Review
Mesodermal induction of pancreatic fate commitment

https://doi.org/10.1016/j.semcdb.2018.08.008Get rights and content

Highlights

  • Pancreatic buds are specified from endoderm via inductive interactions with adjacent mesoderm.

  • Permissive signals from notochord (suppressing Hedgehog expression) render it pancreas-competent.

  • Signalling from the contiguous aortic endothelium is required for dorsal pancreatic fate commitment.

  • Morphogenesis and differentiation is governed by signals from enveloping pancreatic mesenchyme.

  • Pancreas-inductive cues are recapitulated by stem cell-based protocols for diabetic therapy.

Abstract

The pancreas is a compound gland comprised of both exocrine acinar and duct cells as well as endocrine islet cells. Most notable amongst the latter are the insulin-synthesizing β-cells, loss or dysfunction of which manifests in diabetes mellitus. All exocrine and endocrine cells derive from multipotent pancreatic progenitor cells arising from the primitive gut epithelium via inductive interactions with adjacent mesodermal tissues. Research in the last two decades has revealed the identity of many of these extrinsic cues and they include signaling molecules used in many other developmental contexts such as retinoic acid, fibroblast growth factors, and members of the TGF-β superfamily. As important as these inductive cues is the absence of other signaling molecules such as hedgehog family members. Much has been learned about the interactions of extrinsic factors with fate regulators intrinsic to the pancreatic endoderm. This new knowledge has had tremendous impact on the development of directed differentiation protocols for converting pluripotent stem cells to β-cells in vitro.

Introduction

The pancreas is a compound organ serving both exocrine and endocrine functions. The exocrine compartment comprises: 1) rosettes of sac-like acini which synthesize and secrete digestive enzymes and 2) a continuous/connected, ramified ductal tree which secretes bicarbonate and transports secretory products to the duodenum. Embedded within the exocrine tissue and dispersed throughout the pancreas are the heavily-vascularized islets of Langerhans which together comprise the endocrine compartment. Each spheroidal islet contains 100–1000 endocrine cells of five distinct sub-types. Of these, the β-cells (∼75% of islet cells) and α-cells (∼15–20% of islet cells) are most notable, synthesizing and secreting the hormones insulin and glucagon, respectively, into the bloodstream to maintain normoglycemia.

Despite their segregated function and anatomy in the mature organ, the acinar, ductal and endocrine cells share a common ontogeny, all arising from the same embryonic germ layer; the endoderm [1,2]. The pancreas and, likewise, its neighboring organs in the gut (liver, gall-bladder, duodenum and stomach), are derived from the undifferentiated gut endoderm via a sequential restriction of fate commitment (first to organ then specialized organ-specific lineages). This is achieved by inductive interactions between endoderm and adjacent tissues derived from another embryonic germ layer; the mesoderm. These interactions, and how they interact with numerous cell fate regulators intrinsic to the endoderm (e.g. transcription factors (TFs) – and intraepithelial signaling pathways), will be focused upon here. Given the ubiquity of mouse studies in the pursuit of dissecting the molecular and genetic underpinnings of the pancreatic program, pancreas ontogeny in the mouse will form the basis for this review with a few excursions into other species where pertinent.

