Summary statement

TBX3 shows switching expression patterns during embryonic development in the pancreas and leads to fine-tuning of regeneration from acute pancreatitis via limiting proliferation and fibrosis during regeneration.

Background

The family of T-box transcription factors includes five subfamilies and comprises 17 genes in both humans and mice. They all share highly conserved “T” DNA-binding domains and are essential during embryonic development and tissue homeostasis [1,2,3,4].

TBX3, along with TBX2, TBX4, and TBX5, belongs to the TBX2-family [1, 2]. Heterozygous mutations in TBX3 lead to the malformation of multiple structures resulting in ulnar-mammary syndrome in humans [5]. Accordingly, homozygotes for hypomorphic mutations in Tbx3 in murine embryos and adults resulted in lethal maldevelopment of the heart and cardiac arrhythmias [4,5,6].

TBX3 has multiple molecular functions [7] and participates in transcription factor networks that guide organ patterning in multiple tissues. TBX3 and TBX2 are closely entangled in other regulatory programs, such as Wnt and Hedgehog signaling, and both are essential for proper limb [8], lung [9], and ureter development [10]. Interestingly, redundancy between Tbx3 and Tbx2 to compensate for each other has been reported in several organs [9, 10].

Finally, TBX3 is not only critically involved in pluripotency maintenance but also in lineage-specific exit from the pluripotency circuitry in mouse embryonic stem cells [11,12,13,14,15,16,17]. TBX3 is also a putative inhibitor of pancreatic lineage entry in differentiating human pluripotent stem cells [18]. Interestingly, TBX3 was associated with higher aggressiveness in pancreatic cancer via its ability to (i) promote angiogenesis and (ii) induce cancer stem cell properties [19]. Such stem cell-related features may also become relevant during tissue regeneration after organ injury.

Common mechanisms and similar genetic programs between stem cell induction, organ homeostasis, and regeneration from injury have been identified for the pancreas [20, 21]. Among others, augmented Hedgehog, Notch, and Wnt/b-catenin signaling were delineated as mandatory for regeneration in mice after experimental induction of pancreatitis [21,22,23,24]. Interestingly, Tbx3 interacts with certain parts of the Wnt and Hedgehog signaling pathways [8,9,10].

To investigate the functional roles of Tbx3 during pancreatic development, adult pancreatic homeostasis, and pancreatic regeneration after injury, we here re-analyzed publicly available single-cell transcriptomic data sets and employed a Tbx3Venus reporter system as well as a compartment-specific Tbx3 knockout mouse model and a human knockdown induced pluripotent stem cell (iPSC) differentiation model. While TBX3 is expressed in both mesenchyme and epithelium during embryonic development, its deletion does not significantly alter organogenesis. However, loss of Tbx3 leads to overshoot proliferation of acinar cells, accumulation of fibrosis, and enhanced inflammatory stimuli, Il6-Jak-Stat3, and acinar cell-specific NF-κB signaling during pancreatitis.

Results

TBX3 is dynamically expressed during pancreatic organogenesis and in adult pancreatic stellate cells

We first performed an RNA-based analysis to examine the differential expression patterns of Tbx3 in mice by re-analyzing published datasets of mouse development (GSE101099) [25] as well as of adult murine pancreata (GSE109774) [26] revealing dynamic expression patterns over distinct stages (Fig. 1a–c). At E12.5 (embryonic day 12.5) and E14.5, Tbx3 was expressed in various mesenchymal cells and neural crest cells, as indicated by cluster assignment using Col3a1 or Tlx2 [27, 28] (Fig. 1a,b, Additional file 1: Fig. S1a,b). Interestingly, while Tbx3 expression levels were retained in a few cells of the mesenchymal compartment, only a limited number of endothelial cells expressed Tbx3 at E17.5 as indicated by Pecam1 positivity (Fig. 1c, Additional file 1: Fig. S1c). Of note, a few acinar precursor cells marked by Cpa1 demonstrated expression of Tbx3. In the adult pancreas, Tbx3 was detected only in few stellate (Col3a1) and endothelial (Pecam1) cells but not exocrine (Cpa1, Krt19) or endocrine (Ins1, Gcg, Sst) cells (Fig. 1d, Additional file 1: Fig. S1d).

Fig. 1
figure 1

Tbx3 expression during embryonic development of the pancreas. a–d Re-analysis of single-cell transcriptomic data sets [25, 26] from murine pancreata at indicated timepoints. UMAP cell-cluster representation of the re-analyzed single-cell RNA sequencing data (left panels) and expression patterns of Tbx3 in embryonic pancreata as violin plots (middle panel) and featureplots (right panels) at a E12.5 (n=2 mice), b E14.5 (n=3 mice), c E17.5 (n=3 mice), and d in adult pancreata (n=7 mice). UMAP, uniform manifold approximation and projection. Expression levels depicts log-normalized counts

Full size image

To substantiate our analysis across another species, we investigated the expression of human TBX3. First, we chose to re-analyze our recently published bulk transcriptomic datasets (GSE131817) [29, 30] to assess T-box gene expression during in vitro differentiation of human PSCs toward pancreatic progenitor cells (Additional file 1: Fig. S2a,b). While other T-box genes (TBX6, TBX19, EOMES) were highly expressed in human pluripotent stem cells and in definitive endoderm, TBX3 was among the most prominent transcripts at the pancreatic endoderm and pancreatic progenitor stage (Additional file 1: Fig. S2b). As mouse pancreatic progenitors start to arise and specify between E9.5 and E12.5 [31,32,33,34,35], this human analysis enabled us to zoom into earlier time points than those from the murine embryonic pancreata (Fig. 1a–c). To investigate TBX3 expression in the more mature pancreatic lineages [36, 37], we investigated our recently published single-cell transcriptome dataset (GSE162547) monitoring, particularly pancreatic ductal differentiation [37]. Here, we noticed that TBX3 expression was heterogeneously, still robustly expressed throughout the arising lineages, including ductal, endothelial, and endocrine cells, indicative of a widely distributed embryonic expression of TBX3 during human development (Additional file 1: Fig. S2c). Next, we analyzed the time-resolved expression patterns of a set of TBX3 interaction partners [38,39,40] in our published bulk transcriptomic dataset [29, 30] (Additional file 1: Fig. S2d). Here, we observed a decrease in well-known embryonic factors (e.g., NANOG), while several genes demonstrated higher expression levels over time at the pancreatic progenitor stage (e.g., LEF1).

