The pancreas plays a critical role in regulating blood glucose levels by secreting glucagon when blood glucose is low and insulin when blood glucose is high. Disruption of glucose homeostasis leads to diabetes, a disease that is increasingly prevalent worldwide. Although most cases of diabetes are classified as either “type 1” or “type 2”, rare instances occur in which a developmental failure leads to pancreas agenesis (PA). Those born with PA do not have a pancreas and subsequently develop neonatal diabetes along with impaired pancreatic exocrine function. Most commonly, PA is caused by heterozygous mutations in the GATA6 gene, but the mechanism by which these mutations lead to PA remain largely unknown.
Siddharth Kishore, a graduate of the Developmental, Stem Cell, and Regenerative Biology subprogram, focused his thesis work on understanding the role of GATA6 in PA development, and recently published his findings in Cell Stem Cell. As his model for this study, Sid employed human pluripotent stem cells (PSCs). PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) give rise to all cell types and allow the study of human development and disease. As an added bonus, PSCs are easily manipulated using genome editing technologies such as CRISPR/Cas9, making them valuable for studying the role of specific genes in development and disease.
Sid, who completed his thesis work in Dr. Paul Gadue’s lab at CHOP, utilized an iPSC line derived from a PA patient with a four base pair duplication in one allele of GATA6 (iPS+/mut), with the ultimate goal of understanding how this particular mutation affects pancreas development. The mutation was corrected using CRISPR/Cas9 technology to generate a wild- type isogenic control line (iPS+/+). To validate his results, Sid introduced the same four base pair duplication into a second genetic background, the Mel1 ESC line (Mel+/mut) and utilized the unedited Mel1 ESC line as a wildtype isogenic control (Mel+/+).
Once the lines were generated, Sid differentiated the stem cells into pancreatic progenitors (PP). As expected, both iPS+/mut and Mel+/mut differentiated less efficiently to the PP stage than their wildtype counterparts (iPS+/+ and Mel+/+, respectively). Interestingly, the corrected iPS+/+ line generated the same amount of PP cells as the mutant Mel+/mut. This pattern correlated with GATA6 expression: iPS+/+ and Mel+/mut PP cells had similar GATA6 levels while iPS+/mut cells had even lower GATA6 levels. Several other pancreatic markers showed a similar pattern, suggesting that the GATA6 mutation causes a defect in pancreas development.
Graphical abstract from Dr. Kishore’s work on the identification of novel SNPs that affect pan- creatic growth and development. Kishore (2020) Cell Stem Cell.
Interestingly, Sid also observed that PP cells from both the iPS+/mut and Mel+/mut lines had increased expression of SOX2 and IRX2, two genes involved in stomach development. Moreover, the levels of SOX2 and IRX2 were significantly higher in the iPS+/mut line than in the Mel+/ mut line. These findings suggest that in addition to the known duplication in GATA6, this iPSC line may harbor another genetic mutation that also impacts pancreas development.
To identify this second mutation, Sid explored non-coding regulatory regions and discovered a single nucleotide polymorphism (SNP) downstream of the GATA6 gene. The iPSC line was homozygous for the minor allele A at this SNP, and the Mel1 ESC line was homozygous for the major allele G. This SNP was not linked to the mutation in the GATA6 coding region. He examined sequencing data available from additional PA patients and found that they also carry the minor allele A. Interestingly, when the minor allele was on the allele opposite of the GATA6 mutation (i.e. in trans), there was an even greater risk of PA.
To examine the effect of this SNP on PP cell generation, Sid introduced the minor allele A into the Mel1 ESC line. He saw that wildtype ESCs with the introduced minor allele (Mel+/+ | A/A) had lower GATA6 levels and produced fewer PP cells than Mel+/+ cells with the major allele. ESCs with both the introduced GATA6 mutation and minor allele (Mel+/mut | A/A) had even lower GATA6 levels and generated even fewer PP cells than the Mel1+/+ | A/A line. Additionally, Mel+/+ | A/A PP cells displayed in- creased expression of the stomach-related genes SOX2 and IRX2 when compared to Mel+/+ PP cells; in Mel+/mut | A/A cells, this increase was even higher. Together, these results suggest that this particular mutation in GATA6, along with the SNP in the regulatory region, may cause a defect in pancreas development. Furthermore, this developmental defect may be due to a switch in cell fate from pancreas to stomach, as seen by the increased expression of stomach-related genes in the mutant pancreatic progenitor cells.
Finally, to determine the functional impact of the SNP, Sid looked for transcription factor binding domains present in this genomic region. He found that the G>A change at this SNP may potentially disrupt a RORα binding site. RORα is a transcription factor previously suggested to function during pancreas development. Sid examined the ability of RORα to bind this site and found that RORα bound less efficiently in the cell lines with the introduced minor allele (Mel+/+ | A/A and Mel+/mut | A/A). He next added a RORα inhibitor during differentiation and observed reduced GATA6 levels and deceased PP cell yield in lines with the major allele; no effect was seen in lines carrying the minor allele. Sid’s findings suggest that RORα binds to this regulatory region of GATA6 only when the major allele is present, and in doing so, influences pancreas development. “I think it was really cool to find a non-coding genetic modifier for a rare disease using stem cells, and then test that hypothesis in a patient population. The fact that we could use genome editing to then dig deeper into the mechanisms was the icing on the cake!” Sid reflects.
In total, Sid’s work identified a novel SNP in the non-coding regulatory region of GATA6 and demonstrated that the minor allele of this SNP, in conjunction with a mutation in the GATA6 coding region, leads to PA. Many PA patients exhibit mutations in GATA6, but the mutations have variable penetrance. Sid determined that many PA patients carry the minor allele SNP in trans with the GATA6 mutation, explaining the varying degrees of severity of the disease. He hopes that expanding his observations to a wider cohort of patient pedigrees can move the field “to a point where [it can be incorporated] into genetic testing for family planning.”
In the future, Sid would like to identify direct targets of RORα to better understand its role in pancreas development, and to study the role of GATA6 in regulating cell fates. “I find it very interesting that GATA6 almost acts like a morphogen, in that levels of protein decide between [different] cell fates in the developing foregut,” Sid says. The increased expression of stomach-related genes in GATA6 mutants suggests that GATA6 is important for maintaining pancreas cell fate, but the mechanism of GATA6 function remains unclear. Sid’s work gave insight into the biology of PA, and opened up exciting questions to be explored in further studies to enhance the overall understanding of pancreas development.
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