LATS1/2 kinases are required for pancreatic morphogenesis and endocrine cell fate

Because the role of LATS1/2 kinases during pancreas development remains poorly understood, we first characterized the kinetics of LATS1/2 protein expression in the early embryonic pancreas. Immunostaining was performed on wild-type (WT) pancreatic tissue using a previously validated antibody [20] against phospho-LATS1/2 (pLATS1/2). Intriguingly, pLATS1/2 localized to the apical domains adjacent to mucin-1⁺ (MUC1⁺) microlumens in the WT pancreatic bud at embryonic day 9.75 (E9.75; arrowhead, Fig 1A), suggesting pLATS1/2 activity during early pancreatic bud formation. To test whether LATS1/2 are required in the developing pancreas, we performed pancreas-specific deletion of Lats1/2 (Pdx1Cre^early [21];Lats1^f/f;Lats2^f/f, hereafter designated Lats1/2^PanKO). To confirm deletion of Lats1/2 in the Lats1/2 conditional mice [22], we examined pLATS1/2 immunostaining in WT and Lats1/2^PanKO pancreata. In WT pancreatic epithelium, pLATS1 and pLATS2 proteins were expressed at the apical surface of mature lumens at E15.5 (S1A Fig). In Lats1/2^PanKO tissue, pLATS1 and pLATS2 protein were nearly undetectable by E15.5 (S1A Fig), supporting successful Lats1/2 deletion. The Lats1/2 genetic ablation resulted in severe defects in pancreatic epithelial morphogenesis. Lats1/2^PanKO pups died postnatally with a small pancreatic rudiment that lacked all endocrine, ductal, and acinar cells (Fig 1B, 1C, 1D, 1E, 1F, 1G, 1H and 1I). This defect was so severe that we looked at earlier stages to determine the root cause.

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Fig 1. LATS1/2 kinases are required for pancreatic lineage differentiation.

(A) Representative confocal images of pLATS1/2, MUC1, and CDH1 immunostaining of sections of WT pancreas at E9.75 are shown. Arrowhead indicates pLATS1/2 expression adjacent to MUC1⁺ lumens. Scale = 50 μm (left panel), 10 um (right panel). (B) WT and (F) Lats1/2^PanKO pancreata (outlined in black) at P1. Black arrowhead indicates Lats1/2^PanKO pancreatic rudiment. (C–E, G–I) Confocal images of INS, GCG, and CDH1; AMY and MUC1; or DBA and MUC1 immunostaining of WT and Lats1/2^PanKO pancreata at P1. “*” denotes absence of endocrine, acinar, or ductal proteins. Scale = 25 μm. (J) Normalized mRNA expression of endocrine genes Pdx1, Neurog3, Gcg, and Syn; ductal gene Sox9; acinar genes Cpa1 and Nr5a2; Notch ligand Jag1; and YAP1/TAZ/TEAD target [26]. Cyr61 were compared in WT and Lats1/2^PanKO pancreata at E13.5 (n = 3 embryos per genotype). Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05; **p < 0.01; ***p < 0.001). Underlying numerical values can be found in S1 Data. (K, L) Confocal images of GCG and CDH1 immunostaining of WT and Lats1/2^PanKO pancreata at E15.5. Scale = 25 μm. (M–O) Confocal images of CDH1, GCG, and pYAP1 immunostaining of WT, Lats1/2^PanKO, and Pdx1Cre^early;Lats1/2^f/f;Yap1^f/f;Taz^f/wt (Lats1/2/Yap1^PanKOTaz^HET) pancreata at E12.5 (n = 3 embryos per genotype). Arrows indicate loss of pYAP1 expression in Lats1/2-deficient pancreata. Nuclei were counterstained with DAPI (blue). Scale = 25 μm. AMY, amylase; AU, arbitrary units; CDH1, E-cadherin; Cpa1, carboxypeptidase 1; Cyr61, cysteine rich angiogenic inducer 61; DAPI, 4’,6-diamidino-2-phenylindole; DBA, Dolichos biflorus agglutinin; E, embryonic day; GCG, glucagon; INS, insulin; Jag1, jagged1; LATS1/2, large tumor suppressor kinases 1 and 2; MUC1, mucin-1; Neurog3, neurogenin3; Nr5a2, nuclear receptor subfamily 5 group A member 2; P, postnatal day; Pdx1, pancreatic and duodenal homeobox 1; pLATS1/2, phospho-LATS1/2; pYAP1, phospho-yes-associated protein 1; Sox9, sex determining region Y-box 9 protein; Syn, synaptophysin; TAZ, transcriptional coactivator with PDZ-binding motif; TEAD, TEA domain transcription factor; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000382.g001

In WT pancreas, we observed a progressive expansion of epithelial volume accompanied by complex branching morphogenesis between E10.75 and E12.5 (S1B Fig), as previously established [4, 23, 24]. By contrast, differences in organ morphology were already present in Lats1/2-deficient pancreas at E11.5, at which time we observed an abrupt, transient expansion in pancreatic volume (S1B Fig). Strikingly, the mutant pancreas did not initiate branch formation. By E13.5, WT pancreas volume rapidly surpassed that of Lats1/2^PanKO (S1C Fig). Whereas the WT pancreas developed into a characteristic branched gland, the mutant pancreas remained small and rounded, similar to the lung upon loss of Lats1/2 [25]. Together, these data indicate an early, transient increase in epithelial volume and subsequent morphogenetic failure following loss of Lats1/2.

We next examined the onset of pancreatic endocrine cell fate. In WT pancreas, clusters of insulin⁺ (INS⁺) and glucagon⁺ (GCG⁺) (beta + alpha) endocrine cells, intercalated between epithelial branches, were evident at E11.5 and E12.5 (S1D Fig). By contrast, in addition to the failure of the Lats1/2-deficient epithelium to branch, early INS/GCG⁺ cells were dispersed within the abnormally thickened Lats1/2^PanKO epithelium at E11.5 (S1D Fig). By E12.5, INS/GCG⁺ expression was undetectable, and MUC1 expression was decreased (S1D Fig). INS/GCG⁺ endocrine surface volume was significantly lower in Lats1/2^PanKO epithelium than in WT across early bud development (S1E Fig). In fact, endocrine, ductal, and acinar mRNA expression were undetectable in the Lats1/2^PanKO pancreas (Fig 1J), indicating that Lats1/2 deletion leads to loss of pancreatic lineage identity.

At E15.5, the WT pancreas consisted of a branched, ramifying epithelial network intercalated with GCG⁺ alpha cells (Fig 1K). By contrast, we observed defective epithelial architecture without discernible branches nor alpha cells in Lats1/2^PanKO pancreas (Fig 1L). Together, these data point to concurrent failure of epithelial expansion, epithelial branching, and endocrine cell differentiation at the earliest stages of pancreatic differentiation.

LATS1/2 kinase suppression of YAP1/TAZ is required for pancreatic progenitor differentiation and subsequent morphogenesis

Because LATS1/2 kinases are known to phosphorylate the transcriptional coactivators YAP1 and TAZ, resulting in their exclusion from the nucleus, we next tested LATS1/2 activity by examining YAP1 and pYAP1 immunolocalization. In WT pancreas, both nuclear YAP1 and cytoplasmic pYAP1 were present (S1F and S1G Fig). By contrast, in the Lats1/2^PanKO pancreatic bud, increased nuclear YAP1 (arrowhead, S1H Fig) and total loss of cytoplasmic pYAP1 (asterisk, S1I Fig) were observed, indicating efficient deletion of Lats1/2^flox alleles by E10.5. One day later, we observed nuclear TAZ and YAP1 expression in a subset of WT epithelial cells at E11.5 to E12.0 (S1J and S1K Fig). Conversely, increased nuclear TAZ and YAP1 expression were detected in Lats1/2^PanKO pancreas at E11.5, further underscoring abrogation of LATS1/2 kinase function in the early Lats1/2-deficient pancreas (arrowheads, S1J and S1K Fig). Therefore, nuclear localization of YAP1 and TAZ, as well as loss of cytoplasmic YAP1/TAZ, verify inactivity of LATS1/2 in mutant mice by E10.5.

To test whether the inappropriate activation of YAP1 and/or TAZ played a causal role in the Lats1/2^PanKO phenotype, we deleted Yap1 (and one copy of Taz) in addition to Lats1/2 (Pdx1Cre^early;Lats1^f/f;Lats2^f/f;Yap1^f/f;Taz^f/wt or Lats1/2/Yap1^PanKOTaz^HET). We found that the Lats1/2/Yap1^PanKOTaz^HET largely rescued epithelial morphogenesis (Fig 1M, 1N and 1O), as evidenced by the presence of discernable branches (arrow, Fig 1O). Hence, we suggest that LATS1/2 regulate pancreatic differentiation via YAP1/TAZ.

