Quantitative lineage analysis identifies a hepato-pancreato-biliary progenitor niche.

Studies based on single cells have revealed vast cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degrees of plasticity during organogenesis1-5. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including the liver, pancreas, gall bladder and extra-hepatic bile ducts6,7. Experimental manipulation of various developmental signals in the mouse embryo has underscored important cellular plasticity in this embryonic territory6. This is reflected in the existence of human genetic syndromes as well as congenital malformations featuring multi-organ phenotypes in liver, pancreas and gall bladder6. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary and pancreatic structures have not yet been established. Here we combine computational modelling approaches with genetic lineage tracing to accurately reconstruct the hepato-pancreato-biliary lineage tree. We show that a multipotent progenitor subpopulation persists in the pancreato-biliary organ rudiment, contributing cells not only to the pancreas and gall bladder but also to the liver. Moreover, using single-cell RNA sequencing and functional experiments we define a specialized niche that supports this subpopulation in a multipotent state for an extended time during development. Together these findings indicate sustained plasticity underlying hepato-pancreato-biliary development that might also explain the rapid expansion of the liver while attenuating pancreato-biliary growth.

Employing core regulatory circuits to define cell identity.

The interplay between extrinsic signaling and downstream gene networks controls the establishment of cell identity during development and its maintenance in adult life. Advances in next-generation sequencing and single-cell technologies have revealed additional layers of complexity in cell identity. Here, we review our current understanding of transcription factor (TF) networks as key determinants of cell identity. We discuss the concept of the core regulatory circuit as a set of TFs and interacting factors that together define the gene expression profile of the cell. We propose the core regulatory circuit as a comprehensive conceptual framework for defining cellular identity and discuss its connections to cell function in different contexts.

Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions.

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).

Engineering life in synthetic systems.

The second EMBO-EMBL Symposium 'Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture' was held virtually in March 2021, with participants from all over the world joining from the comfort of their sofas to discuss synthetic morphogenesis at large. Leading scientists from a range of disciplines, including developmental biology, physics, chemistry and computer science, covered a gamut of topics from the principles of cell and tissue organization, patterning and gene regulatory networks, to synthetic approaches for exploring evolutionary and developmental biology principles. Here, we describe some of the high points.

In-Depth Analysis of the Pancreatic Extracellular Matrix during Development for Next-Generation Tissue Engineering.

The pancreas is a complex organ consisting of differentiated cells and extracellular matrix (ECM) organized adequately to enable its endocrine and exocrine functions. Although much is known about the intrinsic factors that control pancreas development, very few studies have focused on the microenvironment surrounding pancreatic cells. This environment is composed of various cells and ECM components, which play a critical role in maintaining tissue organization and homeostasis. In this study, we applied mass spectrometry to identify and quantify the ECM composition of the developing pancreas at the embryonic (E) day 14.5 and postnatal (P) day 1 stages. Our proteomic analysis identified 160 ECM proteins that displayed a dynamic expression profile with a shift in collagens and proteoglycans. Furthermore, we used atomic force microscopy to measure the biomechanical properties and found that the pancreatic ECM was soft (≤400 Pa) with no significant change during pancreas maturation. Lastly, we optimized a decellularization protocol for P1 pancreatic tissues, incorporating a preliminary crosslinking step, which effectively preserved the 3D organization of the ECM. The resulting ECM scaffold proved suitable for recellularization studies. Our findings provide insights into the composition and biomechanics of the pancreatic embryonic and perinatal ECM, offering a foundation for future studies investigating the dynamic interactions between the ECM and pancreatic cells.

A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development.

An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the ß-cell lineage, where it plays a central role in ß-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic ß-cell differentiation and increase ß-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFß signaling led to α-cell to ß-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances ß-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and ß-cell development.

Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence.