To further elucidate the cell type-specific expression profiles of these T-box genes in adult pancreata, we performed a single-cell transcriptomic in silico analysis from four published human pancreatic islet donors (GSE84133) [41] (Additional file 1: Fig. S2e,f). In addition to endocrine cells (GCG, INS; SST, PPY), also exocrine cell types (marked by KRT19 and CPA1), stellate (COL3A1), and endothelial (PECAM1) cells were identified (Additional file 1: Fig. S2f). As anticipated from the murine adult pancreata, the expression of TBX3 was restricted to the stellate cells (Additional file 1: Fig. S2e).

Thus, TBX3 is dynamically expressed during the differentiation of pancreatic cells at several developmental stages (PSC-derived pancreatic progenitors, duct-like cells, endothelial-like cells, and endocrine-like cells), and its expression shifts from epithelial cells to stromal cells in the adult human pancreas. Thus, expression in the adult human and murine organisms appears comparable in both species.

TBX3 in site analysis confirms transcriptionally derived expression patterns

Dynamic Tbx3 expression during pancreatic development prompted us to resolve stage-specific protein expression in the pancreatic anlage during embryonic and postnatal development. To visualize the activity of the murine Tbx3 promoter at the protein level, we used a validated Tbx3-Venus (Tbx3tm1(Venus)Vmc) [11, 42,43,44,45,46,47] reporter system (Fig. 2a). In this model, the production of Venus protein reflects Tbx3 promoter activity as detected with anti-GFP antibodies [42, 45]. By using a global knockout of Tbx3 through the replacement of the first three exons by a Cre-knockin [48], we confirmed TBX3 antibody specificity (Additional file 1: Fig. S3a). Similarly, the anti-GFP antibody was licensed to capture the Venus protein in Tbx3-positive cells of the seminal vesicle serving as a positive control (Additional file 1: Fig. S3b). We chose multiple developmental stages (E12.5, E15.5, E18.5, adult) to visualize Venus expression. In addition, we investigated the pancreata of P7 (postpartum day 7 after birth) newborn mice. These experiments revealed a good correlation between protein and the scRNA-seq data (Fig. 1). Venus was significantly expressed in the pancreatic mesenchyme (Fig. 2b), a structure known to mediate inductive cues to pattern the pancreatic epithelium during organogenesis [49]. Specifically, at E12.5 Venus signal was detected around epithelial buds as indicated by co-staining for transcription factors NKX6-1, PDX1, SOX9, FOXA2, and basal membrane proteins such as laminin (LAM) (Fig. 2c). At E15.5, the expression pattern of Venus was virtually the same as at E12.5, demonstrated by colocalization with mesenchymal markers vimentin and collagen IV (COL IV), but not the epithelial markers NKX6-1, PDX1, CDH1, and CD49f (Fig. 2d). Of note, Venus protein was detected both in the mesenchymal and epithelial compartment of the pancreas at E18.5 and P7 whereas RNA expression of Tbx3 was more abundant in mesenchymal and endothelial cells at E17.5 (Figs. 1d and 2e-g). However, concordant to the few acinar precursor cells expressing Tbx3 at E17.5 (Fig. 1c), the epithelial expression was mostly in acinar cells as proven by colocalization of Venus with amylase (AMY) and not in endocrine cells labeled by insulin (INS) and glucagon (GCG). Furthermore, Venus was detected in mesenchymal cells (collagen IV +), and also detected in PECAM-1 expressing endothelial cells. To further validate the expression of Venus in acinar (precursor) cells at E18.5, we performed staining of Venus and TBX3 on consecutive sections of E18.5 pancreata (Fig. 2f) confirming the expression of both markers in acinar structures (Zoom-ins in Fig. 2f). Immunohistochemistry analysis further licensed the acinar expression pattern (Additional file 1: Fig. S3c). In adult mice, concordant with the RNA data, Venus was not detected in pancreas-specific epithelial (CDH-1, AMY, CK-19, INS, GCG) cell types (Figs. 1e and 2h). Thus, TBX3 protein expression, as assayed with the Venus reporter, presents specific and developmentally dynamic expression patterns in both the pancreatic epithelium and the mesenchyme. These patterns correlate mostly with the RNA findings, except the weak but robust acinar expression around E18.5 which appears underestimated in scRNA sequencing data potentially attributed to technical limitations for weakly expressed genes [50,51,52,53,54].

Fig. 2
figure 2

Venus reporter protein expression confirms RNA expression patterns of Tbx3. a Schematic representation of the Tbx3+/Venus [42, 43] reporter mouse model employed to assess TBX3 expression patterns shown in c–h. Venus (VEN) expression is under the control of the Tbx3 promoter and disrupts Tbx3 expression from the knockin allele. b Schematic illustration depicting VEN expression pattern in pancreas during embryonic development and postnatal growth. c Immunofluorescence staining for NKX6-1/LAM (red/white), PDX1/CDH2 (red/white), SOX9/NKX6-1 (red/white), FOXA2/CDH2 (red/white), and Venus (VEN) (green) on E12.5 pancreata (n=3 mice). d Immunofluorescence staining for NKX6-1/COL IV (red/white), PDX1/CD49f (red/white), VIM/CDH1 (red/white), COL IV/CD49f (red/white), and VEN (green) on E15.5 pancreata (n=5 mice). e Immunofluorescence staining for pancreatic amylase (AMY)/INS (red/white), AMY/GCG (red/white), GCG/CDH2 (red/white), PECAM-1/CDH1 (red/white), and VEN (green) on E18.5 pancreata (n=4 mice). f Immunofluorescence staining for VEN (left panels) or TBX3 (right panels), both in green, in combination with CDH1 (red) on consecutive sections in E18.5 pancreata (n=1 mouse). g Immunofluorescence staining for VIM/INS (red/white), AMY/GCG (red/white), AMY/PECAM-1 (red/white), COL IV/PECAM-1 (red/white), and VEN (green) on early postnatal (P7) pancreata (n=4 mice). h Immunofluorescence staining for AMY/GCG (red/white), CDH1/CK-19 (red/white), INS/PECAM-1 (red/white), and VEN (green) on adult pancreata (n=3 mice). Cells were counterstained with DAPI. Scale bars correspond to 25 μm. n≥2 per group. E, exon

