En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

RFX6 regulates human intestinal patterning and function upstream of PDX1.

The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.

Normal Table of Xenopus development: a new graphical resource.

Normal tables of development are essential for studies of embryogenesis, serving as an important resource for model organisms, including the frog Xenopus laevis. Xenopus has long been used to study developmental and cell biology, and is an increasingly important model for human birth defects and disease, genomics, proteomics and toxicology. Scientists utilize Nieuwkoop and Faber's classic 'Normal Table of Xenopus laevis (Daudin)' and accompanying illustrations to enable experimental reproducibility and reuse the illustrations in new publications and teaching. However, it is no longer possible to obtain permission for these copyrighted illustrations. We present 133 new, high-quality illustrations of X. laevis development from fertilization to metamorphosis, with additional views that were not available in the original collection. All the images are available on Xenbase, the Xenopus knowledgebase (http://www.xenbase.org/entry/zahn.do), for download and reuse under an attributable, non-commercial creative commons license. Additionally, we have compiled a 'Landmarks Table' of key morphological features and marker gene expression that can be used to distinguish stages quickly and reliably (https://www.xenbase.org/entry/landmarks-table.do). This new open-access resource will facilitate Xenopus research and teaching in the decades to come.

Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary.

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.

Localized Prox1 Regulates Aortic Valve Endothelial Cell Diversity and Extracellular Matrix Stratification in Mice.

BACKGROUND: Specialized valve endothelial cell (VEC) populations are localized oriented to blood flow in developing aortic and mitral valves, but their roles in valve development and disease are unknown. In the aortic valve (AoV), a population of VECs on the fibrosa side expresses the transcription factor Prox1 together with genes found in lymphatic ECs. In this study, we examine Prox1's role in regulating a lymphatic-like gene network and promoting VEC diversity required for the development of the stratified trilaminar extracellular matrix (ECM) of murine AoV leaflets. METHODS: To determine whether disruption of Prox1 localization affects heart valve development, we generated mice (NFATc1enCre Prox1 gain-of-function) in which Prox1 is overexpressed on the ventricularis side of the AoV beginning in embryonic development. To identify potential targets of Prox1, we performed cleavage under targets and release using nuclease on wild-type and NFATc1enCre Prox1 gain-of-function AoVs with validation by colocalization in vivo using RNA in situ hybridization in NFATc1enCre Prox1 gain-of-function AoVs. Natural induction of Prox1 and target gene expression was evaluated in myxomatous AoVs in a mouse model of Marfan syndrome (Fbn1C1039G/+). RESULTS: The overexpression of Prox1 is sufficient to cause enlargement of AoVs by postnatal day (P)0, as well as a decrease in ventricularis-specific gene expression and disorganized interstitial ECM layers at P7. We identified potential targets of Prox1 known to play roles in lymphatic ECs including Flt1, Efnb2, Egfl7, and Cx37. Ectopic Prox1 colocalized with induced Flt1, Efnb2, and Cx37 expression in NFATc1enCre Prox1 gain-of-function AoVs. Moreover, in Marfan syndrome myxomatous AoVs, endogenous Prox1, and its identified targets, were ectopically induced in ventricularis side VECs. CONCLUSIONS: Our results support a role for Prox1 in localized lymphatic-like gene expression on the fibrosa side of the AoV. Furthermore, localized VEC specialization is required for development of the stratified trilaminar ECM critical for AoV function and is dysregulated in congenitally malformed valves.

Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers.

BACKGROUND: Proteins of the TGFß family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFß signaling. RESULTS: In this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers. CONCLUSIONS: Ultimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.

Vertebrate endoderm development and organ formation.

The endoderm germ layer contributes to the respiratory and gastrointestinal tracts and to all of their associated organs. Over the past decade, studies in vertebrate model organisms, including frog, fish, chick, and mouse, have greatly enhanced our understanding of the molecular basis of endoderm organ development. We review this progress with a focus on early stages of endoderm organogenesis including endoderm formation, gut tube morphogenesis and patterning, and organ specification. Lastly, we discuss how developmental mechanisms that regulate endoderm organogenesis are used to direct differentiation of embryonic stem cells into specific adult cell types, which function to alleviate disease symptoms in animal models.

The Xenopus phenotype ontology: bridging model organism phenotype data to human health and development.

BACKGROUND: Ontologies of precisely defined, controlled vocabularies are essential to curate the results of biological experiments such that the data are machine searchable, can be computationally analyzed, and are interoperable across the biomedical research continuum. There is also an increasing need for methods to interrelate phenotypic data easily and accurately from experiments in animal models with human development and disease. RESULTS: Here we present the Xenopus phenotype ontology (XPO) to annotate phenotypic data from experiments in Xenopus, one of the major vertebrate model organisms used to study gene function in development and disease. The XPO implements design patterns from the Unified Phenotype Ontology (uPheno), and the principles outlined by the Open Biological and Biomedical Ontologies (OBO Foundry) to maximize interoperability with other species and facilitate ongoing ontology management. Constructed in Web Ontology Language (OWL) the XPO combines the existing uPheno library of ontology design patterns with additional terms from the Xenopus Anatomy Ontology (XAO), the Phenotype and Trait Ontology (PATO) and the Gene Ontology (GO). The integration of these different ontologies into the XPO enables rich phenotypic curation, whilst the uPheno bridging axioms allows phenotypic data from Xenopus experiments to be related to phenotype data from other model organisms and human disease. Moreover, the simple post-composed uPheno design patterns facilitate ongoing XPO development as the generation of new terms and classes of terms can be substantially automated. CONCLUSIONS: The XPO serves as an example of current best practices to help overcome many of the inherent challenges in harmonizing phenotype data between different species. The XPO currently consists of approximately 22,000 terms and is being used to curate phenotypes by Xenbase, the Xenopus Model Organism Knowledgebase, forming a standardized corpus of genotype-phenotype data that can be directly related to other uPheno compliant resources.