The pancreas in the mouse is first morphologically discernible as paired dorsal and ventral thickenings of the gut endoderm at embryonic day (E)8.75-9.0 following anterior endoderm closure [[3], [4], [5]]. This is preceded however by a sequence of molecular events driving specification of the pancreatic endoderm or acquisition of the “pancreatic state”, coined the primary transition [3]. These manifest in induction of expression of the TF Pdx1 in the pancreatic domains by E8.0 (somite stages - SS - 8–10) ventrally and E8.5-E8.75 (SS10) dorsally [[6], [7], [8]] with a second TF, Ptf1a, becoming detectable by E8.5–E8.75 in a subset of dorsal and ventral Pdx1+ cells [9,10]. While Pdx1 and Ptf1a specifically demarcate the pancreatic endoderm, their expression is preceded by that of other TFs expressed in a broader endodermal domain, including Foxa2 (Hnf3β), Onecut1 (Hnf6), Hhex (Hex), Hb9, Gata4, Gata6, Hnf1β (Tcf2, vHnf1) and Sox9 [[11], [12], [13], [14], [15], [16], [17], [18]]. The early Pdx1+ columnar epithelium thickens and expands rapidly, forming well-defined dorsal and ventral pancreatic “buds” by E9.0-E9.25. The larger dorsal bud evaginates immediately posterior to the prospective stomach into the mesoderm-derived mesenchyme (embryonic connective tissue) of the adjacent dorsal mesentery overlying the duodenum, the “mesoduodenum”, immediately caudal to the stomach mesentery (the mesogastrium) [19]. Opposite the dorsal bud, the smaller ventral pancreatic bud evaginates ventrally adjacent to the hepatic endoderm into the caudal-most ventral mesentery, the ventral mesoduodenum [19]. Until around E12, both buds are largely composed of multipotent pancreatic progenitors (MPCs), defined by their expression of Pdx1, Ptf1a, Nkx6-1, Sox9, and Hnf1β and which at the population level are competent to differentiate into all three tissue types (acinar, ductal or endocrine) of the mature pancreas [1,[20], [21], [22]]. Recent clonal lineage-tracing studies have confirmed that at a single cell level, some early (E9.5) MPCs are indeed multipotent per se, while other progenitors are duct-/endocrine-bipotent or solely endocrine-committed [2]. MPC maintenance is promoted by the Notch signaling pathway mediated in part by the downstream TF Hes1. MPCs losing the influence of active Notch signaling are able to attain a high level of expression of the pro-endocrine TF Ngn3 (encoded by Neurog3) due to loss of Hes1-mediated repression. Ngn3 is both necessary and sufficient for endocrine differentiation [1,15,23,24] and high expression levels define transient, monopotent endocrine precursors which each give rise to endocrine cells [1,25]. Endocrine progenitor competency changes during development: during the primary transition, glucagon+ α-cells are formed almost exclusively with insulin+ β-cells and PP cells emerging just prior to an explosive wave of differentiation between E13.5-E16.5 known as the secondary transition; somatostatin+ δ-cells are only born after E14.5 [26]. Ngn3+ cells are first detectable in the dorsal pancreas from E8.5 and ventrally from E9.5 with glucagon+ cells first evident one day later, at E9.5 dorsally and E10.5 in the ventral bud [7], consistent with the approximate one day developmental lag of the ventral pancreas [5]. Between E10.5 and E12.5, the pancreatic buds grow parallel to the prospective duodenum and stomach within the dorsal and ventral mesoduodenum [19]. Gut rotation results in migration of the ventral pancreas and hepatopancreatic orifice dorsally toward the dorsal pancreas, culminating in their fusion by E12.5, forming the unified definitive pancreas [27]. Concurrently, multiple cycles of branching morphogenesis between E11.5-E15.5 remodel the epithelium into a complex and highly-organized tubular “plexus” via Cdc42-dependent microlumen expansion and coalescence, remodeling of which forms the ductal tree [28]. During this process, MPCs become segregated physically and commitment-wise into bipotent proximal (“trunk”) duct-/endocrine- progenitors of the plexus expressing Nkx6-1, Hnf1β, Gata6, and Sox9 and distal (“tip”) acinar-committed progenitors expressing Ptf1a and Gata4 [29]. This “proximodistal patterning” is governed via cross-repressive interactions between the pro-trunk TF Nkx6-1 and the tip-favouring TF Ptf1a with Notch signaling acting upstream to promote an Nkx6-1+ state [[30], [31], [32]]. Within the plexus niche, Notch-mediated lateral inhibition of Ngn3, modulated by EGF signaling is thought to regulate lineage segregation of trunk progenitors with those losing Notch induced Hes1 expression being able to attain an Ngn3Hi state and commit to an endocrine fate while those maintaining Hes1 expression will adopt a ductal identity [33,34]. Scattered Ngn3Hi endocrine progenitors exit the cell cycle, undergo terminal endocrine differentiation, and delaminate from the plexus epithelium [35,36]. Coalescence of the distinct endocrine cell sub-types from E16.5 onwards into polyclonal aggregates forms the nascent islets [37,38]. The developmental sequence of the mouse pancreas is outlined in Fig. 1.