Full size image

Compartment-specific deletion of Tbx3 leads to virtually unaltered pancreatic development

To study the functional role of TBX3 in the context of pancreatic organogenesis, two different conditional knockout models targeting Tbx3 in either the pancreatic epithelium or mesenchyme were employed. Embryonic deletion of Tbx3 in the epithelial compartment of the pancreas was achieved by crossing Tbx3flox/+ with Ptf1aCre/+ mice, a model known to target all epithelial lineages of the pancreas, including acinar, ductal, and most endocrine cells starting from E10.5 (Fig. 3a) [35]. Specific recombination in epithelial cells of the pancreas was confirmed by immunohistochemical stainings for tdRFP in Ptf1aCre/+  [35] x LSL-tdRFPKI/KI [55, 56] mice (Additional file 1: Fig. S3d) [56]. Histologic morphology was similar between homozygous knockout mice (Tbx3-KO (epi, epithelial)) and control mice (no knockout of Tbx3) (Fig. 3b). Furthermore, immunofluorescence analysis labeling exocrine pancreatic markers (CK-19, AMY2A; Fig. 3c) revealed virtually no difference between Tbx3-KO (epi) and control mice. This was also valid for the endocrine compartment, where only subtle differences with a trend toward smaller islets in Tbx3-KO (epi) mice was documented while maintaining similar percentages of INS+ beta-cells and GCG+ alpha-cells (Fig. 3d–g).

Fig. 3
figure 3

Tbx3 is largely dispensable for pancreatic development. a Schematic representation of the Tbx3fl/fl; Ptf1aCre/+ mouse model allowing Tbx3 deletion in pancreatic epithelial tissue (Tbx3-KO (epi)). b Hematoxylin and eosin (H&E)-stained histological sections of control and Tbx3-KO (epi) pancreata. Immunofluorescence stainings for c CK-19/AMY2A (red/green) and d INS/GCG (green/red) on control and Tbx3-KO (epi) pancreata. Quantification of e number of cells per islet based on INS/GCG co-stainings, f percentage of insulin expressing cells in islets, and g percentage of glucagon-expressing cells in Langerhans islets. n=14 control (littermates of Tbx3-KO (epi)) pancreata and n=9 Ptf1a-Cre-driven Tbx3-KO (epi) pancreata. h Schematic representation depicting Tbx3fl/fl; Nkx3-2Cre/+ mouse model allowing Tbx3 depletion in pancreatic mesenchyme (Tbx3-KO (mes)). i H&E-stained histological sections of control (littermates of Tbx3-KO (mes)) and Nkx3-2-Cre-driven Tbx3-KO (mes) pancreata. Immunofluorescence stainings for j CK-19/AMY2A (red/green) and k INS/GCG (green/red) on control and Tbx3-KO (mes) pancreata. Quantification of l number of cells per islet area based on INS/GCG co-stainings, m percentage of insulin expressing cells in islets, and g percentage of glucagon-expressing cells in Langerhans islets. n=7 control (littermates of Tbx3-KO (mes)) pancreata and n=4 Nkx3-2-Cre-driven Tbx3-KO (mes) pancreata. Cells were counterstained with DAPI. Scale bars correspond to 50 μm. Data are expressed as individual datapoints and mean ± SEM. Mann-Whitney test was performed to investigate significance levels (no significance reached)

Full size image

Given the essential role of the pancreatic mesenchyme for proper lineage formation [49, 57], Tbx3 was also deleted in this compartment. The Nkx3-2Cre/+ mouse strain was used (Fig. 3h) as it has been employed to demonstrate the relevance of the pancreatic mesenchyme for proper pancreatic development [49]. As with the epithelial compartment, homozygous deletion of Tbx3 (Tbx3-KO (mes, mesenchymal)) in the mesenchyme did not alter pancreatic morphology (assayed by histomorphologic investigation of H&E staining) (Fig. 3i). Additional analysis of exocrine markers (CK-19 and AMY2A) showed no difference between Tbx3-KO (mes) and control mice (Fig. 3j). Interestingly, also the investigation of the endocrine markers insulin and glucagon revealed no significant difference (Fig. 3k–n). The size of islets (Fig. 3l) and the percentages of insulin- and glucagon-expressing cells (Fig. 3m,n) were not significantly different between Tbx3-KO (mes) and control mice. Thus, although Tbx3 displayed specific and dynamic regulation patterns on mRNA and protein levels in both mesenchyme and epithelium, its targeted ablation in either compartment does not relevantly alter proper pancreatic organ formation.

To check whether the well-described homolog TBX2 might compensate for TBX3 loss [9, 10], we performed immunohistochemistry for TBX2 in pancreata of both knockout models (Additional file 1: Fig. S4a,b). However, the overall staining intensity remained similar in control (epi) and Tbx3-KO (epi), as well as control (mes) and Tbx3-KO (mes) pancreata, albeit nuclear and cytoplasmatic protein distribution varied across various animals without a relevant genotype-specific trend.

Finally, we wanted to challenge the relevance of TBX3 in human pancreatic development. Thus, we employed an inducible knockdown of TBX3 by an shRNA in a previously reported iPSC line [12] during in vitro pancreatic progenitor differentiation [58,59,60,61]. We assessed trilineage potential (generation of acinar, ductal, and endocrine cells) in a recently described porcine urinary bladder (PUB) organ culture model for the maturation of pancreatic progenitor cells [62, 63] (Additional file 1: Fig. S5a). Of note, we did observe the generation of morphologically similar (Additional file 1: Fig. S5b), ductal (Additional file 1: Fig. S5c), acinar (Additional file 1: Fig. S5d), and endocrine (Additional file 1: Fig. S5e) structures on the PUB scaffold. Thus, the trilineage potential was not significantly altered by TBX3-knockdown during pancreatic progenitor differentiation. We conclude that TBX3 is dispensable for proper pancreatic organ formation in mice and men.