In addition to phenotypic rescue of morphogenesis, we observed expression of pancreatic TFs hepatocyte nuclear factor 1-beta (HNF1B) and pancreatic and duodenal homeobox 1 (PDX1) (arrowheads, S2A and S2B Fig), as well as normal localization of apicobasal polarity proteins laminin subunit gamma 1 (LAMC1) and mucin 1 (MUC1) and adhesion molecules catenin beta 1 (CTNNB1) and E-cadherin (CDH1) (arrows, S2C and S2D Fig) in Lats1/2/Yap1^PanKOTaz^HET pancreas at E12.5. However, normal expression of sex-determining region Y-box 9 protein (SOX9) was not rescued in these pancreata (S2E Fig), which was expected because Sox9 is a known YAP1 target [27]. Normal levels of cell proliferation were also rescued in Lats1/2/Yap1^PanKOTaz^HET pancreas, as indicated by phospho-histone H3 (pHH3) immunopositivity (S2D Fig). Together, these data show at least partial rescue of Lats1/2-deficient defects.

Lats1/2 deletion leads to progressive loss of pancreatic progenitor gene expression

Given that pancreatic lineage-specific TF mRNA expression was lost by E13.5 in the Lats1/2^PanKO, we assessed earlier progenitor identity of the epithelial cells in the budding pancreas. We focused on the Lats1/2^PanKO bud from E10.75–E12.5, soon after Pdx1Cre-mediated deletion. Pancreas-specific TFs like PDX1, Prospero homeobox 1 (PROX1), SOX9, neurogenin 3 (NEUROG3), and NK6 homeobox 1 (NKX6-1) were expressed as expected in WT pancreata at E10.75 (S3A, S3B, and S3C Fig). In the Lats1/2^PanKO pancreatic bud, we found that PDX1 and PROX1 were also expressed (S3A Fig) with no significant difference in the relative percentage of TF⁺ epithelial cells between WT and mutant tissue at E10.75 (S3B Fig). However, the proportion of NEUROG3⁺ pro-endocrine cells was significantly lower in Lats1/2^PanKO pancreas (S3B Fig) as early as E10.75. In WT branching pancreatic epithelium, PDX1, SOX9, and NKX6-1 expression persisted at E11.5 through E13.5 (S3C and S3D Fig), with slight enrichment of NKX6-1 within the central bipotent (ductal and endocrine progenitor) compartment at E14.5 (arrowhead, S3E Fig). By contrast, PDX1, SOX9, and NKX6-1 were decreased in Lats1/2^PanKO pancreas by E13.5 and later (S3D and S3E Fig). Together, these data show that the pro-endocrine TF NEUROG3 is the first to be down-regulated in Lats1/2^PanKO pancreas, a timeline that corresponds with YAP1’s reported role of repressing Neurog3, as well as restricting progenitor differentiation and endocrine fate [28, 29].

LATS1/2 are required for pancreatic epithelial cell polarity and branching

Following Lats1/2 deletion, we observed a complete failure of epithelial branching. Previous studies have shown that proper evolution of pancreatic epithelial architecture is required for appropriate progenitor cell differentiation [24, 28, 30, 31]. Therefore, we sought to characterize the onset of these early architectural defects by examining the microlumens that normally form and coalesce into a ductal plexus [30]. We performed MUC1 immunostains and found a marked progressive loss of MUC1 protein prior to the secondary transition in Lats1/2^PanKO (S4A Fig). Indeed, Muc1 mRNA was abolished by E13.5 in Lats1/2^PanKO (S4B Fig). Because these observations suggested loss of cell polarity, we examined epithelial apicobasal polarity and cell ultrastructure. Using classical cell apical polarity markers including protein kinase C iota (PKCI) and the Golgi protein golgin A2 (GOLGA2), we found a distinct displacement of apical surfaces, which was accompanied by a progressive hyperfusion of microlumens, an increase in lumen size, and a loss of the expected ductal plexus (S4C Fig).

We next assessed possible defects in cell shape. Measuring and comparing cell apical and basal membranes, we observed a widely variable, increased apical:basal ratio in the Lats1/2^PanKO (S4D Fig). We calculated this ratio based on an analytical method previously published to examine defects in bile duct lumenogenesis [32], in which variable or high apical:basal ratio measurements indicated failure of apical constriction. To assess apical constriction, which is critical for microlumen formation [4, 31], we immunostained for phosphorylated myosin light chain 2 (pMYL2) and found its complete absence at the apical membrane of pancreatic epithelial cells (S4E Fig), suggesting the absence of expected rosette formation and loss of lumen size control. The apicobasal polarity abnormalities displayed in the Lats1/2^PanKO are summarized in the schematic in S4F Fig.

LATS1/2 kinases suppress EMT initiation

Throughout the Lats1/2^PanKO epithelia, we noted that the appearance and shape of mutant cells were altered and that integrity of the epithelium was ultimately lost. In contrast to typical cuboidal epithelial cells in the WT pancreas (Fig 1M), Lats1/2^PanKO cells showed spindle-shaped cells reminiscent of mesenchyme (Fig 1N). We therefore examined intermediate filament protein vimentin (VIM), which is normally enriched in mesenchymal cells [33]. WT pancreas epithelium completely lacks VIM at E11.5 (Fig 2A). By contrast, we observed ectopic high VIM expression in Lats1/2^PanKO pancreas (inset, Fig 2B), suggesting acquired mesenchymal characteristics soon after Lats1/2 deletion. We note that despite increased mesenchymal characteristics, the mutant epithelium retained relatively strong CDH1 expression, indicating that loss of Lats1/2 likely initiates an EMT but that this transition is not complete.

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Fig 2. Lats1/2 deletion initiates EMT transition of pancreatic progenitors.

(A–J) Confocal images of VIM, CDH1, TAGLN, KRT19, and ACTA2 immunostains in WT and Lats1/2^PanKO pancreata at (A–C, G) E11.5, (D, H) E12.5, or (E, F, I, J) E14.5 (n = 3 embryos per stage per genotype). “*” denotes absence of protein expression within pancreas (outlined in white). (G) Arrowhead indicates mosaic ACTA2 expression in pancreas progenitors at E11.5. Nuclei were counterstained with DAPI (blue). Scale = 25 μm. (K) Schematic illustrating partial EMT following Lats1/2 deletion, with acquisition of mesenchymal proteins including VIM, ACTA2, and TAGLN, normally absent from WT pancreas epithelium, and persistent CDH1. ACTA2, alpha smooth muscle actin; CDH1, E-cadherin; E, embryonic day; EMT, epithelial-mesenchymal transition; KRT19, cytokeratin 19; TAGLN, transgelin; VIM, vimentin; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000382.g002

Based on this finding, we assayed additional cytoskeletal proteins: TAGLN and alpha smooth muscle actin (ACTA2). As expected, TAGLN and ACTA2 were absent from WT pancreas epithelia (Fig 2C, 2D, 2E and 2F). However, in E11.5 Lats1/2^PanKO mutant epithelium, we found mosaic TAGLN expression (arrowhead, Fig 2G). Indeed, Lats1/2^PanKO mutant cells lost many epithelial characteristics and increasingly expressed aberrant TAGLN and ACTA2 at E12.5–E14.5 (Fig 2H, 2I and 2J). Given the increase in mesenchymal gene expression upon Lats1/2 deletion, we asked whether the EMT master regulator TF snail2 (SNAI2) was also inappropriately activated in Lats1/2^PanKO pancreata. As previously reported [34, 35], epithelial SNAI2 expression is not grossly detectable in WT pancreatic epithelium at E12.5 by immunofluorescence (IF) (S5A Fig). Surprisingly, we detected no significantly increased SNAI2 expression in Lats1/2^PanKO pancreatic epithelium (S5A Fig). We also examined additional canonical EMT TFs and found no significant difference in Twist2 or Snai1 mRNA expression in the Lats1/2^PanKO pancreas at E13.5, as determined by quantitative polymerase chain reaction (qPCR) analysis (S5B Fig). Twist1 expression, however, was increased in Lats1/2^PanKO compared to WT at E13.5, suggestive of partial EMT activation. The mesenchymal TFs zinc finger E-box binding homeobox 1 (ZEB1) and forkhead box F1 (FOXF1), as well as the mesenchymal cell surface tyrosine kinase receptor platelet derived growth factor receptor beta (PDGFRB), were similarly absent from both WT and Lats1/2^PanKO pancreatic epithelium (S5C and S5D Fig, shown at E14.5 and E12.5, respectively). We propose that full EMT does not occur upon Lats1/2 deletion, as persistent CDH1 expression remained robust in mutant pancreas from E11.5 through P1. These data suggest that CDH1⁺ Lats1/2^PanKO cells acquire a subset of mesenchymal features (Fig 2K), indicating a “partial” or “hybrid” EMT state.

