A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

Abstract

The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.

Keywords: notochord; pancreas development; signaling diffusion; transcriptional regulation; zebrafish.

Graphical abstract

Figure 1

nog2 Is Required for Proper Pancreas Development (A) ED301 zebrafish line corresponds to an ED transposon integration containing a potent enhancer blocking insulator (yellow) and mapped 2 kb downstream of nog2. The loxP sequences are depicted as black triangles (see also Figure S1). Scale bar represents 2 kilobases. (B) In the ED301 zebrafish line, nog2 expression is downregulated, as detected by qPCR performed in three biological replicates of 30 embryos each (error bars represent SD; ^∗p < 0.05). (C) In situ hybridization of insulin in 48 hpf representative embryos from an outcross of nog2^ED301/ED301 and nog2^+/mut lines compared with control embryos. Scale bar represents 80 μm. (D) Quantification of the insulin expression area detected by in situ hybridization in control (n = 194), nog2 ^ED301/ED301 (n = 93), nog2^ED301/ED301 injected with Cre recombinase (n = 32), nog2^+/mut incross (n = 115), and nog2^ED301/ED301, nog2^+/mut outcross embryos (n = 36) at 48 hpf. Error bars represent SD; ^∗∗∗∗p < 0.0001. (E) Representative confocal images of 48 hpf zebrafish embryos counterstained with a DAPI nuclear marker (blue), an anti-insulin antibody marking β cells (red), and an anti-Nkx6.1 antibody marking pancreatic progenitor cells (green). Images represent the maximum-intensity z projection of several focal planes obtained in a Leica Sp5 confocal microscope using a 40× objective. Scale bars represent 20 μm. (F) Quantification of the number of insulin-expressing cells in nog2^ED301/ED301 and nog2^ED301/ED301, nog2^+/mut outcross embryos compared with controls (n ≥ 30). Error bars represent SD; ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01. (G) Quantification of the number of nkx6.1-expressing cells in nog2 ^ED301/ED301 and nog2^ED301/ED301, nog2^+/mut outcross embryos compared with controls (n ≥ 18). Error bars represent SD; ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001.

Figure 2

nog2 Genomic Regulatory Landscape Contains a Notochord-Specific Enhancer (A) Genomic landscape of nog2, represented in the University of California Santa Cruz Genomics Institute (UCSC) genome browser (Kent et al., 2002), comprising more than 100 kb. The black track represents chromatin interaction points detected by 4C-seq using the nog2 promoter as the viewpoint (red asterisk). Scale bar represents 25 kilobases. (B) Zoom-in in the locus of nog2 comprising approximately 25 kb. Representation of the ED transposon integration 2 kb downstream of nog2. Purple (top), brown (middle), and blue (bottom) tracks represent the ATAC-seq and chromatin immunoprecipitation sequencing (ChIP-seq) signal for H3K27ac and H3K4me1, respectively, at three developmental times: 24 hpf, 80% epiboly (80 epi), and dome. The black track represents the 4C-seq signal at 24 hpf using the nog2 promoter as the viewpoint. Pale blue boxes highlight selected sequences for enhancer activity assays (E1 to E3). Two replicates of 4C data are shown in Figure S3. Scale bar represents 5 kilobases. (C) Representative images of GFP reporter lines for enhancers E1 to E3 (see also Figure S1). Scale bars represent 100 μm.

Figure 3

Nog2 Diffuses from the Notochord to the Pancreatic Progenitor Domain (A) Representative confocal image of a 18 hpf zebrafish embryo injected with the nog2E3:GFPnog2 construct. Of 20 embryos, 16 (80%) showed colocalization of GFPNog2 aggregates (green) with pancreatic progenitor cells, labeled with anti-Nkx6.1 antibody (white). Details of the colocalization can be observed in the inset (white box). (B) Representative confocal image of a 18 hpf zebrafish embryo injected with nog2E3:GFP (control), in which the pattern of GFP expression (green) is restricted to the notochord. None of 18 analyzed embryos displayed colocalization of GFP aggregates with Nkx6.1-labeled cells. Embryos were counterstained with the nuclear marker DAPI (blue), and the endocrine progenitor domain is delimited by a yellow dashed line (see also Figure S2). Images represent the maximum-intensity z projection of several focal planes obtained in a Leica Sp5 confocal microscope using a 40× objective. Scale bars represent 30 μm.