Loss of Tbx3 results in increased fibrosis and excessive proliferation during recovery from acute pancreatitis

Since pancreatic development shares common regulatory patterns with pancreatic repair, we challenged Tbx3-KO (epi) mice and control mice with caerulein-induced acute pancreatitis to investigate the potential relevance of Tbx3 under exogenous stress conditions (Fig. 4a). Notably, no significant differences between control and Tbx3-KO mice were detected based on the amount of acinar-to-ductal metaplasia (ADM) as well as edema and inflammatory infiltration scores (Fig. 4b–e). As expected, a substantial increase of ADMs at 72h preceded by increased edema and infiltration of inflammatory cells (24h) was documented after induction of caerulein-driven acute pancreatitis in both groups of animals. The damage was largely repaired 7 days after the first injection. However, when assessing the proliferation rates of acinar cells with normal morphological configuration by KI-67 quantification, we noticed a significantly higher amount of KI-67+ cells per field in the Tbx3-KO (epi) mice compared to the control counterpart at 72h (4.3 cells/per field vs 1.7 cells/field) and 168h (3.0 cells/field vs 1.2 cells/field) (Fig. 4f–h). Interestingly, increased acinar proliferation was accompanied by significantly more fibrosis as quantified upon Sirius Red stainings at 72h in Tbx3-KO (epi) mice (Fig. 4i,j). The amount of fibrosis was reflected by the presence of ACTA2-positive cells around ADM structures (Fig. 4k). Interestingly, gene expression analysis of Acta2 at 72h after the onset of pancreatitis confirmed the significant difference between Tbx3-KO (epi) and control mice (Fig. 4l). Finally, we checked if apoptosis at 72h after induction of pancreatitis might play a role in the altered regeneration (Fig. 4m,n). However, no significant differences were observed concerning the rate of apoptotic cells. Thus, despite the fact that regeneration was achieved in Tbx3-KO (epi) mice, prolonged proliferation in acinar cells and accumulation of fibrosis suggest that Tbx3 is involved in proper regeneration from organ injury, but its absence is well compensated by other genes.

Fig. 4
figure 4

Tbx3 loss leads to overshoot proliferation of acinar cells and accumulation of fibrosis. a Schematic representation of the caerulein-induced acute pancreatitis assay shown in b–l. Mice were euthanized at the indicated time points (arrows in blue). b Hematoxylin and eosin (H&E)-stained histological sections of control littermates of Tbx3-KO (epi) and Tbx3fl/fl; Ptf1aCre/+ (Tbx3-KO (epi)) pancreata after treatment with caerulein or vehicle. c Quantification of acinar-to-ductal metaplasia (ADM), d edema score, f inflammatory infiltration score per field in pancreata from caerulein-induced acute pancreatitis assay shown in a (n≥6 per group). f Representative immunohistochemistry stainings for KI-67 and g immunofluorescence co-staining of KI-67/AMY2A (red/green) at indicated timepoints. h Quantification of KI-67-positive acinar cells per field in pancreata from caerulein-induced acute pancreatitis assay shown in a (n≥6 per group, detailed in Additional file 3). i Picrosirius red stained histological sections of pancreata from caerulein-induced acute pancreatitis assay shown in a. j Quantification of picrosirius red-positive area as ratio to hematoxylin-based whole pancreas surface in pancreata from caerulein-induced acute pancreatitis assay shown in a (n≥6 per group). k Immunofluorescence staining for ACTA2 (red) in pancreata at 72h after induction of the acute pancreatitis. l Relative mRNA expression of Acta2 at 72h after induction of acute pancreatitis. m Representative immunohistochemistry staining for caspase-3 (CASP3) at the 72h timepoint and n respective quantifications. Cells were counterstained with DAPI for immunofluorescence analysis. Graphs present individual data points and mean with SEM. Two-way ANOVA with Sidak’s post-test was performed for graphs with multiple time points. Mann-Whitney test was performed to assess significance at single time points for apoptosis and Acta2. *, p < 0.05; **, p < 0.01. Scale bars represent 100 μm

Full size image

Loss of Tbx3 results in increased susceptibility of acinar cells to fibroinflammatory stimuli

To further specify the subtle differences across the two genotypes during regeneration (Tbx3-KO (epi) vs. control (epi) mice), we performed RNA sequencing for the two most interesting time points (72 and 168h). Interestingly, we noticed a clear difference in the principal component analysis (PCA) of whole transcriptomes from Tbx3-KO (epi) mice compared to their respective controls at 72h, while the differences at 168h were virtually absent (Fig. 5a). We could identify 1152 differentially expressed genes (DEG) at 72h (762 up, 390 down in Tbx3-KO (epi)), while only 1 DEG was detected at 168h (Fig. 5b). Subsequently, we performed gene set enrichment analysis (GSEA) for hallmark (HM) gene sets and one acinar-specific NF-κB response gene set (Additional file 2: Supplementary Table 1) adapted from [64] (Fig. 5c). Interestingly, the significantly enriched gene sets included the Il6-Jak-Stat3-, Il2-Stat5-signaling, Inflammatory-Response, E2F-Targets, G2M-Checkpoint, and the acinar-specific NF-κB response. Notably, most gene sets are related to the immune system. We also checked other well-known gene sets relevant to pancreatic organ regeneration and detected no significant enrichment for the HM-Hedgehog, Wnt, Tgf-β, and Notch signaling. Concerning depleted gene sets, we noticed a non-significant depletion of Pancreatic-Beta-Cell-signature and Protein-Secretion-Signature, the latter being potentially indicative of normal acinar function.