Understanding how distinct cell types arise from multipotent progenitor cells is a major quest in stem cell biology. The liver and pancreas share many aspects of their early development and possibly originate from a common progenitor. However, how liver and pancreas cells diverge from a common endoderm progenitor population and adopt specific fates remains elusive. Using RNA sequencing (RNA-seq), we defined the molecular identity of liver and pancreas progenitors that were isolated from the mouse embryo at two time points, spanning the period when the lineage decision is made. The integration of temporal and spatial gene expression profiles unveiled mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the noncanonical Wnt pathway as a potential developmental regulator of this fate decision and capable of inducing the pancreas program in endoderm and liver cells. Our study offers an unprecedented view of gene expression programs in liver and pancreas progenitors and forms the basis for formulating lineage-reprogramming strategies to convert adult hepatic cells into pancreatic cells.

The histone methyltransferase Setd7 promotes pancreatic progenitor identity.

Cell fate specification depends on transcriptional activation driven by lineage-specific transcription factors as well as changes in chromatin organization. To date, the interplay between transcription factors and chromatin modifiers during development is not well understood. We focus here on the initiation of the pancreatic program from multipotent endodermal progenitors. Transcription factors that play key roles in regulating pancreatic progenitor state have been identified, but the chromatin regulators that help to establish and maintain pancreatic fate are less well known. Using a comparative approach, we identify a crucial role for the histone methyltransferase Setd7 in establishing pancreatic cell identity. We show that Setd7 is expressed in the prospective pancreatic endoderm of Xenopus and mouse embryos prior to Pdx1 induction. Importantly, we demonstrate that setd7 is sufficient and required for pancreatic cell fate specification in Xenopus Functional and biochemical approaches in Xenopus and mouse endoderm support that Setd7 modulates methylation marks at pancreatic regulatory regions, possibly through interaction with the transcription factor Foxa2. Together, these results demonstrate that Setd7 acts as a central component of the transcription complex initiating the pancreatic program.

Xenopus as a model system for studying pancreatic development and diabetes.

Diabetes is a chronic disease caused by the loss or dysfunction of the insulin-producing ß-cells in the pancreas. To date, much of our knowledge about ß-cells in humans comes from studying rare monogenic forms of diabetes. Importantly, the majority of mutations so far associated to monogenic diabetes are in genes that exert a regulatory role in pancreatic development and/or ß-cell function. Thus, the identification and study of novel mutations open an unprecedented window into human pancreatic development. In this review, we summarize major advances in the genetic dissection of different types of monogenic diabetes and the insights gained from a developmental perspective. We highlight future challenges to bridge the gap between the fast accumulation of genetic data through next-generation sequencing and the need of functional insights into disease mechanisms. Lastly, we discuss the relevance and advantages of studying candidate gene variants in vivo using the Xenopus as model system.

Rho signalling restriction by the RhoGAP Stard13 integrates growth and morphogenesis in the pancreas.

The development of functional organ architecture relies on coordinated morphogenesis and growth. In the developing pancreas, the branching epithelium is organised in discrete domains, delineating one specific domain of progenitor cells at the tip of the branches. The molecular mechanisms underlying the coordinated action of branching and proliferation in organ formation are largely unknown. Here, we identify the RhoGAP protein Stard13 as an essential regulator of pancreas tissue architecture in the mammalian embryo. Conditional ablation of Stard13 expression in the pancreas disrupts epithelial morphogenesis and tip-domain organisation, resulting in hampered proliferation of tip progenitors and subsequent organ hypoplasia. Stard13 acts by regulating Rho signalling spatially and temporally during pancreas development. Our findings provide new insights into the mechanisms that shape pancreatic epithelium to create a mature organ and establish a functional link between Rho-mediated control of epithelial remodelling and organ size determination, involving reciprocal interaction of actin-MAL/SRF and MAPK signalling pathways.

Glimpse into Hox and tale regulation of cell differentiation and reprogramming.

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming.

Acinar-ductal cell rearrangement drives branching morphogenesis of the murine pancreas in an IGF/PI3K-dependent manner.