Overactivated YAP1 triggers a partial transition to a mesenchymal state

To determine whether activated YAP1 alone phenocopied the EMT initiation observed in the Lats1/2^PanKO, we expressed a constitutively active Yap1^GFP allele (S112A) in pancreas progenitors. We used the tetO-Yap1-GFP transgene (hereafter designated Yap1^GFP), combined with a Ptf1a-rtTA driver (Pancreas specific transcription factor, 1a) [36]. Ptf1a expression initiates at E9.5 [37–40], and we activated Yap1^GFP expression from E9.5 through E13.5 by administering doxycycline (dox). To confirm that YAP1 was activated in the Yap1^GFP transgenic mouse embryo, we examined YAP1 protein colocalization with green fluorescent protein (GFP). As expected, WT pancreatic epithelium did not express GFP (S6A Fig), while robust colocalization of high YAP1 and GFP (arrowheads, S6A Fig) was observed in Yap1^GFP embryonic pancreas at E12.5, verifying constitutive YAP1 activation in this model. We found that, similar to the Lats1/2 ablation, Yap1^GFP led to abnormal morphogenesis (S6B Fig). The Yap1^GFP pancreas exhibited elongated morphology with few branches (S6B Fig) and a significantly lower epithelial volume (S6C Fig) by E13.5. This Yap1^GFP morphology differed from Lats1/2^PanKO, likely due to the mosaicism of the transgene, since only about 50% of epithelial cells in the Yap1^GFP pancreas express the transgene (S6D Fig). Like the Lats1/2^PanKO mutant, however, excess YAP1 expressed by the transgene led to abnormal TAGLN expression in pancreatic epithelial cells (arrowhead in S6E, and S6F Fig) at E13.5. These findings support a model in which excess YAP1 downstream of Lats1/2 inactivation promotes a mesenchymal program.

Loss of LATS1/2 kinases initiates an EMT transcriptional program and loss of apicobasal polarity in pancreatic epithelium

To investigate the molecular mechanisms disrupting pancreatic identity in the Lats1/2^PanKO pancreas, we carried out RNA sequencing (RNA-seq) of E11.0 mutant pancreata (Fig 3A), soon after Pdx1Cre^early-driven deletion [21]. RNA-seq data were analyzed by comparing amplified total RNA from individual Lats1/2^PanKO (n = 3) and WT pancreas buds (n = 5). The validity of the RNA-seq data was confirmed, as the Hippo signaling pathway was identified within the top 10 up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Fig 3B). The most highly up-regulated pathway identified was the maturity onset diabetes of the young (MODY) gene set. YAP1 and TAZ transcriptional coactivators frequently bind TEA domain TFs (TEAD) to drive gene expression [7, 41]. Differential expression of established TEAD target genes in RNA-seq data, summarized in S1 Table, was analyzed using RNA in situ hybridization (ISH) at E11.0 (S7A Fig).

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Fig 3. LATS1/2 kinases are required to suppress Vnn1 in early pancreatic progenitors.

(A) RNA isolation from pancreas buds at E11.0 (39–40 somites) for RNA-seq of total RNA from individual WT (n = 5) and Lats1/2^PanKO (n = 3) embryos. Pancreatic bud is outlined (black). The heat map was generated using genes that were differentially expressed by at least 2-fold in Lats1/2^PanKO (mutant) compared to WT (control). Gene down-regulation and up-regulation are indicated in green and red, respectively. (B) Selected pathways from KEGG pathway analysis are shown. The x-axis indicates (-log₁₀) fold change, and the vertical red line designates significance, with a threshold of 1.5-fold change. Underlying data analysis can be found in S1 Data. (C, D) RNA ISH of Vnn1 antisense probe on WT and Lats1/2^PanKO pancreata (outlined in black) at E11.5 (n = 3 embryos per genotype). “*” denotes undetectable Vnn1 mRNA in WT pancreas epithelium. Scale = 50 μm. (E) Normalized Vnn1 mRNA expression was compared in Lats1/2^PanKO and WT pancreata at E13.5 (n = 3 embryos per genotype). Data are shown as mean ± SEM. Statistical significance was determined by Student t test (**p < 0.01). Underlying numerical values can be found in S1 Data. (F, G) Confocal images of VNN1 and CDH1 immunostains of WT and Lats1/2^PanKO pancreata at E11.5 (n = 3 embryos per genotype). Scale = 10 μm. (H) TEAD motif analysis in the Vnn1 promoter. (I, J) Confocal images of GFP, VNN1, and CDH1 immunostains of WT and Ptf1a-rtTA;Yap1-GFP (Yap1^GFP) pancreata at E12.5 (n = 3 embryos per genotype). Arrowheads denote immunopositivity for both GFP and VNN1. Arrow points to cell with low GFP and no VNN1 expression. Scale = 20 μm. AU, arbitrary units; C, control; CDH1, E-cadherin; CTNNB1, catenin beta 1; E, embryonic day; GFP, Green Fluorescent Protein; ISH, in situ hybridization; KEGG, Kyoto Encyclopedia of Genes and Genomes; M, mutant; RNA-seq, RNA sequencing; TEAD, TEA domain transcription factor; TSS, transcription start site; Vnn1, vanin1; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000382.g003

KEGG pathway analysis of our RNA-seq data also indicated abnormal up-regulation of cell adhesion molecule and tight junction pathways in the Lats1/2^PanKO pancreas (Fig 3B), likely a compensatory response to the observed defects in EMT, apicobasal polarity, and cell shape. Indeed, Hallmark pathway analysis confirmed a significant activation of EMT gene expression (S7B Fig). Loss of cell polarity and changes in cell shape are necessary steps in EMT [42]. Previous studies from our lab and many others have shown that polarity, cell movement, differentiation, and proliferation must be precisely controlled to coordinate proper pancreas organ formation, supporting the idea that Lats1/2 deletion hijacks these developmental mechanisms, ultimately impeding morphogenesis.

LATS1/2 kinases are required to suppress Vnn1 via YAP1 in the developing pancreas

To address what mechanisms might be driving these cellular defects, we analyzed genes altered in the Lats1/2^PanKO. The most highly up-regulated gene in Lats1/2^PanKO pancreas was Vnn1 (approximately 32-fold), which encodes a pantetheinase, a glycolipid-bound ectoenzyme that converts pantetheine into pantothenic acid and cysteamine [43]. While Vnn1 mRNA was undetectable in WT pancreas bud at E11.5 (asterisk, Fig 3C) and only low protein expression was detected by IF (Fig 3F), strong Vnn1 mRNA expression was observed in Lats1/^PanKO pancreas at E11.5 and E13.5 (Fig 3D and 3E). We also observed significantly increased VNN1 protein levels in Lats1/2^PanKO pancreas at E11.5 and E13.5 (Fig 3G, S7C and S7D Fig). We asked whether YAP1/TAZ interaction with TEAD could drive Vnn1 expression. Indeed, motif analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data show a TEAD binding site within 500 bp surrounding the Vnn1 transcription start site (TSS), suggesting a potential YAP1/TAZ/TEAD TF complex driving Vnn1 expression (Fig 3H). These data strongly suggest that loss of Lats1/2 triggers aberrant VNN1 expression in the early pancreatic epithelium.

We asked whether Lats1/2 loss acted via YAP1 to induce VNN1 expression by testing the effect of constitutively active YAP1 expression on VNN1 activity. In E12.5 WT pancreas, VNN1 was detected at low levels at the apical membrane lining lumens (Fig 3I). Like Lats1/2 depletion, YAP1 activation led to robust VNN1 expression in GFP⁺ cells observed in Yap1^GFP pancreas at E12.5 (arrowheads, Fig 3J). VNN1 localized to the plasma membrane, while YAP1^GFP expression was nuclear. These findings suggest that aberrantly increased YAP1/TAZ activity leads to excess VNN1 expression in the cells that express YAP1 and that, normally, LATS1/2 maintain VNN1 at low levels via suppression of nuclear YAP1 in the developing pancreas.