Fig. 5
figure 5

Whole transcriptome analysis uncovers a potential role of Tbx3 to fine-tune fibroinflammatory stimuli. a Principal component analysis of n=3 mice per genotype (control littermates of Tbx3-KO (epi) and Tbx3fl/fl; Ptf1aCre/+ (Tbx3-KO (epi))) at 72 and 168h after induction of acute pancreatitis. b Differentially expressed genes (DEG) in Tbx3-KO (epi) mice at indicated timepoints. c Gene set enrichment analysis (GSEA) in Tbx3-KO (epi) of different hallmark (HM) gene sets and one acinar-specific upregulated gene set in response to NF-KB (Supplementary Table 1) [64] at 72h. d Volcano plot of the enriched IL-6-JAK-STAT3-Signaling in Tbx3-KO (epi) mice at 72h. e GSEA of immune cell signatures [65] in Tbx3-KO (epi) mice at 72h. f Immunohistochemistry staining for B220+ B cells at 72h after induction of acute pancreatitis . g Immunohistochemistry staining for MPO+ neutrophils at 72h after induction of acute pancreatitis. h Quantification of percentages of B220+ B cell area per field of view in n=7 control (littermates of Tbx3-KO (epi)) pancreata and n=6 Ptf1a-Cre-driven Tbx3-KO (epi) pancreata. i Quantification of MPO+ neutrophils per field of view in n=8 control (littermates of Tbx3-KO (epi)) pancreata and n=9 Ptf1a-Cre-driven Tbx3-KO (epi) pancreata. Graphs present individual data points and mean with SEM. Mann-Whitney test was performed to assess significance. *, p < 0.05. j Heatmap of log2 fold changes of Tbx genes at 72h in Tbx3-KO (epi) pancreata compared to control pancreata. k Heatmap of log2 fold changes of known interaction partners of Tbx3 at 72h in Tbx3-KO (epi) pancreata compared to control pancreata. l DNA footprint analysis of putative DNA binding sites in the promoter of Lef1 at −18bp from TSS. PWM-based score 0.82, p-value=0.0009. m Proposed mechanism for Tbx3-related fine-tuning of acute pancreatitis. *, adjusted p < 0.05. Scale bars represent 100 μm. MPO, myeloperoxidase.

Full size image

Fitting with the enrichment of the Il6-Jak-Stat3 gene signature, a variety of immune cell-related genes (e.g., Cxcl9, Il2rg) were significantly overexpressed at 72h post pancreatitis (Fig. 5d), which prompted us to investigate specific immune cell signatures (Additional file 2: Supplementary Table 2 [65]; (Fig. 5e). Interestingly, signatures for B cells and neutrophils were significantly upregulated. However, validation on protein level could only confirm an increased abundance of B cells in Tbx3-KO (epi) pancreata as compared to control (epi) counterparts at 72h after induction of pancreatitis (Fig. 5f,h). Neutrophil granulocyte quantification revealed only a trend (Fig. 5g,i). Infiltrating T cells and macrophages remained similar across the two genotypes (Additional file 1: Fig. S6a-d). To identify potential compensators for Tbx3 loss in the epithelial compartment, we analyzed the expression of all expressed Tbx genes at 72h (Fig. 5j). Interestingly, most Tbx genes remained unaltered, with Tbx18 being significantly downregulated and Tbx21 overexpressed in the Tbx3-KO (epi) mice. Next, a set of Tbx3 target genes were investigated [38] (Fig. 5k). Amongst these, the sole strongly and significantly upregulated gene was Lef1. Accordingly, we calculated the putative binding score and significance using the palindromic TBX3 binding site (18 bp) for the Lef1 promoter consensus sequence. Indeed, the relative similarity score was highly significant (score = 0.82, p = 0.0009), indicating direct TBX3 binding to the Lef1 promoter, most likely triggering its transcriptional repression (Fig. 5l).

Discussion

In this study, we (i) determined the expression of TBX3 during pancreatic organogenesis, (ii) demonstrated expression switching between mesenchymal and epithelial cells during pancreatic development, and (iii) revealed expression in the stromal compartment of the adult pancreas. Surprisingly, epithelial and mesenchymal knockout of Tbx3 did not lead to statistically significant phenotypic alterations in the murine pancreas, and knockdown of TBX3 in a human iPSC-based system for pancreatic differentiation did not alter ex vivo pancreatic development. It is likely that TBX3 function may be compensated by other genes in these compartments during development. Driven by the fact that developmental programs can become re-activated during regeneration from injury, we investigated the relevance of Tbx3 for regeneration after caerulein-induced acute pancreatitis. Our findings indicate that epithelial deletion of Tbx3 does not significantly impact overall organ regeneration but does prolong proliferation and increase fibrosis. Again, this lack of a major effect in the face of Tbx3 ablation is likely attributable to other factors, while Tbx3 in itself is required for the fine-tuning of the regenerative process. Whole transcriptome analysis finally revealed putative mechanisms that alter the regeneration via enhanced immune cell-acinar interactions, fibroinflammatory stimuli, and dysregulated proliferation (Fig. 5m). Altogether, while Tbx3 is not a master regulator of pancreatic development and organ regeneration, our data show a relevant but subtle contribution of this factor to adult pancreatic organ homeostasis and development.

Interestingly, Tbx3 RNA expression levels demonstrated a virtually lower percentage of Tbx3+ cells than assayed in immunofluorescence analysis. This discrepancy was higher for acinar cells than for mesenchymal or stromal cells, which may be related to either technical limitations of single-cell RNA-seq [51,52,53,54] with a bias to filter out genes with low expression [50] and eventually boosted by the fact that the protease- and ribonuclease-rich acinar cells raise difficulties to isolate intact RNA [66,67,68]. Another potential explanation would be that Tbx3 expression levels in mesenchymal/stromal cells are, in general, higher (also evident in the violin plots of Fig. 1), which eases the detection.

Nevertheless by employing a knockout-validated antibody and a Venus reporter system [11, 42,43,44,45,46,47], the expression of TBX3, or Venus, respectively, was proven on the protein level. In addition, the evidence of human TBX3 expression in an in vitro iPSC differentiation in the epithelial compartment highlights that TBX3 is indeed expressed during embryonic pancreatic development.

Indeed, important roles for TBX3 have been shown in lung branching morphogenesis and ureter organogenesis [9, 10]. In both cases, TBX3 was located in the mesenchyme, and depletion of Tbx3 (together with Tbx2, which otherwise would have compensated Tbx3) resulted in defective organ patterning [9, 10]. Based on such recent publications, we chose to not only perform a knockout of Tbx3 in the epithelial compartment by Ptf1a-Cre but also in the mesenchymal compartment by Nkx3-2-Cre. Other than our anticipation from the expression level profiles and the recent publications [9, 10], neither of the knockout models led to a strong phenotype. TBX2 and 3 are members of the same T-box subfamily [1]. Their partially redundant roles in lung or ureter development [5, 9, 10] raise the question if this leads to redundant function in pancreatic evolution. However, our solely on gene and protein quantification relying study was underpowered to detect evidence for TBX2 compensating during pancreatic development as TBX2 remained unchanged in both the two knockout models and the pancreatic injury model. To ultimately clarify this hypothesis, a double-knockout of Tbx2 and Tbx3 in both pancreatic compartments would be necessary. However, this was clearly beyond the scope of the current study.

