Endoderm Development Research Articles

CellRank 2: unified fate mapping in multiview single-cell data

Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies.

Integrative physiology and transcriptome reveal salt-tolerance differences between two licorice species: Ion transport, Casparian strip formation and flavonoids biosynthesis

BackgroundGlycyrrhiza inflata Bat. and Glycyrrhiza uralensis Fisch. are both original plants of ‘Gan Cao’ in the Chinese Pharmacopoeia, and G. uralensis is currently the mainstream variety of licorice and has a long history of use in traditional Chinese medicine. Both of these species have shown some degree of tolerance to salinity, G. inflata exhibits higher salt tolerance than G. uralensis and can grow on saline meadow soils and crusty saline soils. However, the regulatory mechanism responsible for the differences in salt tolerance between different licorice species is unclear. Due to land area-related limitations, the excavation and cultivation of licorice varieties in saline-alkaline areas that both exhibit tolerance to salt and contain highly efficient active substances are needed. The systematic identification of the key genes and pathways associated with the differences in salt tolerance between these two licorice species will be beneficial for cultivating high-quality salt-tolerant licorice G. uralensis plant varieties and for the long-term development of the licorice industry. In this research, the differences in growth response indicators, ion accumulation, and transcription expression between the two licorice species were analyzed.ResultsThis research included a comprehensive comparison of growth response indicators, including biomass, malondialdehyde (MDA) levels, and total flavonoids content, between two distinct licorice species and an analysis of their ion content and transcriptome expression. In contrast to the result found for G. uralensis, the salt treatment of G. inflata ensured the stable accumulation of biomass and total flavonoids at 0.5 d, 15 d, and 30 d and the restriction of Na+ to the roots while allowing for more K+ and Ca2+ accumulation. Notably, despite the increase in the Na+ concentration in the roots, the MDA concentration remained low. Transcriptome analysis revealed that the regulatory effects of growth and ion transport on the two licorice species were strongly correlated with the following pathways and relevant DEGs: the TCA cycle, the pentose phosphate pathway, and the photosynthetic carbon fixation pathway involved in carbon metabolism; Casparian strip formation (lignin oxidation and translocation, suberin formation) in response to Na+; K+ and Ca2+ translocation, organic solute synthesis (arginine, polyamines, GABA) in response to osmotic stresses; and the biosynthesis of the nonenzymatic antioxidants carotenoids and flavonoids in response to antioxidant stress. Furthermore, the differential expression of the DEGs related to ABA signaling in hormone transduction and the regulation of transcription factors such as the HSF and GRAS families may be associated with the remarkable salt tolerance of G. inflata.ConclusionCompared with G. uralensis, G. inflata exhibits greater salt tolerance, which is primarily attributable to factors related to carbon metabolism, endodermal barrier formation and development, K+ and Ca2+ transport, biosynthesis of carotenoids and flavonoids, and regulation of signal transduction pathways and salt-responsive transcription factors. The formation of the Casparian strip, especially the transport and oxidation of lignin precursors, is likely the primary reason for the markedly higher amount of Na+ in the roots of G. inflata than in those of G. uralensis. The tendency of G. inflata to maintain low MDA levels in its roots under such conditions is closely related to the biosynthesis of flavonoids and carotenoids and the maintenance of the osmotic balance in roots by the absorption of more K+ and Ca2+ to meet growth needs. These findings may provide new insights for developing and cultivating G. uralensis plant species selected for cultivation in saline environments or soils managed through agronomic practices that involve the use of water with a high salt content.