LATS1/2 kinases suppress a pro-oxidant program in the developing pancreas

Because Vnn1 was the most highly up-regulated gene in the Lats1/2^PanKO and was absent from early WT progenitors, we reasoned that aberrant VNN1 might drive abnormalities downstream of Lats1/2 deletion. VNN1 and its pantetheinase product cysteamine are known to promote oxidative stress [44]. Consistent with this idea, further RNA-seq analysis revealed that multiple Gene Ontology (GO) processes regulating ROS metabolism and biosynthesis were significantly increased in Lats1/2^PanKO total RNA compared to WT (S8A Fig).

To interrogate cellular response to the absence of LATS1/2, we measured ROS and the oxidative stress response. We used a Cre inducible knockout system Pdx1Cre^ERT2;Lats1^f/f;Lats2^f/f (hereafter designated Lats1/2^i-PanKO) and carried out daily tamoxifen (tmx) induction of Lats1/2^i-PanKO from E8.5 to E12.5. WT and Lats1/2^i-PanKO pancreata were explanted at E12.5 and cultured in vitro to facilitate ROS visualization in live tissue (rather than fixed pancreas sections). After 24 hours, explants were analyzed using CellROX, which fluoresces when oxidized by ROS. Low levels of ROS were observed in WT pancreas explants (Fig 4A). By contrast, ROS were robustly increased in explanted Lats1/2^i-PanKO pancreata (Fig 4B). Overall, the percent area of CellROX⁺ pixels was approximately 22-fold higher in Lats1/2^i-PanKO pancreas explants than in WT (Fig 4C). Furthermore, oxidative stress response gene heme oxygenase 1 [45–47] was significantly up-regulated in Lats1/2^PanKO pancreas at E13.5 (Fig 4D), indicating an active response to increased ROS in Lats1/2-deficient pancreata.

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Fig 4. Lats1/2 deletion, or the VNN1 product cysteamine, induces ROS.

(A, B) Compressed Z stack confocal images of CellROX, a green fluorogenic probe that detects ROS, on WT and Pdx1Cre^ERT2;Lats1^f/f;Lats2^f/f (Lats1/2^i-PanKO) pancreas explants (n = 5 explants per genotype). Daily tmx induction was performed on WT and Lats1/2^i-PanKO embryos from E8.5 to E11.5. Lower panels depict overlay of CDH1 immunostaining (outlined in white). Scale = 100 μm. (C) The percent area covered by CellROX⁺ pixels within the pancreatic CDH1⁺ progenitor epithelium was quantified in WT and Lats1/2^i-PanKO explants. (D) Normalized mRNA expression of oxidative stress-responsive Hmox1 was compared in WT and Lats1/2^PanKO pancreas at E13.5 (n = 3 embryos per genotype). (E–H) Compressed Z stack confocal images of CellROX plus CDH1 immunostains of WT pancreas explants treated with 4 mM cysteamine and/or 10 μM NAC (n = 3–7 explants per treatment group). (F) Cysteamine provoked ROS in WT epithelium and mesenchyme (arrowheads). Scale = 150 μm. (I) The percent area covered by CellROX⁺ pixels (above a set threshold) within the pancreatic epithelium was quantified using ImageJ. (J–M) Compressed Z stack confocal images of CellROX plus CDH1 and TAGLN immunostaining on WT and Lats1/2^i-PanKO pancreas explants, treated O/N with antioxidant NAC or vehicle control (n = 3 explants per genotype per treatment). Daily tmx induction was performed on WT and Lats1/2^i-PanKO embryos from E8.5 to E11.5. Scale = 100 μm. (N) The percent area covered by TAGLN⁺ pixels within the pancreatic CDH1⁺ progenitor epithelium was quantified in WT and Lats1/2^i-PanKO explants. Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05; **p < 0.01; ***p < 0.001). Underlying numerical values can be found in S1 Data. AU, arbitrary units; CDH1, E-cadherin; E, embryonic day; Hmox1, heme oxygenase 1; NAC, N-acetyl-cysteine; O/N, overnight; ROS, reactive oxygen species; TAGLN, transgelin; tmx, tamoxifen; VNN1, vanin1; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000382.g004

As proof of principle that the Lats1/2^i-PanKO recapitulates that of the Lats1/2^PanKO pancreas, we performed mosaic deletion of Lats1/2^flox alleles in the Lats1/2^i-PanKO system. Deletion was induced using one-quarter of the normal tmx dosage at E8.5 only, and pancreata were examined at E13.5. While cytoplasmic and nuclear YAP1 expression was observed in WT littermates, robust nuclear YAP1 immunostaining was observed in Lats1/2 mutant clones (Lats1/2^{mosaic i-PanKO}) at E13.5 (S8B Fig). A TAGLN⁺ artery was observed in WT pancreas section (arrow), while the clone exhibited robust TAGLN immunopositivity, colocalizing with the high YAP1⁺ cells (arrowhead, S8B Fig). These data support the idea that the Lats1/2 ^i-PanKO system indeed recapitulates that of Lats1/2^PanKO.

To pinpoint the earliest time point at which oxidative stress was triggered following Lats1/2 deletion, pancreata were explanted at E10.5, and CellROX activation was quantified 2 days later (since pancreatic explants grow more slowly in ex vivo culture compared to in vivo development [48]). WT and Lats1/2^PanKO pancreas explants exhibited similarly low levels of CellROX fluorescence (S8C and S8D Fig), indicating that oxidative stress is triggered later than E11.5 (approximately, given cultured explant). Therefore, oxidative stress is temporally downstream of Vnn1 up-regulation, which occurs at E11.0.

To determine whether the ROS increase was primarily in Lats1/2^PanKO epithelial cells or the result of increased immune cell infiltration, as seen in disease contexts [49], we asked whether immune cells invaded the mutant tissue, which could in turn spur the oxidative stress response [50]. To test this idea, we assessed leukocyte infiltration in the Lats1/2^PanKO pancreas prior to morphogenetic failure. We found no infiltrating protein tyrosine phosphatase receptor type C (PTPRC)⁺ leukocytes within either WT or Lats1/2^PanKO pancreas epithelium at E11.5 (S8E and S8F Fig). We found only a few PTPRC⁺ leukocytes outside the Lats1/2^PanKO bud epithelial periphery (arrows, S8E Fig), suggesting initiation of a cell-autonomous epithelial cell response at E11.5. By E14.5, however, leukocyte infiltrate could be detected within the epithelium and was significantly increased in Lats1/2^PanKO pancreas relative to WT (S8G and S8H Fig). These results showed that the oxidative stress response preceded any immune infiltration, indicating that pancreatic defects in Lats1/2^PanKO are not secondarily caused by recruitment of inflammatory cells and that initial increased ROS levels are epithelial cell autonomous.

Studies have shown that VNN1 increases oxidative stress through its product cysteamine [51]. To mimic a cellular environment high in VNN1 [52], independent of LATS1/2 kinase activity, we treated WT pancreas epithelium with cysteamine and asked whether this affected redox homeostasis in WT pancreata. Such a finding would suggest that ROS response detected upon loss of LATS1/2 may largely be mediated by elevated VNN1 and its product cysteamine. Indeed, we detected robust increase in ROS following cysteamine treatment of WT pancreas epithelium (Fig 4E and 4F). We confirmed the specificity of the assay by treatment with the antioxidant N-acetyl-cysteine (NAC) as a control (Fig 4G and 4H). Quantification of the ROS levels showed an 81-fold increase upon cysteamine treatment (Fig 4I). Thus, this experimentally elevated VNN1-cysteamine signaling promotes a pro-oxidative state in WT pancreas, mimicking the effect of depleting LATS1/2.

To determine whether aberrant ROS signaling was causing partial EMT, we treated E12.5 explants with an antioxidant to inhibit ROS and measured TAGLN expression. TAGLN was negligibly expressed within WT pancreatic explants, with or without NAC (Fig 4J and 4K). As expected, TAGLN⁺ pixels were robustly increased in Lats1/2^i-PanKO explants (Fig 4L) compared to WT. NAC, however, did not down-regulate TAGLN expression in Lats1/2^i-PanKO explants (Fig 4M and 4N), indicating that 16 hour antioxidant treatment alone does not rescue the mesenchymal phenotype in Lats1/2-deficient pancreata. Overall, these data strongly suggest that LATS1/2 normally suppress VNN1-cysteamine signaling and the downstream pro-oxidative gene program in pancreatic progenitors during normal homeostasis and development but that either parallel pathways drive EMT (TAGLN expression) or short-term NAC treatment is insufficient to block these deleterious effects.