Even though we did not observe a gross impact of Tbx3 on recovery from acute pancreatitis, we observed (i) higher amounts of fibrosis, (ii) prolonged proliferation in acinar cells in its absence, and (iii) higher variance in some investigated parameters of regeneration of Tbx3-KO mice. Of note, we assessed the putative compensation by other Tbx genes during pancreatitis. Here we observed an upregulation of Tbx21. However, as Tbx21 (T-bet) and also Eomes (increased expression, though not significant) also play relevant roles in immune cell signaling, we attributed the enhanced expression to the increased inflammatory signature. Considering the downregulation of Tbx18, we pinpoint a recent publication of cardiac pacemaker differentiation where Tbx3 and Tbx18 are co-regulated, implying that a similar program could occur during pancreatic regeneration [69].

Based on the fact that Tbx3 is involved in the regulation of Wnt signaling in embryonic development [9] and Wnt signaling controls the proliferation of acinar cells during organ regeneration [24], Tbx3 may have a regulatory role on Wnt signaling during acinar cell regeneration. By demonstrating that Lef1, a central mediator of the Wnt signaling axis [24, 70,71,72,73], is not only significantly upregulated in Tbx3-KO (epi) mice but also possesses a putative DNA binding site for TBX3 in its promoter region, we postulate a previously unknown potential suppressive function of TBX3 on Lef1 building the following hypothetical model: The absence of Tbx3 in the pancreatic epithelium could trigger excessive proliferation eventually involving Lef1, a known member of the Wnt signaling [74, 75], and acinar-specific upregulated NF-KB-pathway members leading to abnormal proliferation of acinar cells [64]. Such proliferation pulse is accompanied by an increased fibroinflammatory response resulting in transient accumulation of B cell-infiltrated fibrotic tissue (Fig. 5m). This suggests an ongoing homeostatic process during pancreatic regeneration from an injury involving only Tbx3 for fine-tuning without the generation of substantial alterations upon genetic loss.

Conclusions

In summary, Tbx3 shows switching expression patterns in the developing pancreas. Although Tbx3 seems dispensable for proper pancreatic development and lineage entry, its absence results in altered organ regeneration after induction of acute pancreatitis marked by enhanced fibrosis and inflammation.

Methods

Single-cell RNA sequencing re-analysis

Expression patterns of Tbx3 on a single-cell level were derived from two different murine datasets: GSE101099 [25] for embryonic pancreata at E12.5, E14.5, and E17.5, as well as GSE109774 for adult pancreata [26]. Expression of TBX3 in a recently published human pancreatic duct-like cell differentiation approach from pluripotent stem cells was performed as recently described in detail (GSE162547) [37]. The expression of TBX3 in adult pancreata of four human donors was interrogated in a published dataset: GSE84133 [41]. Before expression analysis, preprocessing steps were conducted in RStudio with the version 4.0.4 and the “dplyr”, “Seurat” version 4.0.6 [76,77,78,79], and “patchwork” packages. For the murine datasets, cells with less than 1000 expressed genes, more than 4000 genes for E12.5, E14.5, and more than 5000 genes for E17.5 and genes expressed in fewer than 3 cells were filtered out before analysis. For GSE109774, cells expressing more than 5% ERCC RNA spike-ins were additionally removed. For human datasets, cells expressing either less than 300 genes or more than 4000 genes and genes expressed in fewer than 10 cells were filtered out. Data were merged for E12.5 for GSM2699156_E12_B2 and GSM3140915_E12_v2, for E14.5 for GSM2699154_E14_B1, GSM2699155_E14_B2, and GSM3140916_E14_v2, and for E17.5 for GSM2699157_E17_B2, GSM3140917_E17_1_v2, and GSM3140918_E17_2_v2 of the respective dataset. Human datasets were also merged. Batch effects were corrected with the FindIntegrationAnchor() and the IntegrateData() function in the reciprocal PCA approach of the Seurat workflow. Datasets were Log-normalized, and the top 2000 highly variables genes were identified by the “vst” method for the murine datasets. For the human datasets, similar to our recent work [59], the top 4000 highly variable genes were identified. Data were scaled. A standard Seurat workflow was performed. Single-cell neighborhood was calculated with the first 30 principal components and clustering was performed with the Louvain algorithm with a resolution of 0.015 for E12.5, 0.5 for E14.5, 0.5 for E17.5, 0.2 for the adult murine pancreas, and 0.5 for human datasets. Cluster identity was annotated by specific marker genes.

Bulk RNA expression re-analysis from publicly available RNA sequencing data sets

RNA expression levels of different T-Box transcription factors in embryonic stem cells, definitive endoderm, pancreatic endoderm, and pancreatic progenitors were derived as FPKM (fragments per kilobase of transcript per million mapped reads) from a previously published dataset (GSE131817, wildtype samples) [29, 30]. Data were analyzed in RStudio with the R version 4.0.4. The heatmap was generated with the “pheatmap” package (version 1.0.12), scaling was set to columns, and clustering followed the “ward.D2” method. A list of described TBX3 interaction partners was derived from [38]. A heatmap was generated as indicated above from the same dataset (GSE131817) [29, 30]. The scale was set to the row factor.

Ethics statement

All animal care and procedure were conducted in compliance with the German legal regulations and were previously approved by the local governmental review board of the state of Baden-Württemberg (Permission no. 1477, O.195-4, O.195-6, O.195-10, and O.195-12) or conducted in compliance with the local government of Bavaria. All mouse work aspects were performed according to acknowledged guidelines of the Society of Laboratory Animals (GV-SOLAS) and of the Federation of Laboratory Animal Science Associations (FELASA).