New insights into mesoderm and endoderm development, and the nature of the onychophoran blastopore

BackgroundEarly during onychophoran development and prior to the formation of the germ band, a posterior tissue thickening forms the posterior pit. Anterior to this thickening forms a groove, the embryonic slit, that marks the anterior–posterior orientation of the developing embryo. This slit is by some authors considered the blastopore, and thus the origin of the endoderm, while others argue that the posterior pit represents the blastopore. This controversy is of evolutionary significance because if the slit represents the blastopore, then this would support the amphistomy hypothesis that suggests that a slit-like blastopore in the bilaterian ancestor evolved into protostomy and deuterostomy.ResultsIn this paper, we summarize our current knowledge about endoderm and mesoderm development in onychophorans and provide additional data on early endoderm- and mesoderm-determining marker genes such as Blimp, Mox, and the T-box genes.ConclusionWe come to the conclusion that the endoderm of onychophorans forms prior to the development of the embryonic slit, and thus that the slit is not the primary origin of the endoderm. It is thus unlikely that the embryonic slit represents the blastopore. We suggest instead that the posterior pit indeed represents the lips of the blastopore, and that the embryonic slit (and surrounding tissue) represents a morphologically superficial archenteron-like structure. We conclude further that both endoderm and mesoderm development are under control of conserved gene regulatory networks, and that many of the features found in arthropods including the model Drosophila melanogaster are likely derived.

ScRNA-seq analysis of cells comprising the amphioxus notochord

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway.

Normal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs). The 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated. A highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2. Our results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.

Do differentially expressed genes explain high myopia in congenital stationary night blindness?

Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signalling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON‐bipolar cell signal transmission defect and is also associated with high myopia. The purpose of this work was to identify the molecular cause of myopia present in cCSNB. A whole transcriptome sequencing (RNA‐seq) approach was performed using retina from three mouse lines with cCSNB, Gpr179−/−, Lrit3−/− and Grm6−/− and the expression data compared to data of age‐matched wild‐type littermates (n = 5). DEGs with fold changes of at least 1.2, P‐values of ≤0.01, an expression value of at least 5 transcripts per million reads and appearing in at least two cCSNB mouse lines were investigated in detail. A meta‐analysis was performed by a) comparing our data with published transcriptome data from purified retinal cells and single cell RNA‐Seq data, b) pathway analyses, c) myopia databases and publications concerning their role in normal vision. Several of the DEGs were validated by RT‐qPCR experiments and analysed by western blot and immunolocalization studies. More than 50 DEGs were found in at least two cCSNB mouse lines. Expression of those was found in different retinal cell types. The difference in expression was independent of the number of nuclei present in wild‐type and mutant inner retina. Pathway analysis revealed that mitogen‐activated protein kinase pathways, synaptic signalling, G protein‐coupled receptor ligand binding pathways, and proteins implicated in eye, endoderm and connective tissue development were affected in cCSNB. More than half of the genes were already associated with myopia. Our study reveals DEGs in all retinal cell types of cCSNB models, previously associated with myopia and novel candidates. These studies combined with pathway analyses provide the basis for the development of pharmacological and optical myopia therapies.

Human RFX6 regulates endoderm patterning at the primitive gut tube stage.

Transcriptional factor RFX6 is known to be a causal gene of Mitchell-Riley syndrome (MRS), an autosomal recessive neonatal diabetes associated with pancreatic hypoplasia and intestinal atresia/malformation. The morphological defects are limited to posterior foregut and mid-hindgut endodermal lineages and do not occur in the anterior foregut lineage; the mechanism remains to be fully elucidated. In this study, we generated RFX6+/eGFP heterozygous knockin and RFX6eGFP/eGFP homozygous knockin/knockout human-induced pluripotent stem cell (hiPSC) lines and performed in vitro endoderm differentiation to clarify the role of RFX6 in early endoderm development. RFX6 expression was found to surge at the primitive gut tube (PGT) stage in comparison with that in the undifferentiated or definitive endoderm stage. At the PGT stage, the expression of PDX1 and CDX2, posterior foregut and mid-hindgut master regulators, respectively, was decreased by the RFX6 deficit. PDX1+ and CDX2+ cells were mostly green fluorescent protein (GFP)+ in RFX6+/eGFP hiPSCs, but their cell number was markedly decreased in RFX6eGFP/eGFP hiPSCs. The expression of SOX2, an anterior foregut marker, was not affected by the RFX6 deficit. In addition, we found a putative RFX6-binding X-box motif using cap analysis of gene expression-seq and the motif-containing sequences in the enhancer regions of PDX1 and CDX2 bound to RFX6 in vitro. Thus, RFX6 regulates the ParaHox genes PDX1 and CDX2 but does not affect SOX2 in early endodermal differentiation, suggesting that defects in early stage endoderm patterning account for the morphological pathology of MRS.