Lats1/2 deletion stimulates NFκB1 and RELA

VNN1 is known to activate NFκB family member RELA proto-oncogene, NF-KB subunit (RELA) [44], as is Leucine Rich Adaptor Protein 1 Like (Lurap1l) [53]. The latter was also significantly up-regulated (approximately 8-fold) in the Lats1/2^PanKO. Presence of both Vnn1 and Lurap1l (S9A and S9B Fig) within the top 8 most up-regulated genes in the Lats1/2^PanKO in our RNA-seq suggested that NFκB signaling might be one of the detrimental factors downstream of Lats1/2 depletion (S1 Table). Therefore, we asked whether Lats1/2 deletion in pancreas progenitors abnormally activated NFκB. In the E12.0 WT pancreas, we observed that a subset of cells expressed activated phospho-nuclear factor kappa B subunit 1 (pNFκB1; p105/p50) (arrowhead, Fig 5A), indicating normal but infrequent homeostatic pNFκB1 signaling in the developing bud. By contrast, pNFκB1 was observed in a significantly higher proportion of cells in Lats1/2^PanKO pancreas (Fig 5B and 5C), with almost double the number of pNFκB1⁺ cells per section. This implied aberrantly elevated pNFκB1 activity following Lats1/2 deletion.

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Fig 5. Lats1/2 deletion or ROS stimulates NFκB1 and RELA.

(A, B) Confocal images of pNFκB1 and CDH1 immunostains of WT and Lats1/2^PanKO pancreata at E12.0 (n = 3 embryos per genotype). Scale = 20 μm. (C) The average percentage of pNFκB1⁺ epithelial cells/total epithelial cells per pancreas section at E12.0 was calculated. (D, E) Confocal images of pNFκB1 and pHH3 immunostaining of WT and Lats1/2^PanKO pancreata at E14.5 (n = 3 embryos per genotype). Scale = 20 μm. (F) Western blotting from E12.5 WT and Lats1/2^PanKO pancreata using pRELA and GAPDH antibodies (n = 3 embryos per genotype). (G, H) Confocal images of CDH1 and pRELA immunostained WT pancreas explants treated with 200 μM H₂O₂ for 2 hours (n = 4 explants per treatment group). Arrowheads in (H) indicate pRELA⁺ nuclei. (I) The average number of pRELA⁺ nuclei per field of view were quantified. Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05). Underlying numerical values can be found in S1 Data. (J) Lats1/2 deficiency increases expression of the pantetheinase VNN1, which produces cysteamine, followed by stimulation of ROS and subsequent NFκB activation. CDH1, E-cadherin; E, embryonic day; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; pHH3, phospho-histone H3; pNFκB1, phospho-nuclear factor kappa B subunit 1; pRELA, phospho-RELA proto-oncogene, NF-KB subunit; ROS, reactive oxygen species; VNN1, vanin1; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000382.g005

Noting that both WT and mutant pNFκB1⁺ cells were enlarged and rounded (arrowheads, Fig 5A and 5B), which are hallmarks of epithelial mitotic cells [54], we asked whether pNFκB1⁺ cells were proliferating. Indeed, approximately 49% of pNFκB1⁺ progenitor cells were pHH3⁺ in both WT and Lats1/2^PanKO pancreata at E14.5 (arrowheads, Fig 5D and 5E). This confirmed that NFκB1 was activated in proliferating pancreatic cells during development, and this relationship was maintained in the Lats1/2^PanKO. Yet the overall proportions of pHH3⁺ epithelial cells were higher in Lats1/2^PanKO pancreata compared to WT at E10.75, pointing to an abnormally increased rate of cell proliferation in the early Lats1/2-deficient pancreas (S9C and S9D Fig). This proliferative abnormality correlated with the increased size of the mutant bud at E11.5 (S1B and S1C Fig). However, the effect was transient, as WT and Lats1/2^PanKO rates of cell proliferation equalized by E11.5 (S9C and S9D Fig). Together these data implicate pNFκB1 activity in cell proliferation in WT and Lats1/2-deficient pancreata.

Because pNFκB1 activity persisted, though aberrantly increased cell proliferation did not, we next addressed why the Lats1/2^PanKO pancreas failed to expand in size as development progressed. We examined cell death by cleaved caspase3 (CASP3) immunostaining, because NFκB signaling can promote proliferation or cell death depending on the cellular context [55]. Few CASP3⁺ epithelial cells were observed in WT pancreas at E11.5–E14.5, and there was no significant difference in the proportion of CASP3⁺ epithelial cells in Lats1/2^PanKO pancreas at E11.5 (S9E Fig), a time point when the Lats1/2^PanKO pancreatic bud was enlarged relative to WT. Beginning at E12.5, however, the rate of cell death increased significantly in Lats1/2-deficient pancreas compared to WT, and this rate continued to increase from E13.5 to E14.5 (S9E and S9F Fig). In addition, because the mutant bud at these stages failed to branch, and pancreatic vasculature is normally closely entwined with WT branching epithelium [56] (arrows, S10A Fig), we predicted that vascularization would fail in Lats1/2^PanKO. Indeed, endothelial cells positive for platelet and endothelial cell adhesion molecule 1 (PECAM1) and endomucin (PE⁺) failed to associate at all with Lats1/2^PanKO CDH1⁺ cells at E13.5 (arrowheads, S10A Fig). Because there was a dearth of blood vessel intercalation, we tested whether mutant cells at the center of the pancreas were experiencing hypoxia. Indeed, by E15.5 we observed increased hypoxia inducible factor 1 subunit alpha (HIF1A) immunopositivity in the Lats1/2^PanKO relative to WT (S10B Fig), confirming increased hypoxia. Together, these cell death, absent vascularization, and hypoxic phenotypes occurring at E12.5 and later likely explain the failed bud expansion following Lats1/2 deficiency.

Total phospho-RELA (pRELA) protein was also significantly increased in Lats1/2^PanKO pancreata relative to WT (Fig 5F), further demonstrating NFκB up-regulation following Lats1/2 deletion. In addition, the NFκB direct downstream target genes Nfkbia and Mmp9 [57, 58] were significantly increased in Lats1/2^PanKO pancreata (S10C Fig). To further test our model, we asked whether oxidative stress, such as that induced by VNN1, causes increased NFκB activity [44]. Our findings predict that exogenous stimulation of oxidative stress should induce NFκB activity in pancreas cells. Indeed, H₂O₂ provoked RELA phosphorylation and nuclear translocation in WT pancreas explants, thereby mimicking the loss of Lats1/2 (arrowheads, Fig 5G, 5H, and 5I). These findings show that an oxidative state activates NFκB, even in WT pancreas. Overall, these data reinforce the model that LATS1/2 kinases are central factors in suppressing VNN1 activity, which in turn increases both oxidative stress and NFκB activity (Fig 5J). Together, these data validate the hypothesis that LATS1/2 kinases normally restrict cell-intrinsic and deleterious excessive NFκB signaling in the developing pancreas.

NFκB is required to initiate pancreatic EMT downstream of Lats1/2 deletion

Our findings suggest that NFκB hyperactivity is a major mechanism driving epithelial defects in Lats1/2^PanKO pancreas. Therefore, we next asked whether NFκB activity is required or sufficient to induce the phenotypic abnormalities of Lats1/2^PanKO. To test for this requirement, we inhibited NFκB in Lats1/2^PanKO and examined the effects on EMT initiation. Indeed, treatment of the Lats1/2^PanKO epithelium with the NFκB inhibitor anatabine suppressed ectopic VIM expression, suggesting that NFκB is required for EMT initiation downstream of Lats1/2 deletion (Fig 6A, 6B, 6C, 6D and 6E). (N.B. anatabine treatment also lessened endogenous mesenchymal VIM expression; asterisks, Fig 6C and 6D). We measured the expression of transcriptional targets Nfkbia and Mmp9 following anatabine treatment of WT pancreatic explants and found significant down-regulation of both NFκB target genes Nfkbia and Mmp9 (S10D Fig), thereby confirming the efficacy of NFκB inhibition ex vivo. These data show that Lats1/2 depletion leads to EMT initiation (as measured by ectopic VIM expression) via activated NFκB.

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Fig 6. NFκB is necessary and sufficient to initiate pancreatic EMT.