Mice

Ptf1aCre (Ptf1atm1(cre)Cvw) mouse strain [35] was previously described [80, 81]. Nkx3-2Cre (Nkx3-2tm1(cre)Wez) was a kind gift of Warren E. Zimmer (Texas A&M University) [82]. Tbx3flox (Tbx3tm3.1Moon) (kind gift of Anne M. Moon) [4], and Tbx3Venus (Tbx3tm1(Venus)Vmc) mouse (kind gift of Vincent M. Christoffels) [42] strain were maintained on a complex C57BL/6×129/Sv genetic background. Mice were housed and bred in a conventional health status-controlled animal facility. All animal care and procedures followed German legal regulations and if applicable were previously approved by the governmental review board of the state of Baden-Württemberg. All the aspects of the mouse work were carried out following strict guidelines to insure careful, consistent, and ethical handling of mice.

Analysis of Ptf1aCre-specific recombination in adult pancreata

Formalin-fixed paraffin-embedded (FFPE) tissue sections of pancreata of Ptf1aCre [35] x R26-LSL-tdRFPKI [55] mice which were previously described [56] were a kind gift of Patrick Hermann (Ulm University Hospital, Department of Internal Medicine I).

Knockout validation of TBX3-antibodies

TBX3 antibody was analyzed by immunofluorescence in archived Tbx3Cre/Cre at E13.5 or Tbx3wt/wt. [48].

Mouse embryos and adult pancreata preparation

Embryos were collected at 12.5, 15.5, and 18.5 days post-coitum. Newborn mice were euthanized at post natal day 7 (P7). Pancreata were fixed in 4% PFA for 2 h at room temperature or for 16 h at 4°C, cryopreserved, and embedded in optimal cutting temperature compound (OCT compound). Adult mice were at least 8 weeks old. Pancreata were fixed in cold 4% PFA for 16 h at 4°C and embedded in paraffin for histological analysis.

Model of Caerulein-induced acute pancreatitis

Acute pancreatitis in Tbx3-KO (epi) and control mice was induced in adult animal (≥ 8 weeks) by hourly injections (8 times) of 50 μg caerulein /kg bodyweight (Sigma-Aldrich) dissolved in PBS (vehicle). Mice were sacrificed 24, 72, and 168 h after the first caerulein injection. Corresponding volumes of vehicle were injected in Tbx3-KO (epi) and control mice, and pancreata were isolated 24 h after the first injection.

Differentiation of pancreatic progenitor cells and organ culture model

Maintenance culture and differentiation to pancreatic progenitors of a well-described iPSC line with a doxycycline-inducible shRNA against TBX3 [12] was performed as described recently in a step-by-step protocol [58, 59] with slight modifications. For shRNA expression, 3 μg/mL doxycycline was added to the cells. The medium was changed on a daily basis. After the differentiation of pancreatic progenitor cells, organ culture of porcine urinary bladders was performed as described recently [62, 63]. Briefly, porcine urinary bladders (PUB) were cleaned, de-epithelialized, and sterilized with 0.1% peroxy-acetic acid. In total, 500,000 cells per ring were seeded in 30 μL of 50% Matrigel and 50% basal medium as in [58, 59], including 5% FCS and 10 μM Y-27632. The medium of PUBs was changed once a week, and doxycycline was added twice a week freshly. After 2 weeks, PUBs were fixed in 3.7% formaldehyde and processed for histology as described recently [62, 63].

Histology

All histological experiments on FFPE tissue were performed as previously described [20, 59, 62]) following standard procedures. Four-micrometer-thin sections were rehydrated. Heat-mediated antigen retrieval was either performed with citrate-based buffer (pH=6) or TRIS-based buffer (pH=9). Blocking was performed in 5% normal donkey serum in 0.1% Triton X-100 in PBS for 30 min at RT. Primary and secondary antibodies and respective dilution factors are listed in Additional file 2: Supplementary Table 3. Bright-field images were acquired using a Leica DM5500B microscope (Leica) equipped with a Leica DMC5400 camera and Leica Application Suite software (Leica) or by using a Zeiss Axioscope2 microscope (Carl Zeiss) ZEN3.1 imaging software (Carl Zeiss). Immunofluorescence images were obtained with a Zeiss Axioscope2 microscope equipped with an Axiocam 702 (Carl Zeiss). Acquired pictures were subsequently analyzed using ImageJ software (National Institutes of Health).

Immunofluorescence stainings of cryosections were performed following standard protocols. Briefly, 10-μm-thick cryo-sectioned pancreata were rehydrated in PBS for 30 min, washed twice with PBS containing 0.1 % Tween 20 (PBS-T), and permeabilized using 0.1 M glycine (Merck) and 0.1% Triton X-100 (for NKX6.1: 0.5% Triton X-100; Merck) in MilliQ water for 15 min at RT and blocked using 0.1% Tween - 20, 10% heat inactivated fetal calf serum (FCS), 0.1% BSA, and 3% donkey serum in PBS for 1 h at RT. Incubation with primary antibodies diluted in blocking solution occurred overnight at 4°C or 1 h at RT. The slides were washed 3× with PBS-T for 10 min each, and subsequently, secondary antibody solution was added for 2 h at RT. DAPI/PBS solution was added for 20 min before washing of slides 3× with PBS for 10 min each. Finally, the slides were mounted with Elvanol and kept 24 h at RT to dry.

Histological quantifications

The total number of cells per Langerhans islets, as well as the number of insulin and glucagon-expressing cells, was counted manually from at least 3 different islets per pancreas on one section. Acinar-to-ductal metaplasias (ADMs) were quantified by counting at least ten 200× fields. Edema and immune cell infiltration were scored from 0 to 3 (0, no evident edema to 3, maximal degree of edema; 0, no immune infiltration to 3 maximal degree of immune infiltration) as previously described [20]. Proliferation was quantified by counting KI-67-positive cells in immunohistochemistry from ten 400× fields of each pancreas. KI67-positive cells within only clearly identifiable acinar structures were considered. Immune cells were excluded from the quantification. For picrosirius red-positive area/hematoxylin-based whole pancreas surface ratio quantification, pictures from at least five 100× fields were loaded into ImageJ software (National Institutes of Health) to perform color deconvolution. Areas covered by picrosirus red (red) and hematoxylin (yellow) were quantified automatically. Immune cell populations were quantified by counting the absolute number of positive cells per field in at least ten 200× fields. Caspase-3 (CASP3)-positive cells per field of view were quantified manually by counting the number of positive cells in each pancreas in at least six 100× fields. Monocytes, neutrophils, and T cells were manually quantified in at least five different 200× fields. B cells were quantified in at least 8 different 200× fields by quantifying the B220 positive area per field in ImageJ.