MCPIP1 functions as a safeguard of early embryonic development

Monocyte chemoattractant protein-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase that has been described as a key negative modulator of inflammation. MCPIP1 also controls numerous tumor-related processes, such as proliferation, apoptosis and differentiation. In this study, we utilized a zebrafish model to investigate the role of Mcpip1 during embryogenic development. Our results demonstrated that during embryogenesis, the expression of the zc3h12a gene encoding Mcpip1 undergoes dynamic changes. Its transcript levels gradually increase from the 2-cell stage to the spherical stage and then decrease rapidly. We further found that ectopic overexpression of wild-type Mcpip1 but not the catalytically inactive mutant form resulted in an embryonic lethal phenotype in zebrafish embryos (24 hpf). At the molecular level, transcriptomic profiling revealed extensive changes in the expression of genes encoding proteins important in the endoplasmic reticulum stress response and in protein folding as well as involved in the formation of primary germ layer, mesendoderm and endoderm development, heart morphogenesis and cell migration. Altogether, our results demonstrate that the expression of zc3h12a must be tightly controlled during the first cell divisions of zebrafish embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper embryo development.

Loss of TAZ after YAP deletion severely impairs foregut development and worsens cholestatic hepatocellular injury.

We previously showed that loss of yes-associated protein 1 (YAP) in early liver development (YAPKO) leads to an Alagille syndrome-like phenotype, with failure of intrahepatic bile duct development, severe cholestasis, and chronic hepatocyte adaptations to reduce liver injury. TAZ, a paralog of YAP, was significantly upregulated in YAPKO hepatocytes and interacted with TEA domain family member (TEAD) transcription factors, suggesting possible compensatory activity. We deleted both Yap1 and Wwtr1 (which encodes TAZ) during early liver development using the Foxa3 promoter to drive Cre expression, similar to YAPKO mice, resulting in YAP/TAZ double knockout (DKO) and YAPKO with TAZ heterozygosity (YAPKO TAZHET). We evaluated these mice using immunohistochemistry, serum biochemistry, bile acid profiling, and RNA sequencing. DKO mice were embryonic lethal, but their livers were similar to YAPKO, suggesting an extrahepatic cause of death. Male YAPKO TAZHET mice were also embryonic lethal, with insufficient samples to determine the cause. However, YAPKO TAZHET females survived and were phenotypically similar to YAPKO mice, with increased bile acid hydrophilicity and similar global gene expression adaptations but worsened the hepatocellular injury. TAZ heterozygosity in YAPKO impacted the expression of canonical YAP targets Ctgf and Cyr61, and we found changes in pathways regulating cell division and inflammatory signaling correlating with an increase in hepatocyte cell death, cell cycling, and macrophage recruitment. YAP loss (with or without TAZ loss) aborts biliary development. YAP and TAZ play a codependent critical role in foregut endoderm development outside the liver, but they are not essential for hepatocyte development. TAZ heterozygosity in YAPKO livers increased cell cycling and inflammatory signaling in the setting of chronic injury, highlighting genes that are especially sensitive to TAZ regulation.