(A–D) Compressed Z stack images of VIM⁺ immunostains of vehicle- or anatabine-treated WT and Lats1/2^PanKO pancreas explants (n = 3–6 explants per genotype per treatment group). Scale = 50 μm. (B) Arrowhead indicates ectopic VIM expression in Lats1/2^PanKO epithelium. (C, D) Anatabine also decreased mesenchymal VIM expression (asterisks). (E) The percent area covered by VIM⁺ pixels within the pancreatic epithelium (above a set threshold) was quantified in anatabine- and vehicle-treated Lats1/2^PanKO and WT explants. Data are shown as mean ± SEM. Statistical significance was determined by Student t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (F, G) Compressed Z stack confocal images of CDH1 and VIM immunostains of WT pancreas explants treated with 10 μg/mL PMA (n = 3 explants per treatment). Masked VIM (VIM within CDH1⁺ progenitor surface volume) was used to exclude mesenchymal VIM. Scale = 50 μm. (F’,G’) Colocalization (white) was calculated above common thresholds and visualized using Imaris. (H) The percent area of VIM+CDH1 colocalization pixels was quantified using ImageJ. (I, J) Mean VIM⁺ pixel intensity and the percent area of VIM⁺ pixels (above a common threshold) within WT pancreatic epithelium were quantified using ImageJ. Underlying numerical values can be found in S1 Data. (K, L) Slice view through Z stack shows VIM (green) + CDH1 (red). (L) Arrowheads indicate VIM within the CDH1⁺ epithelial layer. (M) PMA hyperactivates NFκB, which initiates EMT in pancreatic progenitors. CDH1, E-cadherin; EMT, epithelial-mesenchymal transition; NFκB, nuclear factor kappa light chain enhancer of activated B cells; PMA, Phorbol 12-myristate 13-acetate; VIM, vimentin; WT, wild type.

https://doi.org/10.1371/journal.pbio.3000382.g006

We asked whether NFκB inhibition is sufficient to rescue the Lats1/2 mutant phenotype. We measured endocrine cell differentiation in pancreatic tissue, which was explanted at E11.5 and treated with anatabine for 2 days. As expected, INS+/GCG+ endocrine cells were decreased in vehicle-treated Lats1/2^PanKO explants compared to WT (S10E Fig). Surprisingly, NFκB inhibition did not affect endocrine differentiation in Lats1/2-deficient endocrine cells (S10E and S10F Fig), indicating that blocking NFκB signaling does not rescue the differentiation phenotype in Lats1/2^PanKO pancreas.

Lastly, to test for sufficiency of NFκB activity in initiating EMT, we treated WT E10.5 pancreas explants with the NFκB activator phorbol 12-myristate 13-acetate (PMA) [59, 60]. In WT controls, VIM was never expressed in epithelial cells but was readily observed in the mesenchyme surrounding the epithelium in vehicle-treated explants (Fig 6F and 6F’). By contrast, exogenous NFκB activation by PMA treatment triggered robust VIM expression in the WT CDH1⁺ pancreas epithelium (Fig 6G), with a striking 39-fold increase in colocalization area (white, Fig 6G’ and 6H). Overall, VIM expression within the progenitor epithelium was significantly higher following exogenous NFκB activation (Fig 6I and 6J), visualized by slice view through vehicle- (Fig 6K) and PMA-treated explants (arrowheads, Fig 6L). NFκB activation also increased VIM in mesenchymal cells surrounding epithelial branches, where VIM was normally expressed albeit at lower levels (S10G Fig). Exogenous NFκB activation similarly triggered expression of another mesenchymal protein, TAGLN, normally absent from WT pancreas epithelium (S10H Fig). Together, these data confirm that hyperactivated NFκB is sufficient to initiate abnormal EMT in pancreatic progenitors (Fig 6M).

Collectively, these findings show that LATS1/2 provide an endogenous protective mechanism to suppress NFκB signaling and to maintain epithelial integrity by suppressing EMT pathways. Triggering NFκB or removing protective LATS1/2 suppression of NFκB activity leads to deleterious progression toward EMT and hinders developmental epithelial morphogenesis. This work underscores the role of the Hippo pathway acting as a rheostat to control cell-intrinsic NFκB signaling in pancreatic epithelium.

Ethics statement

All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act, as well as protocols approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee (IACUC; approval number 2017–102243, approval date 26 September 2017). All animals were observed daily, and appropriate care was provided by the veterinary staff of the UTSW Animal Resource Center, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Care, International (Unit Number 000673) and by the NIH Office of Laboratory Animal Welfare (Assurance Number D16-00296). Dams were euthanized via IACUC-approved humane methods, using carbon dioxide asphyxiation and secondary cervical dislocation.

Mice

E10.0–E18.5 embryos and postnatal mice (Mus musculus) were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA)/PBS at 4°C for 3 hours for section and whole-mount IF or overnight (O/N) for RNA ISH. Tissue was then washed, dehydrated to 70% ethanol (EtOH), and stored at −20°C.

CD1 mice (Charles River Laboratories, Houston, TX) were used for WT experimental analyses, including drug treatment experiments. Otherwise, Lats1^flox;Lats2^flox [22], Yap1^flox;Taz^flox [92], Pdx1Cre^early, Pdx1Cre^ERT2 [21], Yap1^GFP [2], Ptf1a-rtTA[38], R26^TdTOM [93], and R26^YFP [94] were used for experiments described herein. Pancreata from mutant embryos were compared to WT or heterozygous tissue from littermate embryos. Genotypes were determined by PCR after O/N digestion using DirectPCR (Tail) Lysis buffer (Viagen, Los Angeles, CA) per manufacturer’s instructions. tmx (Sigma Aldrich, St. Louis, MO) induction was performed as previously described [30]. Briefly, inducible simultaneous deletions were obtained using Lats1^f/f;Lats2^f/f females with Lats1^f/f;Lats2^f/f;Pdx1^CreERT2 (Lats1/2^i-PanKO) males; daily tmx induction was performed by oral gavage from E8.5 to E13.5. To obtain mosaic Lats1/2-deficient clones, pregnant mothers were gavaged with 0.75 mg/40 g tmx at E8.5 and examined at E13.5. Oxidative stress was analyzed in pancreas explants using Pdx1Cre^ERT2;Lats1^f/f;Lats2^f/f embryos. To induce transgene expression in Ptf1a^rtTA;tetO-Yap1-GFP (Yap1^GFP) [2] embryos, dox was provided from E8.5 until dissection. Ptf1a expression begins at E9.5 [37]; however, given the possible delay between dox induction and transgene activation, we initiated dox treatment at E8.5.

Section IF

For paraffin sectioning, tissues were embedded as described previously with some modifications [30]. Briefly, tissues were dehydrated to 100% EtOH (2 × 30 minutes), washed in xylene (2 × 10 minutes), and then incubated in 1:1 xylene:paraffin for 30 minutes at 65°C. After incubating for at least 4 hours at 65°C with paraffin replacement approximately every hour, tissues were embedded in paraffin and sectioned at 10 μm.

Paraffin sections were baked at 60°C for 10 minutes and de-paraffinized in xylene and rehydrated through EtOH to PBS. All washes were performed using PBS. After washing, tissue sections were permeabilized using 0.3% Triton-X/PBS for 10 minutes. Antigen retrieval was performed under pressure using acidic buffer A (Electron Microscopy Sciences, Hatfield, PA). For standard immunostaining, sections were washed, blocked in CAS block (Invitrogen, Carlsbad, CA), and incubated in primary antibodies (diluted in CAS Block, S2 Table) using hybridization chambers O/N at 4°C. The next day, sections were washed and incubated in Alexa Fluor series antibodies (Invitrogen, Carlsbad, CA; 1:200) for 2 hours at room temperature (RT). Sections were then washed, incubated in 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear stain (Sigma Aldrich, St. Louis, MO) (1:10,000), rinsed, and mounted in Prolong Gold Anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA). Images were obtained using Nikon A1R confocal or LSM710 Meta Zeiss confocal microscopes. Unless stated otherwise, representative images from 3 experiments are shown.

Tyramide signal amplification immunostaining was performed per manufacturer’s protocol (Life Technologies, Carlsbad, CA). Briefly, after antigen retrieval, sections were washed, blocked using tyramide blocking reagent for at least 1 hour at RT, and incubated in primary antibodies (diluted in tyramide block, S2 Table) O/N at 4°C. Sections were then washed and incubated in donkey anti-rabbit horseradish peroxidase (HRP; 1:200) or Donkey anti-Mouse HRP (1:200) (Santa Cruz, Dallas, TX) diluted in tyramide block for 1 hour at RT. Sections were again washed, labeled using tyramide 488 (diluted 1:100 in tyramide amplification buffer) for 10 minutes at RT, washed, and then incubated in remaining secondary (Alexa Fluor) series antibodies (diluted 1:200 in tyramide block) for 2 hours at RT. Nuclear staining, mounting, and imaging were performed as described above.