RNA extraction, cDNA synthesis, qRT-PCR

RNA extraction was performed as described previously [20] with the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was determined with a NanoDrop. cDNA synthesis was performed with the iScript™ cDNA Synthesis Kit (Bio-Rad) following the manufacturer’s instruction. Briefly, 1 μg of RNA was transcribed. cDNA was diluted 15-fold. PCR was performed at the Rotor-Gene-Q (Qiagen) using 4 μL of diluted cDNA with 5 μL of Green Master Mix (Genaxxon) and 0.4 μM forward and reverse primer (each 0.5μL). Gene expression was normalized to ribosomal protein S18. Primer (Biomers) sequences are as follows: Acta2: fwd 5′-GTTCAGTGGTGCCTCTGTCA-3′, rev 5′-ACTGGGACGACATGGAAAAG-3′, S18: fwd 5′-GTAACCCGTTGAACCCCATT-3′, rev 5′- CCATCCAATCGGTAGTAGCG-3′.

RNA sequencing

The amount of total RNA was quantified using the Qubit 2.0 Fluorometric Quantitation system (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA integrity number (RIN) was determined using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). RNA-seq libraries were prepared with the NEBNext® Ultra™ II Directional RNA sample preparation kit (New England Biolabs, Inc., Ipswich, MA, USA). Library concentrations were quantified with the Qubit 2.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, CA, USA), and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts.

Next-generation sequencing and raw data acquisition

Expression profiling libraries were sequenced on a HiSeq 3000 instrument (Illumina, San Diego, CA, USA) following a 50-base-pair, single-end recipe. Raw data acquisition (HiSeq Control Software, HCS, HD 3.4.0.38) and base calling (Real-Time Analysis Software, RTA, 2.7.7) was performed on-instrument, while the subsequent raw data processing off the instruments involved two custom programs (https://github.com/epigen/picard/) based on Picard tools (2.19.2) (https://broadinstitute.github.io/picard/). In a first step, base calls were converted into lane-specific, multiplexed, unaligned binary alignment map (BAM) files suitable for long-term archival (IlluminaBasecallsToMultiplexSam, 2.19.2-CeMM). In a second step, archive BAM files were demultiplexed into sample-specific, unaligned BAM files (IlluminaSamDemux, 2.19.2-CeMM).

Transcriptome analysis

Next-generation sequencing (NGS) reads were mapped to the Genome Reference Consortium GRCm38 assembly via “Spliced Transcripts Alignment to a Reference” (STAR, 2.7.9a) [83] utilising the “basic” Ensembl transcript annotation from version e100 (April 2020) as reference transcriptome. Since the mm10 assembly flavour of the University of California, Santa Cruz (UCSC) Genome Browser was preferred for downstream data processing with Bioconductor packages for entirely technical reasons, Ensembl transcript annotation had to be adjusted to UCSC Genome Browser sequence region names. STAR was run with options recommended by the ENCODE project. NGS read alignments overlapping Ensembl transcript features were counted with the Bioconductor (3.14) GenomicAlignments (1.30.0) package via the summarizeOverlaps function in Union mode, ignoring secondary alignments and alignments not passing vendor quality filtering. Since the NEBNext® Ultra™ II Directional RNA protocol leads to sequencing of the first strand, all alignments needed inverting before strand-specific counting in feature (i.e., gene, transcript, and exon) orientation. Transcript-level counts were aggregated to gene-level counts, and the Bioconductor DESeq2 (1.34.0) [84] package was used to test for differential expression based on a model using the negative binomial distribution.

An initial exploratory analysis included principal component analysis (PCA), multidimensional scaling (MDS), sample distance, and expression heatmap plots, all annotated with variables used in the expression modelling (ggplot2 [85], 3.3.6, and Bioconductor ComplexHeatmap [86], 2.10.0), as well as volcano plots (Bioconductor EnhancedVolcano [87] , 1.12.0). Biologically meaningful results were extracted from the model, and log2-fold values were shrunk with the CRAN ashr [88] (2.2.-54) package, while two-tailed p-values obtained from Wald testing were adjusted with the Bioconductor Independent Hypothesis Weighting [89] (IHW, 1.22.0) package.

Gene set enrichment analysis

GSEA was performed using the hallmark data sets from the Molecular signatures database v7.4 (MSigDB, Broad Institute; http://software.broadinstitute.org/gsea/msigdb) and the GSEA software version 2.4.3 [90, 91]. Additional GSEA was performed for an acinar-specific NF-κB gene set. Genes upregulated with a significant fold change of least 2 from [64] were chosen to generate the reference gene list (Additional file 2: Supplementary Table 1). Immune cell gene sets were derived from [65] (Additional file 2: Supplementary Table 2). Significant enrichments were defined false discovery rate <0.25.

Putative TBX3-DNA binding analysis

All the analysis was performed using R and Bioconductor (R Core Team, 2022). Lef1 promoter sequence was retrieved from M. musculus UCSC genome version mm10 known Gene database. The position weight matrix (PWM) was generated using the 8 entries of Lef1 promoter regions and was subsequently used for the sequence logo. Mouse TBX3 palindromic binding site was obtained from Uniprot (https://www.uniprot.org/uniprotkb/P70324/entry). Sequence logos, relative similarity score, correlation, Euclidean distance, and p-value were calculated using Transcription Factors binding sites tools in R.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 9.3.1 software. Data are expressed as individual data points and mean ± SEM (indicated in figure legends). Significance levels for islet size, insulin+, and glucagon+ cells were calculated with a Mann-Whitney test. Significance was evaluated with a two-way ANOVA and a Sidak post-test for the investigation of number of ADM per field, edema score, infiltration, score, proliferation, and fibrosis in the acute pancreatitis model. Significance levels for apoptosis, Acta2 gene expression, and immune cell infiltrations at 72h after onset of the pancreatitis were assessed by Mann-Whitney test. Significance levels were defined as the following: p < 0.05 = *, p < 0.01 = **.