Interleukin-6 stimulates in vitro development of late-stage ovine embryos

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72–192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0–192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8–16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ± 1.52%) compared to the control (21.1 ± 1.11%), and 5 ng/ml (25.9 ± 2.98%) or 25 ng/ml (16.5 ± 0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ± 1.57%) and blastocyst formation (14.5 ± 1.28%) compared to the control group (65.8 ± 1.35% and 21.5 ± 0.97%, respectively). In 8–16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

The Haematopoietically-expressed homeobox transcription factor: roles in development, physiology and disease.

The Haematopoietically expressed homeobox transcription factor (Hhex) is a transcriptional repressor that is of fundamental importance across species, as evident by its evolutionary conservation spanning fish, amphibians, birds, mice and humans. Indeed, Hhex maintains its vital functions throughout the lifespan of the organism, beginning in the oocyte, through fundamental stages of embryogenesis in the foregut endoderm. The endodermal development driven by Hhex gives rise to endocrine organs such as the pancreas in a process which is likely linked to its role as a risk factor in diabetes and pancreatic disorders. Hhex is also required for the normal development of the bile duct and liver, the latter also importantly being the initial site of haematopoiesis. These haematopoietic origins are governed by Hhex, leading to its crucial later roles in definitive haematopoietic stem cell (HSC) self-renewal, lymphopoiesis and haematological malignancy. Hhex is also necessary for the developing forebrain and thyroid gland, with this reliance on Hhex evident in its role in endocrine disorders later in life including a potential role in Alzheimer's disease. Thus, the roles of Hhex in embryological development throughout evolution appear to be linked to its later roles in a variety of disease processes.

Response of Prolyl 4 Hydroxylases, Arabinogalactan Proteins and Homogalacturonans in Four Olive Cultivars under Long-Term Salinity Stress in Relation to Physiological and Morphological Changes.

Olive (Olea europeae L.) salinity stress induces responses at morphological, physiological and molecular levels, affecting plant productivity. Four olive cultivars with differential tolerance to salt were grown under saline conditions in long barrels for regular root growth to mimic field conditions. Arvanitolia and Lefkolia were previously reported as tolerant to salinity, and Koroneiki and Gaidourelia were characterized as sensitive, exhibiting a decrease in leaf length and leaf area index after 90 days of salinity. Prolyl 4-hydroxylases (P4Hs) hydroxylate cell wall glycoproteins such as arabinogalactan proteins (AGPs). The expression patterns of P4Hs and AGPs under saline conditions showed cultivar-dependent differences in leaves and roots. In the tolerant cultivars, no changes in OeP4H and OeAGP mRNAs were observed, while in the sensitive cultivars, the majority of OeP4Hs and OeAGPs were upregulated in leaves. Immunodetection showed that the AGP signal intensity and the cortical cell size, shape and intercellular spaces under saline conditions were similar to the control in Arvanitolia, while in Koroneiki, a weak AGP signal was associated with irregular cells and intercellular spaces, leading to aerenchyma formation after 45 days of NaCl treatment. Moreover, the acceleration of endodermal development and the formation of exodermal and cortical cells with thickened cell walls were observed, and an overall decrease in the abundance of cell wall homogalacturonans was detected in salt-treated roots. In conclusion, Arvanitolia and Lefkolia exhibited the highest adaptive capacity to salinity, indicating that their use as rootstocks might provide increased tolerance to irrigation with saline water.

RBM47 regulates intestinal injury and tumorigenesis by modifying proliferation, oxidative response, and inflammatory pathways.