Whole-mount IF

Whole-mount immunostaining was performed as described previously [31]. Briefly, after fixation, tissues were washed, dehydrated to 100% methanol (MeOH), and stored at −20°C. Prior to immunostaining, tissues were bleached in 4:1:1 MeOH:DMSO:H₂O₂ solution for 2 hours at RT, placed in fresh MeOH for 1 hour, and then rehydrated through MeOH series to PBS. Block solution was prepared using 0.5% blocking reagent powder (Perkin Elmer, Waltham, MA) in 1M Tris (pH 7.5) and heated at 65°C to dissolve. Tissues were blocked for 2 hours at RT and then incubated in primary antibodies (S2 Table) O/N at 4°C. The next day, tissues were washed for 5–8 hours, refreshing PBS approximately every hour, and then incubated in secondary (Alexa Fluor) antibodies (diluted 1:200 in Blocking Reagent) O/N at 4°C. Tissues were then washed in PBS 3 times for 20 minutes, dehydrated to 100% MeOH, equilibrated in a 1:2 benzyl alcohol:benzyl benzoate (BABB) solution, and mounted in BABB using 1.6 mm concavity slides (Electron Microscopy Sciences, Hatfield, PA). Images of whole-mount immunostaining were obtained using a LSM710 Meta Zeiss confocal microscope.

Total RNA-seq

RNA was isolated from pancreas buds at E11.0 (39–40 somites). Embryos were dissected on ice in cold PBS. Mesenchyme was manually removed using forceps, and pancreas epithelia were homogenized using a 20-gauge needle in RLT buffer (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol (Fisher Scientific, Hampton, NH). The amount of 35 ng total RNA was isolated from each pancreas bud using RNeasy Microkit (Qiagen, Hilden, Germany) following manufacturer instructions. RNA sample quantity and quality were measured using Bioanalyzer with RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA), and samples with RIN < 8.8 were excluded.

cDNA libraries were constructed by using KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KR1151-v3.15, Kapa Biosystems, Pleasanton, CA). Amplified cDNA was validated and quantified on an Agilent Bioanalyzer with the High Sensitivity DNA chip. The purified libraries were normalized, pooled together, denatured, and diluted at final concentration of 1.8 pM. Furthermore, 1.3 mL of diluted pool was used to perform cluster generation, followed by 2 × 75 bp sequencing on NextSeq500 (Illumina, San Diego, CA). From each sample, we obtained about 31 million reads.

Differential gene expression from RNA-seq

For gene expression analysis, reads for E11.0 pancreata were aligned to the reference genome (UCSC GRCm38/mm10 genome assembly) using TopHat version 2.1.0 and Bowtie version 2.6 (https://ccb.jhu.edu/software/tophat/index.shtml) with the following parameters (—no-coverage-search—library-type fr-firststrand–G). GFF files were obtained from Genome Reference Consortium Mouse Build 38 [95, 96]. The distribution of alignments was analyzed using the Cufflinks version 2.2.1 (http://cole-trapnell-lab.github.io/cufflinks/), and FPKM values were quantile normalized. To identify differentially expressed genes, transcript abundance from Lats1/2^PanKO pancreata (n = 3) was compared to that from WT pancreata (n = 5). Differential expression testing was performed using the Cuffdiff version 2.2.1 application. All up- and down-regulated genes were subjected to GO and KEGG analysis using the Functional Enrichment Analysis unit of HOMER version 3 (http://homer.ucsd.edu/homer/).

Gene set enrichment analysis

Gene set enrichment analyses using multiple algorithms were performed using the EGSEA [97] package. Specifically, the EGSEA analysis included the camera, safe, gage, padog, plage, zscore, gsva, ssgsea, globaltest, ora, and fry algorithms. Gene sets from the h, c2, and c5 collections of the Molecular Signatures Database [98] (MSigDB) and the KEGG [99] pathways included in the EGSEAdata package were indexed. p-Values were combined using Wilkinson’s method followed by application of the Benjamini-Hochberg [100] algorithm to control the false discovery rate (FDR). Results were ordered hierarchically by vote rank, with a bin width of 5, followed by FDR.

Motif analysis

A motif library was generated by concatenating the Tead1, Tead2, Tead3, and Tead4 motif files from the HOMER [101] motif database (http://homer.ucsd.edu/homer/). The genomic region extending from 400 bp upstream to 100 bp downstream of the Vnn1 TSS was scanned using findMotifs.pl from the HOMER tool suite. Sequence logos were generated from position weight matrices using the ggseqlogo [102] Bioconductor R package (http://bioconductor.org). Annotation information, including motif locations, was plotted along genomic coordinates with the Gviz [103] package.

Real-time qPCR

Real-time qPCR was performed as described previously [104]. Briefly, 240 ng of total RNA was isolated from individual E13.5 Lats1/2^PanKO pancreata using RNeasy Microkit (Qiagen, Hilden, Germany), and cDNA was synthesized using Super-Script III (Invitrogen, Carlsbad, CA). The amount of 1 μL of cDNA in Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) was used for qPCR analysis of gene expression (CFX96, BioRad, Hercules, CA). Primers for Cpa1, Nr5a2, Neurog3, Mmp9, Pdx1, Gcg, Sox9, Cyclophlin, and GAPDH have been described previously [1, 31, 105–109]. Primers for Hmox1, Nox4, Synaptophysin, Cyr61, Muc1, Nfkbia, Vnn1, and Lurap1l were designed to span exon-exon junctions (S3 Table) and were tested using E15.5 WT mouse pancreas cDNA. Gene expression levels were determined by PCR reactions (30 seconds at 95°C, 30 seconds at 62°C, and 30 seconds at 72°C for 35 cycles), and fluorescence was measured at 72°C. Gene expression levels were normalized to Cyclophilin or Gapdh, and the ΔΔC_t method was used to calculate fold change. Data were collected from individual embryos (n = 3 embryos per genotype), and samples were analyzed in triplicate. Data are presented as mean ± SEM.

Digoxigenin-labeled RNA probes and ISH

Digoxigenin-labeled probes and RNA ISH were performed as described previously [110]. Briefly, plasmids (GE Dharmacon, Lafayette, CO) were linearized using restriction enzymes listed in S4 Table. Probes were synthesized as described previously [111]. Briefly, antisense DigoxigeninUTP-labeled RNA probes were synthesized at 37°C for approximately 2 hours using RNA DIG labeling mix per the manufacturer’s instructions (Roche, Basel, Switzerland) using RNA polymerase (S4 Table). After incubation with RQ1 DNase I (Promega, Madison, WI), RNA was purified using Micro Bio-Spin columns (BioRad, Hercules, CA). Probes were resuspended at 10X concentration (10 μg/mL) in pre-hybridization stock solution [111].

ISH was performed as previously described [110]. Briefly, fixed E11.0 embryos were dehydrated, embedded in paraffin, and sectioned as described above. Paraffin sections (10 μm thick) were de-paraffinized in xylene, rehydrated, treated with 15 μg/mL proteinase K (Sigma Aldrich, St. Louis, MO) for 15 minutes at RT, washed in PBS, post-fixed in 4% PFA/PBS for 15 minutes at RT, washed, and incubated in pre-hybridization solution for 30 to 45 minutes at RT. Probe hybridization (1 μg/mL) was performed O/N at 65°C. The next day, slides were washed in 0.2X SSC and then MBST, followed by blocking in 2% blocking reagent (Roche, Basel, Switzerland) for 2 hours at RT. Sections were incubated in Anti-Digoxigenin (1:4,000, Roche, Basel, Switzerland) antibody O/N at 4°C. The next day, slides were washed in MBST followed by NTMT. Substrate color reaction using BM purple (Roche, Basel, Switzerland) was performed at 37°C for 4–24 hours. Slides were then post-fixed in 4% PFA/PBS for approximately 1 hour, washed, dehydrated, and mounted using Permount (Fisher Scientific, Hampton, NH). Images were obtained using Zeiss Axiovert 200 M inverted microscope and DP-70 camera (Olympus, Shinjuku, Japan).