Figure 4

The nog2 Notochord Enhancer Is Required for Proper Pancreas Development (A) Representation of the WT nog2E3 enhancer (above) and somatic deletions (below) generated by the injection of the Cas9 protein, together with sg1 and sg2 targeting two regions of the sequence 237 bp apart (see also Figure S3). (B) In situ hybridization of insulin in 48 hpf representative embryos, injected with the Cas9 protein, and two sgRNAs compared with control embryos, injected with only Cas9. Scale bars represent 80 μm. (C) Quantification of the insulin expression area detected by in situ hybridization in injected embryos at 48 hpf compared with controls. Error bars represent SD; ^∗∗∗∗p < 0.0001. In all cases, n ≥ 97. (D) Diagram of the nog2E3:GFPnog2 construct used to achieve notochord-specific overexpression of GFPnog2 and the respective control. (E) In situ hybridization of insulin in 48 hpf representative embryos injected with nog2E3:GFPnog2 compared with control embryos. Scale bars represent 80 μm. (F) Quantification of the insulin expression area detected by in situ hybridization in injected embryos at 48 hpf compared with controls. Error bars represent SD; ^∗∗∗p < 0.001. In all cases, n ≥ 38. (G) Representative confocal images of 48 hpf zebrafish embryos counterstained with a DAPI nuclear marker (blue), an anti-insulin antibody marking β cells (red), and an anti-Nkx6.1 antibody marking pancreatic progenitor cells (green). Images represent the maximum-intensity z projection of several focal planes obtained in a Leica Sp5 confocal microscope using a 40× objective. Scale bars represent 20 μm. (H) Quantification of the number of insulin-expressing cells in nog2E3^del1/del2 embryos compared with controls (n ≥ 19). Error bars represent SD; ^∗p < 0.05. (I) Quantification of the number of nkx6.1-expressing cells in nog2E3^del1/del2 embryos compared with controls (n ≥ 13). Error bars represent SD; ^∗∗∗p < 0.001.

Figure 5

The Expression of Nog in the Notochord Is Conserved in Vertebrates and Is Mediated by Equivalent Notochord Enhancers (A) Genomic landscape of human NOG, comprising approximately 150 kb. Black vertical lines represent SNPs associated with diabetes from the Diabetes Genetics Replication and Meta-analysis (DIAGRAM) Consortium (p < 0.02) (Morris et al., 2012; Pasquali et al., 2014). The dark blue track represents vertebrate conservation and shows multiple alignments of 100 vertebrate species and measurements of evolutionary conservation (phyloP) (Pollard et al., 2010). Dashed line boxes are putative enhancer sequences selected for enhancer assays. Sequences in pale blue shaded boxes showed reproducible expression patterns. D10 (green shaded box) showed enhancer activity in the notochord. Scale bar represents 50 kilobases. (B) Representative images of GFP reporter lines for enhancers D5, D6, D9, and D10 (see also Figure S5). Scale bars represent 100 or 200 μm. (C) Genomic landscape of the human D10 sequence, showing vertebrate (blue track) and mouse conservation (green track) (above). Genomic landscape of mouse Nog, including the conserved mouse D10 (mD10) sequence, located approximately 150 kb downstream of the promoter (see also Figures S5 and S6). Scale bars represent 500 basepairs or 50 kilobases. (D) Representative image of the GFP reporter line for the mouse enhancer mD10. Scale bar represents 100 μm.

Figure 6

Proper Pancreas Development Requires the Expression of Nog2 in the Notochord (A) Diagram of the nog2 notochord enhancer driving expression of nog2. Nog2 diffusion from the notochord, located in the midline of the embryo, counteracts the signaling of Bmp2b from the lateral plate mesoderm, allowing the proper placement and proper size of the endocrine pancreas. (B) When the activity of the nog2 notochord enhancer is disrupted, nog2 expression in the notochord is reduced. In this case, Bmp2b signaling is expanded, impairing β cell development and reducing pancreas size.