RNA-binding protein 47 (RBM47) is required for embryonic endoderm development, but a role in adult intestine is unknown. We studied intestine-specific Rbm47-knockout mice (Rbm47-IKO) following intestinal injury and made crosses into ApcMin/+ mice to examine alterations in intestinal proliferation, response to injury, and tumorigenesis. We also interrogated human colorectal polyps and colon carcinoma tissue. Rbm47-IKO mice exhibited increased proliferation and abnormal villus morphology and cellularity, with corresponding changes in Rbm47-IKO organoids. Rbm47-IKO mice adapted to radiation injury and were protected against chemical-induced colitis, with Rbm47-IKO intestine showing upregulation of antioxidant and Wnt signaling pathways as well as stem cell and developmental genes. Furthermore, Rbm47-IKO mice were protected against colitis-associated cancer. By contrast, aged Rbm47-IKO mice developed spontaneous polyposis, and Rbm47-IKO ApcMin/+ mice manifested an increased intestinal polyp burden. RBM47 mRNA was decreased in human colorectal cancer versus paired normal tissue, along with alternative splicing of tight junction protein 1 mRNA. Public databases revealed stage-specific reduction in RBM47 expression in colorectal cancer associated independently with decreased overall survival. These findings implicate RBM47 as a cell-intrinsic modifier of intestinal growth, inflammatory, and tumorigenic pathways.

Abstract 1352: SOX17 plays a critical role in immune evasion of colorectal cancer

Abstract Colorectal cancers (CRCs) are a leading cause of cancer-related death. Most CRCs are immune cold, and do not respond to checkpoint blockade therapy except for microsatellite-high CRCs harboring high mutational burdens. Deciphering the mechanism by which CRC cells evade immune surveillance has the potential to dramatically improve the prognosis of CRC patients. During tumor evolution of CRCs, epigenetic changes play critical roles in addition to accumulation of genetic mutations. However, distinct mutational patterns and patient backgrounds render it challenging to distinguish the driver epigenetic alterations from passenger epigenetic alterations induced by gene mutations or other environmental factors. Identification of the driver epigenetic alterations may provide novel mechanistic insights into colon cancer biology including how they evade immune surveillance. Here, we utilized the colon cancer organoid orthotopic transplantation approach to establish colon cancer organoids from different stages of the tumors, and performed comprehensive epigenomic and transcriptomic analyses to understand the epigenetic alterations during tumor evolution. We found that in vivo environment induces epigenetic alterations that converge on SOX17, a transcription factor that is required for endoderm development. SOX17 is re-expressed in colon cancers in vivo, but not in the in vitro organoid culture, and reprograms tumor cell fate with fetal intestinal gene expressions. Importantly, SOX17 knockout leads to tumor rejection in immunocompetent mice, but not in immunodeficient mice, by turning immune cold tumors into hot tumors with robust intratumoral infiltration of activated CD8+ T cells. Mechanistically, SOX17 directly downregulates Ifngr1 expression and mitigates MHC-I expression to evade CD8+ T cell-mediated tumor cell killing. Together, our result reveals that SOX17 is a master transcription factor that induces the in vivo epigenetic reprograming of tumors, which contributes to the immune evasion of colon cancers. Citation Format: Norihiro Goto, Saori Goto, Peter Westcott, Shinya Imada, Judith Agudo, Omer Yilmaz. SOX17 plays a critical role in immune evasion of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1352.

Congenital hyperinsulinism and panhypopituitarism: a rare combination.

Neonatal hypoglycemia is a serious condition that can have a major impact on the growing neonatal brain. The differential diagnosis of neonatal hypoglycemia is broad and includes hyperinsulinism as well as panhypopituitarism. The FOXA2 gene has been involved in the development of the pancreas as well as the pituitary gland. Six cases have been reported thus far with FOXA2 mutations presenting with variable degrees of hypopituitarism, and only two patients had permanent hyperinsulinism; other cases have been reported with microdeletions in 20p11, the location that encompasses FOXA2, and those patients presented with a wider phenotype. A full-term female infant presented with severe hypoglycemia. Critical sampling showed an insulin of 1 mIU/mL, suppressed beta-hydroxybutyric acids, and suppressed free fatty acids. Blood glucose responded to glucagon administration. Growth hormone (GH) stimulation test later showed undetectable GH in all samples, and cortisol failed to respond appropriately to stimulation. Gonadotropins were undetectable at 1 month of life, and MRI showed ectopic posterior pituitary, interrupted stalk, hypoplastic anterior pituitary, cavum septum pellucidum, and diminutive appearance of optic nerves. Whole-exome sequencing revealed a likely pathogenic de novo c.604 T>C, p.Tyr202His FOXA2 mutation. We expand the known phenotype on FOXA2 mutations and report a likely pathogenic, novel mutation associated with hyperinsulinism and panhypopituitarism. FOXA2 has been shown to play an important role in the neuroectodermal and endodermal development. FOXA2 mutation may lead to the rare combination of hyperinsulinism and panhypopituitarism. Patients reported so far all responded well to diazoxide. Dysmorphology may be subtle, and liver functions should be monitored.

