells, were counted in all Z sec-tions of four embryos for each category. **P < 0.005; ***P < 0.002.

Fig. 7. lnx2a-MO injection results in impaired cell proliferation in the ven-tral pancreas. (A) Tg(ptf1a:EGFP) embryos were incubated in 10 mM BrdUfrom 46 to 47 hpf, fixed, stained with anti-BrdU and anti-Numb antibodies,and analyzed by confocal microscopy. For clarity, the same plane of the BrdUchannel is shown separately in addition to the merged images. (Scale bar:10 μm.) (B) The average number of ptf1:EGFP+, Numb+ and ptf1:EGFP+/BrdU+

double-positive cells in the pancreas of 10 uninjected and lnx2a-MO–injectedembryos are shown (***P < 0.001).

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precursor cells. Loss of Lnx2 activity in the lnx2aΔ329 mutants orlnx2a morphants stabilizes Numb to inhibit Notch in cells whereit is normally active, ultimately interfering with the normal de-velopmental progression of these cells. We suggest that in nor-mal development, the regulation of Numb protein stability byLnx2a/b is an important component of the system that controlsthe levels of Notch activity during pancreas formation and theassignment of cells to different pancreatic lineages.

Morphant Versus Mutant Phenotypes. Recent publications (21, 40)and much informal discussions have raised doubts about thevalidity of morphant phenotypes. Although this suspicion is un-doubtedly well founded in certain instances, we show here thatfailure of a null mutant to replicate a morphant phenotype mayhave complex and ultimately insightful reasons. It should benoted that Kok et al. (21) considered the possibility of functionalredundancy of paralogs and possible effects of exon skipping inthe interpretations. Our example emphasizes the need to con-sider these and possibly other effects in evaluating mutant versusmorphant phenotypes.

Materials and MethodsAnimals. Zebrafish AB* were maintained as described (41). Embryos weretreated with 0.003% Phenylthiourea (Sigma) at 22–24 hpf to preventpigmentation. Transgenic lines were as follows: Tg(ptf1a:EGFP) (42),

Tg(Tp1bglob:H2BmCherry)S939 abbreviated Tg(Tp1:H2BmCherry), andTg(Tp1bglob:venusPest)S940 abbreviated Tg(Tp1:venusPest) (13).

TALEN Construction. TALENs were designed by using the TAL Effector-Nucleotide Targeter (TALE-NT) 2.0 (43) and assembled with Golden gate kits(22). Modified destination vectors, pCS2TAL3 KKR/ELD, were constructed byreplacing RR and DD FokI domains in pCS2TAL3 RR/DD vectors (44) with KKRand ELD FokI domains by using site-directed mutagenesis of pMLM 290/292(KK/EL) vectors (45) (Addgene nos. 21872/21873).

MO Injection. MOs were purchased from Gene Tools: lnx2a splice MO, TTGA-GAACTTTACCTGTGTTTGAGA, 5 ng per embryo; lnx2b splice MO, TGCAG-CATGCACTAACCTGTTGTGC, as described (20); numb translation MO, AAC-TCTGCCGTAGCTTATTCATCGC, 5 ng per embryo. Random sequence MO fromGene Tools was used for control injections, and we also used uninjectedembryos for comparison.

For additional materials and methods, see SI Appendix, SI Materialsand Methods.

ACKNOWLEDGMENTS. We thank Greg Palardy and Damian Dalle Nogare foradvice on microscopy; John Gonzales and Allisan Aquilina-Beck for help withfish husbandry; T.-Y. Choi and Jin-Gu Lee for helpful discussion; and T.-Y. Choi,N. Ninov, D. Y. Stainier, C.V. Wright, and L. Godinho for reagents and zebrafishlines. This work has been supported by the Intramural Research Program of theNational Institute of Child Health and Human Development, National Institutesof Health, and by the Basic Science Research Program of the National ResearchFoundation of Korea, Ministry of Education (NRF-2015R1D1A4A01016532).

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