Pancreas explant cultures

Pancreata were explanted as described previously [30]. Briefly, WT and Lats1/2^PanKO pancreata were explanted at E10.5–E11.5 onto fibronectin-coated plastic dishes in complete DMEM (American Type Culture Collection [ATCC], Manassas, VA), containing 1% penicillin/streptomycin, 10% fetal bovine serum (ATCC, Manassas, VA), and 10 μg/ml gentamicin (ThermoFisher Scientific, Waltham, MA). The next day, explants were treated in complete medium with anatabine [112] (Cayman Chemicals, Ann Arbor, MI), cysteamine [52], PMA [59, 60], NAC [113] (Sigma Aldrich, St. Louis, MO), or H₂O₂ [114] (Fisher Scientific, Hampton, NH). Experimental cysteamine treatment at 4 mM was utilized based on a previous dose response study, which showed that 7 mM cysteamine induced cell death and that 2 mM cysteamine failed to induce ROS [52]. See S5 Table for chemical information. EtOH (vehicle for anatabine), dimethyl sulfoxide (DMSO, vehicle for SN50 [115] and PMA), and PBS (vehicle for cysteamine) were used as controls. Media were replenished every 1 to 2 days. Explants were fixed after 2 to 4 days. Explant immunostaining was carried out as previously described [30]. Briefly, explants were fixed in 4% PFA/PBS at RT for 15 minutes, washed in PBS, permeabilized in 0.3% Triton-X/PBS for 1 hour, washed in PBS, and blocked for 1 hour in CAS Block (Invitrogen, Carlsbad, CA). Primary antibodies (S2 Table) were incubated O/N at 4°C. For the second day, explants were washed in PBS and then incubated in secondary antibodies O/N at 4°C. Explants were washed the next day and mounted flat on a glass slide with Prolong Gold Anti-Fade (Invitrogen, Carlsbad, CA). Images were obtained using Nikon A1R confocal or LSM710 Meta Zeiss confocal microscopes. Representative compressed Z stack images from 3 independent experiments are shown.

CellROX

CellROX Green Reagent (ThermoFisher Scientific, Waltham, MA) oxidative stress assay was performed as described [113], according to the manufacturer’s instructions. Briefly, CellROX Green Reagent was added to explant media at a final concentration of 5 μM and incubated at 37°C for 30 minutes. (This and all subsequent steps were performed in the dark.) Pancreas explants were then washed, fixed in 4% PFA/PBS at RT for 15 minutes, washed, permeabilized in 0.3% Triton-X/PBS for 1 hour at RT, washed, blocked in CAS block (Invitrogen, Carlsbad, CA) for 1 hour at RT, incubated in primary antibodies for 2 hours at RT, washed, incubated in secondary antibodies O/N at 4°C, washed, and mounted as described above. Explants were imaged using Nikon A1R confocal microscope within 24 hours of CellROX incorporation assay.

Western blot on E12.5 pancreata

Lats1^f/f;Lats2^f/f were mated into Lats1^f/+;Lats2^f/f;Pdx1^Cre or Lats1^f/f;Lats^f/f;Pdx1^CreERT2;R26^TdTOM (tamoxifen-induced daily at E8.5–E11.5) to obtain E12.5 Lats1/2^iPanKO and control embryos. When using R26^TdTOM male, embryo genotypes were determined based on reporter expression. Otherwise, when using Lats1/2^PanKO tissue, quick genotyping was carried out using the AccuStart II mouse genotyping kit (Quantabio, Beverly, MA).

The amount of 30 μL of lysis buffer was prepared per sample (lysis buffer: 10 mL PBS + 10 μg/ml aprotinin [final] + 10 μg/ml leupeptin [final] + 10 μg/ml pepstatin [final] + 1 phosSTOP tablet from Roche, Basel, Switzerland). All dissection steps were performed in PBS in a Sylgard dish placed in a larger Petri dish with ice, under the dissecting microscope. Stomachs along with the pancreas were dissected and transferred to a clean dish on ice. Once all stomachs were dissected, when available, reporter expression was used to determine the genotype (Lats1/2^PanKO versus all others as control). Otherwise, stomachs were kept on ice until genotyping was complete. When fewer than 4 stomachs were obtained per genotype, pancreata were dissected and snap-frozen for later use.

Pancreata were dissected away from the stomach, and remaining stomach or spleen pieces were removed (note that the pancreatic mesenchyme was not removed). Pancreata were transferred to a clean dish on ice using a glass pipette. Once all pancreata were dissected, same-genotype pancreata were transferred into a small Sylgard dish along with a drop of PBS using a glass pipette; 20 μL of lysis buffer was placed next to the PBS, and forceps were used to quickly transfer the pancreata from PBS into the lysis buffer. Each pancreas was excised into 4–5 pieces as fast as possible, and the pieces were transferred using a pipette (2–20 μL) into a microcentrifuge tube with 10 μL lysis buffer on ice (total volume = 30 μL). Once all genotypes were complete, homogenization was carried out by pipetting up and down the contents of each tube, until no chunks remained. Then, 3 μL of 10% Triton-X (prediluted in PBS) was added into each tube, and the contents were mixed by pipetting. Samples were then frozen at −80°C to pellet the cellular debris for 20 minutes (up to O/N). The tubes were centrifuged at 10,000g for 5 minutes, and the supernatant was transferred into a fresh tube on ice.

For measuring protein concentration, Low Range Assay of the Pierce BCA protein assay kit (ThermoFisher Scientific, Waltham, MA) microplate procedure was used according to the kit protocol. Samples were diluted 1:5. The concentration of each sample was equalized to that of the sample with lowest concentration by diluting in lysis buffer; 6X sample buffer was added to a final concentration of 1X into each sample, and samples were boiled at 95°C for 5 minutes. Any kD Mini-PROTEAN TGX Precast Protein Gel (Biorad, Hercules, CA) was used to run the samples at 150 V for 45 minutes. The transfer was performed at 150 V for 1 hour. Washes were carried out in TBST (TBS + 0.1% Tween) and the membrane was blocked in 5% milk for 30 minutes at RT. The membrane was incubated in primary antibodies O/N at 4°C, and in secondary antibodies for 2 hours at RT. Representative images of membranes from 3 independent experiments are shown. Antibody information is provided in S2 Table.

Quantification and statistical analysis

Proliferative (pHH3⁺/CDH1⁺) cells were assessed on sections of E10.25, E10.75, E11.5, E12.0, E12.5, E13.5, and/or E14.5 pancreata. In WT and Lats1/2^PanKO embryos, the number of CDH1⁺ cells expressing pHH3⁺ was quantified. Values were normalized to the total number of CDH1⁺ cells per section, and at least 5 sections were quantified per pancreas (n = 3 embryos per stage per genotype). pNFκB1 activity was assessed on sections of WT and Lats1/2^PanKO pancreata at E12.0. pNFκB1⁺/CDH1⁺ cells (above the set threshold) were counted, and the proportion of pNFκB1⁺/CDH1⁺ cells, normalized to the total number of CDH1⁺ cells per pancreas section, was calculated. At least 5 sections were quantified per pancreas (n = 3 per genotype). Apical and basal widths were measured using the freehand line tool (Fiji, https://fiji.sc/) to calculate apical:basal ratios. To quantify epithelial VIM expression in pancreas explants, pancreas CDH1⁺ epithelial volume was delineated using Imaris x64 9.0.2 (Bitplane AG, Zurich, Switzerland) surface function analysis of Z stack images, and the VIM channel was masked, to restrict analysis to CDH1⁺ epithelium. Compressed Z stack images of the VIM channel were analyzed (above the set threshold), and the percent area of VIM expression within the pancreas epithelium was measured using Fiji Analyze Particles function. Mean VIM pixel intensity within the epithelium was measured using Fiji Measure function. To quantify the number of pHH3⁺ cells per explant, pancreas CDH1⁺ epithelial volume was isolated using Imaris surface function analysis of pHH3/CDH1 immunostained explants, and the pHH3 channel was masked. These isolated epithelial pHH3⁺ cells were analyzed using Imaris Surface function, with background subtraction and seed point diameter set to 12 μm. Consistent channel threshold and voxel quality settings were used within each experiment for analysis. To quantify fluorogenic CellROX, the CellROX channel was masked based on pancreas CDH1⁺ epithelial volume, isolated using Imaris Surface function analysis. Compressed Z stack images of the masked CellROX green fluorescent channel were then color thresholded using Fiji, and the percent area of CellROX⁺ pixels within the pancreatic epithelium was measured using Fiji Analyze Particles functions. The color threshold was set to 80–255 for every CellROX image analyzed (n = 3–5 images per genotype per treatment group.) Data are presented as mean ± SEM. Unless otherwise noted, statistical significance was calculated using two-tailed unpaired Student t test or Mann-Whitney test in GraphPad Prism 7 software. p < 0.05 was considered statistically significant.