Development's Inaugural Outstanding Paper Prize.

Development's Inaugural Outstanding Paper Prize.

Plpp3, a novel regulator of pluripotency exit and endodermal differentiation of mouse embryonic stem cells.

In recent decades the study of the actions of bioactive lipids such as Lysophosphatidic Acid (LPA) and Sphingosine-1-Phosphate (S1P) has importantly increased since they are involved in regulating many processes, including self-renewal of embryonic stem cells, embryo development and cancer. The Phospholipid Phosphatase type 3 (PLPP3) has been shown key player in regulating the balance of these lipids, and in consequence their signaling. Different lines of evidence suggest PLPP3 could play a role in endoderm development, to approach this hypothesis we use mouse embryonic stem cells (ESC) as a model to study Plpp3 function in self-renewal and the transition towards differentiation. We found that lack of PLPP3 mainly affects endoderm formation during suspension embryoid bodies differentiation. PLPP3-deficient ESC strongly decreases the amount of FOXA2 expressing cells and fail to properly downregulate the expression of pluripotency factors when subjected to an endoderm directed differentiation protocol. Impaired endoderm differentiation correlated with a transient reduction in nuclear localization of YAP1. These phenotypes were rescued by transiently restoring the expression of catalytically active hPLPP3. In conclusion, PLPP3 plays a role in downregulating pluripotency-associated factors and in endodermal differentiation. PLPP3 regulates proper lipid/YAP1 signaling required for endodermal differentiation.

Shedding light on myopia by studying complete congenital stationary night blindness

AbstractPurpose: Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signalling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON‐bipolar cell signal transmission defect and is also associated with high myopia. The purpose of this work was to identify the molecular cause of myopia present in cCSNB.Methods: A whole transcriptome sequencing (RNA‐seq) approach was performed using retina from three mouse lines with cCSNB, Gpr179−/−, Lrit3−/− and Grm6−/− and the expression data compared to data of age‐matched wild‐type littermates (n = 5). DEGs with fold changes of at least 1.2, p‐values of ≤0.01, an expression value of at least 5 transcripts per million reads and appearing in at least two cCSNB mouse lines were investigated in detail. A meta‐analysis was performed by (a) comparing our data with published transcriptome data from purified retinal cells and single cell RNA‐Seq data, (b) pathway analyses, (c) myopia databases and publications concerning their role in normal vision. Several of the DEGs were validated by RT‐qPCR experiments and analysed by western blot and immunolocalization studies.Results: More than 50 DEGs were found in at least two cCSNB mouse lines. Expression of those was found in different retinal cell types. The difference in expression was independent of the number of nuclei present in wild‐type and mutant inner retina. Pathway analysis revealed that mitogen‐activated protein kinase pathways, synaptic signalling, G protein‐coupled receptor ligand binding pathways, and proteins implicated in eye, endoderm and connective tissue development were affected in cCSNB. More than half of the genes were already associated with myopia.Conclusions: Our study reveals DEGs in all retinal cell types of cCSNB models, previously associated with myopia and novel candidates. These studies combined with pathway analyses provide the basis for the development of pharmacological and optical myopia therapies.

