Daniel O. Kechele

Multiple signaling pathways and transcription factors (TFs) establish organ domains in the developing gastrointestinal tract. How these are integrated into spatial-temporal networks to regulate organogenesis and how disruptions to those networks lead to congenital syndromes remain poorly understood. Using human pluripotent stem cell cultures and Xenopus embryos, we demonstrate that retinoic acid (RA) from the lateral plate mesoderm directly activates expression of the TF rfx6 in posterior foregut endoderm. Rfx6 subsequently promotes posterior foregut identity while suppressing Wnt-dependent hindgut and Bmp-dependent pharyngeal fates through direct and indirect mechanisms. Rfx6 can directly activate the expression of several key foregut TFs (onecut1 and pdx1) and Wnt antagonists (sfrp2/5) while indirectly restricting expression of Wnt and Bmp ligands. Rfx6 also directly suppresses transcription of the Wnt-dependent hindgut TF cdx2 and the Bmp-dependent pharyngeal TFs nkx2-5 and nkx2-6. Thus, Rfx6 acts at multiple levels to integrate RA, Wnt and Bmp activity into a network with lineage-promoting TFs to control gut tube patterning. These results provide insight into the molecular basis of Mitchell–Riley Syndrome congenital anomalies, which are caused by RFX6 mutations.

The goal of engineering increasingly complex human organoid models is to more accurately model human organogenesis and disease. Recently, human antral gastric organoids (hAGOs) were engineered to contain splanchnic mesenchyme (SM) and enteric neural crest cells (NCCs), resulting in functional three germ layer gastric organoids. To determine the robustness of hAGOs and how additional germ layers impact development, we benchmarked these hAGOs with the developing human stomach. Human AGOs in vitro were most similar to 7 week fetal stomach with the epithelium comprising primarily mucous precursors. The SM and NCCs added to hAGOs formed fetal gastric-like mesenchymal or neuroglial precursors, respectively. Incorporation of SM and NCCs did not drastically alter the cellular diversity of the epithelium in vitro. Following transplantation, hAGOs with SM matured into tissue more like the 2nd trimester stomach. Bioinformatic inference confirmed known signaling crosstalk between germ layers and identified new signaling candidates that may regulate tissue assembly. Together, three germ layer hAGOs faithfully model the multilayer complexity of the fetal stomach at the single-cell transcriptomic level and provide insight into human stomach development.

Summary: To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm. The cellular composition in multilineage organoids resembles that of human fetal organs. Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics. Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.

Pancreas and gastrointestinal (GI) tract development is guided by several transcription factors, including pancreatic and duodenal homeobox gene-1 (PDX1). While it is well known that PDX1is essential for pancreatic development and beta cell function, its role in GI development and function is not fully understood. We identified a patient with a homozygous point mutation in PDX1 causing total loss of PDX1 protein resulting in pancreatic agenesis. We differentiated induced pluripotent stem cells from the patient into human antral gastric (HAGO) and intestinal (HIO) organoids as a novel diagnostic approach to identify new GI pathologies. Immunofluorescence staining revealed that portions of the PDX1-null HAGOs lost the gastric marker claudin18 and increased intestinal villin1 expression, signifying that regions of the antrum are converting to an intestinal phenotype. Similarly, PDX1-null HIOs lost the intestinal marker cadherin17 and increased claudin18, indicating that regions of the intestine are undergoing gastric metaplasia. Examination of functional markers revealed an increase in acid producing parietal cells, complete lack of gastrin producing endocrine cells in HAGOs, and an increase in gastric surface mucin in HIOs. These in vitro results were further validated using endoscopy biopsies from two patients with PDX1 mutations, which showed a similar disruption in gastric and intestinal identity. CRISPR/Cas9-mediated correction of the PDX1 mutation reversed the observed PDX1-null metaplasic phenotypes in HAGOs and HIOs and also allowed for the successful generation of pancreatic tissue indicating unambiguously that the PDX1 mutation is responsible for these pathologies. Based on these findings, these PDX1-null patients will have additional screening to monitor the progression of metaplasia into gastric and intestinal cancer. This study further demonstrates the utility of organoids in improving patient diagnoses and treatment. Disclosure M. Krishnamurthy: None. T.R. Broda: None. D.O. Kechele: None. X. Zhang: None. J.J. Palermo: None. M.H. Collins: None. I.H. Thomas: None. A. Heider: None. A. Dauber: None. J. Wells: None. Funding American Diabetes Association (1-19-PDF-036 to M.K.)

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein–coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin–CLR–RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure–function insights of this signaling axis for human physiology.

Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor–like receptor ( Calcrl ), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrl ^fl/fl /Prox1-CreER ^T2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrl ^fl/fl /Prox1-CreER ^T2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl /adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the Apc^Min mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling–induced proliferation, particularly during regeneration and adenoma formation.

RAMPs (receptor activity–modifying proteins) serve as oligomeric modulators for numerous G-protein–coupled receptors, yet elucidating the physiological relevance of these interactions remains complex. Ramp2 null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor signaling. We aimed to genetically rescue the Ramp2^−/− lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with family B G-protein–coupled receptors in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2^−/− mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2^−/−Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking calcitonin receptor-like receptor. Yet, the hearts of Ramp2^−/−Tg animals displayed dysregulation of family B G-protein–coupled receptors, including parathyroid hormone and glucagon receptors, as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional G-protein–coupled receptor pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/calcitonin receptor-like receptor signaling, in which future studies could elucidate novel and pharmacologically tractable pathways for treating cardiovascular diseases.