During organ formation, progenitor cells need to acquire different cell identities and organize themselves into distinct structural units. How these processes are coordinated and how tissue architecture(s) is preserved despite the dramatic cell rearrangements occurring in developing organs remain unclear. Here, we identified cellular rearrangements between acinar and ductal progenitors as a mechanism to drive branching morphogenesis in the pancreas while preserving the integrity of the acinar-ductal functional unit. Using ex vivo and in vivo mouse models, we found that pancreatic ductal cells form clefts by protruding and pulling on the acinar basement membrane, which leads to acini splitting. Newly formed acini remain connected to the bifurcated branches generated by ductal cell rearrangement. Insulin growth factor (IGF)/phosphatidylinositol 3-kinase (PI3K) pathway finely regulates this process by controlling pancreatic ductal tissue fluidity, with a simultaneous impact on branching and cell fate acquisition. Together, our results explain how acinar structure multiplication and branch bifurcation are synchronized during pancreas organogenesis.

Heterozygous missense variant in GLI2 impairs human endocrine pancreas development.

Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we apply CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in two siblings with early-onset and insulin-dependent diabetes of unknown cause. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic ß-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. We establish and study a set of isogenic iPSC lines harbouring the missense variant for their ability to differentiate into pancreatic ß-like cells. Interestingly, iPSCs carrying the missense variant show altered GLI2 transcriptional activity and impaired differentiation of pancreatic progenitors into endocrine cells. RNASeq and network analyses unveil a crosstalk between Hedgehog and WNT pathways, with the dysregulation of non-canonical WNT signaling in pancreatic progenitors carrying the GLI2 missense variant. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a gene variant that may lead to diabetes.

Where do you come from; where do you go? Pluripotency, differentiation and malfunction of stem cells.

The international conference 'Stem Cells in Development and Disease' took place in September 2011 at the Max-Delbrück-Center for Molecular Medicine (MDC) in Berlin. It brought together scientists working on different types of stem cell and covered the latest findings in stem cell biology, including the genetic and epigenetic mechanisms of reprogramming, maintenance of pluripotency and differentiation.

3D multicellular systems in disease modelling: From organoids to organ-on-chip.

Cell-cell interactions underlay organ formation and function during homeostasis. Changes in communication between cells and their surrounding microenvironment are a feature of numerous human diseases, including metabolic disease and neurological disorders. In the past decade, cross-disciplinary research has been conducted to engineer novel synthetic multicellular organ systems in 3D, including organoids, assembloids, and organ-on-chip models. These model systems, composed of distinct cell types, satisfy the need for a better understanding of complex biological interactions and mechanisms underpinning diseases. In this review, we discuss the emerging field of building 3D multicellular systems and their application for modelling the cellular interactions at play in diseases. We report recent experimental and computational approaches for capturing cell-cell interactions as well as progress in bioengineering approaches for recapitulating these complexities ex vivo. Finally, we explore the value of developing such multicellular systems for modelling metabolic, intestinal, and neurological disorders as major examples of multisystemic diseases, we discuss the advantages and disadvantages of the different approaches and provide some recommendations for further advancing the field.

Understanding cell fate acquisition in stem-cell-derived pancreatic islets using single-cell multiome-inferred regulomes.

Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem-cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, serotonin-producing pre-ß cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro ß cell maturation and identify sex hormones as drivers of ß cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem-cell-derived islets and a framework for manipulating cell identities and maturity.

Functional genomics and the future of iPSCs in disease modeling.

Induced pluripotent stem cells (iPSCs) are valuable in disease modeling because of their potential to expand and differentiate into virtually any cell type and recapitulate key aspects of human biology. Functional genomics are genome-wide studies that aim to discover genotype-phenotype relationships, thereby revealing the impact of human genetic diversity on normal and pathophysiology. In this review, we make the case that human iPSCs (hiPSCs) are a powerful tool for functional genomics, since they provide an in vitro platform for the study of population genetics. We describe cutting-edge tools and strategies now available to researchers, including multi-omics technologies, advances in hiPSC culture techniques, and innovations in drug development. Functional genomics approaches based on hiPSCs hold great promise for advancing drug discovery, disease etiology, and the impact of genetic variation on human biology.

Identification and implication of tissue-enriched ligands in epithelial-endothelial crosstalk during pancreas development.

Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. BMP7 and WNT7B, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and SEMA6D, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced BMP7 limited development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of BMP7 in the intercellular communications contributing to vessel development and organization during pancreas organogenesis.