Fate mapping using the fluorescent lipophilic dye DiI and whole embryo culture has shown that in mice, the dorsal and ventral pancreas arise from distinct endodermal domains: while dorsal progenitors originate from medial endoderm abutting somites 2–4 between SS2-11, the ventral pancreas derives from two fields: 1) the left and right lateral endoderm caudal to the anterior intestinal portal (AIP) by SS6 and 2) the ventral midline of the endoderm lip (VMEL) [39]. Thus, prospective dorsal and ventral pancreatic endoderm cells are adjacent to distinct mesodermal tissues and, so, exposed to different inductive milieus during pancreatic specification. While the dorsal pancreas-forming medial endoderm is initially in extended contact with the notochord followed by the dorsal aorta, prospective ventral pancreatic endoderm is exposed to the splanchnic layer of the lateral plate mesoderm and, subsequently, to signals emanating from cardiac mesoderm and the septum transversum, formed from condensation of mesenchyme within the caudal ventral mesentery at around E8.5 [19]. Both buds are also in close proximity to endothelial cells of major blood vessels [40]. All of these tissues serve as important sources of inductive and permissive cues as will be expanded upon below (Fig. 2). It is striking that despite their contrasting niches and signaling requirements, the transcriptional programs of the dorsal and ventral pancreata are so similar despite the ventral developmental lag. This is especially so given the apparent lack of “cross-talk” between the two early developing pancreatic anlagen.

Section snippets

Suppression of hedgehog expression delimits the pancreatic domains

Seminal studies two decades ago first revealed the inhibitory effect of Hedgehog signaling on pancreatic induction. Of the three mammalian Hedgehog genes encoding a family of secreted proteins, Sonic hedgehog (Shh) as well as Indian hedgehog (Ihh) are expressed throughout the early gut endoderm with the notable exception of prospective dorsal and ventral pancreatic domains. Likewise, and consistent with its induction by Shh, the gene encoding the transmembrane receptor Patched1 (Ptch1) (which

Pancreatic mesenchyme promotes evagination and expansion of committed pancreas endoderm

Once pancreas fate has been induced in the dorsal and ventral foregut endoderm, morphogenesis of buds, expansion of pancreatic progenitors and their differentiation towards endocrine and exocrine lineages is governed by signals from the enveloping pancreatic mesenchyme. Mesenchymal cells coalesce around the gut endoderm around E9.5, concomitant with evagination of the pancreatic buds. Using embryonic tissue explants, seminal studies [4,45] revealed that dissected pancreatic rudiments can

Therapeutic applications

The past two decades have seen remarkable progress in the identification of mesoderm-derived factors that regulate pancreatic development in mice. These advances have enabled the development of in vitro protocols for generation of pancreatic cell types, including insulin-producing β-cells from human pluripotent stem cells as a cell replacement therapy in the management of diabetes mellitus. Despite rapid progress, however, such β-cell-like cells are still poorly-functional compared with their

Acknowledgments

P.S. is supported by the Novo Nordisk Foundation and the Juvenile Diabetes Research Foundation International. The Novo Nordisk Foundation Center for Stem Cell Biology is supported by a Novo Nordisk Foundation grant number NNF17CC0027852. We thank our colleagues whose work this review inevitably builds upon and apologize to those whose studies were omitted for reasons of brevity.

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