Species-specific rewiring of definitive endoderm developmental gene activation via endogenous retroviruses through TET1-mediated demethylation

Transposable elements (TEs) are the major sources of lineage-specific genomic innovation and comprise nearly half of the human genome, but most of their functions remain unclear. Here, we identify that a series of endogenous retroviruses (ERVs), a TE subclass, regulate the transcriptome at the definitive endoderm stage with invitro differentiation model from human embryonic stem cell. Notably, these ERVs perform as enhancers containing binding sites for critical transcription factors for endoderm lineage specification. Genome-wide methylation analysis shows most of these ERVs are derepressed by TET1-mediated DNA demethylation. LTR6B, a representative definitive endoderm activating ERV, contains binding sites for FOXA2 and GATA4 and governs the primate-specific expression of its neighboring developmental genes such as ERBB4 in definitive endoderm. Together, our study proposes evidence that recently evolved ERVs represent potent de novo developmental regulatory elements, which, in turn, fine-tune species-specific transcriptomes during endoderm and embryonic development.

Abstract A003: Developmental parallels in pancreatic cancer subtypes revealed by single cell transcriptomics

Abstract Recent research into the transcriptional subtypes in pancreatic ductal adenocarcinoma (PDA) has demonstrated their importance in predicting survival and therapeutic response. The Basal-like subtype is enriched in metastases and generally predicts poorer prognosis, while the Classical subtype is defined by gene expression reflective of pancreatic progenitors. The two-subtype scheme can be further subdivided into five subtypes that help better reflect the vast transcriptional heterogeneity found in PDA tumors. Basal-like A tumors fare worse than Basal-like B tumors. KRAS wildtype tumors are enriched in Classical B, which have the best prognosis, while Classical A is more reflective of the two-subtype Classical program. Hybrid tumors, generally a mix of Basal-like B and Classical A, are intermediate in prognosis between the Classical and Basal-like subtypes. Despite the importance of the subtypes, we have yet to fully understand how subtype programs within single cells of a tumor are intrinsically regulated, and how they contribute to tumor-level transcriptional observations. To investigate, we generated and leveraged a single cell RNA sequencing dataset enriched for malignant tumor cells spanning both primary and metastatic disease. Along with the richly annotated COMPASS dataset featuring bulk RNA sequencing of over 300 laser captured samples, we developed a single sample classifier utilizing gene pairs for assigning Basal-like A, Basal-like B, Hybrid, Classical A or Classical B to bulk tumors as well as individual cells, allowing interrogation of the subtypes across modalities. We find that the bulk subtype of the tumor is generally reflected in the dominant cell subtype. However, there are frequently cells of other subtypes within individual tumors, and substantial differences in cell subtype composition across tumors of the same subtype. In addition, we find that the proportion of Basal-like cells is a significant risk prognosticator. Investigating deeper into the transcriptional drivers of the subtypes reveals differences in expression of programs linked to endodermal development. Transcription factors such as FOXC1 and IRX3 generally related to foregut development are enriched in Basal-like A, forming a gradient of high expression to lower expression from Basal-like A, Basal-like B, Hybrid, Classical A to Classical B. Interestingly, midgut transcription factors such as CDX2 and ELF3 are expressed in the reverse order. Integrating data from single cell endoderm RNA sequencing further supports this finding, suggesting that the subtypes may arise from tumor cells exploiting pathways normally expressed early in development. This work provides a stepping stone to better understanding the mechanisms behind transcriptional subtypes in PDA. Citation Format: Sabrina Ge, Gun Ho Jang, Michelle Chan-Seng-Yue, Eugenia Flores Figueroa, Karen Ng, Stephanie Ramotar, Anna Dodd, Grainne M. O'Kane, Julie M. Wilson, Jennifer J. Knox, Sandra E. Fischer, Steven Gallinger, Faiyaz Notta. Developmental parallels in pancreatic cancer subtypes revealed by single cell transcriptomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A003.