Developmental basis of trachea-esophageal birth defects.

Trachea-esophageal defects (TEDs), including esophageal atresia (EA), tracheoesophageal fistula (TEF), and laryngeal-tracheoesophageal clefts (LTEC), are a spectrum of life-threatening congenital anomalies in which the trachea and esophagus do not form properly. Up until recently, the developmental basis of these conditions and how the trachea and esophagus arise from a common fetal foregut was poorly understood. However, with significant advances in human genetics, organoids, and animal models, and integrating single cell genomics with high resolution imaging, we are revealing the molecular and cellular mechanisms that orchestrate tracheoesophageal morphogenesis and how disruption in these processes leads to birth defects. Here we review the current understanding of the genetic and developmental basis of TEDs. We suggest future opportunities for integrating developmental mechanisms elucidated from animals and organoids with human genetics and clinical data to gain insight into the genotype-phenotype basis of these heterogeneous birth defects. Finally, we envision how this will enhance diagnosis, improve treatment, and perhaps one day, lead to new tissue replacement therapy.

The homeodomain transcription factor Ventx2 regulates respiratory progenitor cell number and differentiation timing during Xenopus lung development.

Ventx2 is an Antennapedia superfamily/NK-like subclass homeodomain transcription factor best known for its roles in the regulation of early dorsoventral patterning during Xenopus gastrulation and in the maintenance of neural crest multipotency. In this work we characterize the spatiotemporal expression pattern of ventx2 in progenitor cells of the Xenopus respiratory system epithelium. We find that ventx2 is directly induced by BMP signaling in the ventral foregut prior to nkx2-1, the earliest epithelial marker of the respiratory lineage. Functional studies demonstrate that Ventx2 regulates the number of Nkx2-1/Sox9+ respiratory progenitor cells induced during foregut development, the timing and level of surfactant protein gene expression, and proper tracheal-esophageal separation. Our data suggest that Ventx2 regulates the balance of respiratory progenitor cell expansion and differentiation. While the ventx gene family has been lost from the mouse genome during evolution, humans have retained a ventx2-like gene (VENTX). Finally, we discuss how our findings might suggest a possible function of VENTX in human respiratory progenitor cells.

Xenbase: deep integration of GEO & SRA RNA-seq and ChIP-seq data in a model organism database.

Xenbase (www.xenbase.org) is a knowledge base for researchers and biomedical scientists that employ the amphibian Xenopus as a model organism in biomedical research to gain a deeper understanding of developmental and disease processes. Through expert curation and automated data provisioning from various sources Xenbase strives to integrate the body of knowledge on Xenopus genomics and biology together with the visualization of biologically significant interactions. Most current studies utilize next generation sequencing (NGS) but until now the results of different experiments were difficult to compare and not integrated with other Xenbase content. Xenbase has developed a suite of tools, interfaces and data processing pipelines that transforms NCBI Gene Expression Omnibus (GEO) NGS content into deeply integrated gene expression and chromatin data, mapping all aligned reads to the most recent genome builds. This content can be queried and visualized via multiple tools and also provides the basis for future automated 'gene expression as a phenotype' and gene regulatory network analyses.

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus.

The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.

Genomic integration of Wnt/ß-catenin and BMP/Smad1 signaling coordinates foregut and hindgut transcriptional programs.

Digestive system development is orchestrated by combinatorial signaling interactions between endoderm and mesoderm, but how these signals are interpreted in the genome is poorly understood. Here we identified the transcriptomes of Xenopus foregut and hindgut progenitors, which are conserved with mammals. Using RNA-seq and ChIP-seq we show that BMP/Smad1 regulates dorsal-ventral gene expression in both the endoderm and mesoderm, whereas Wnt/ß-catenin acts as a genome-wide toggle between foregut and hindgut programs. Unexpectedly, ß-catenin and Smad1 binding were associated with both transcriptional activation and repression, with Wnt-repressed genes often lacking canonical Tcf DNA binding motifs, suggesting a novel mode of direct repression. Combinatorial Wnt and BMP signaling was mediated by Smad1 and ß-catenin co-occupying hundreds of cis-regulatory DNA elements, and by a crosstalk whereby Wnt negatively regulates BMP ligand expression in the foregut. These results extend our understanding of gastrointestinal organogenesis and of how Wnt and BMP might coordinate genomic responses in other contexts.

Xenbase: a genomic, epigenomic and transcriptomic model organism database.

Xenbase (www.xenbase.org) is an online resource for researchers utilizing Xenopus laevis and Xenopus tropicalis, and for biomedical scientists seeking access to data generated with these model systems. Content is aggregated from a variety of external resources and also generated by in-house curation of scientific literature and bioinformatic analyses. Over the past two years many new types of content have been added along with new tools and functionalities to reflect the impact of high-throughput sequencing. These include new genomes for both supported species (each with chromosome scale assemblies), new genome annotations, genome segmentation, dynamic and interactive visualization for RNA-Seq data, updated ChIP-Seq mapping, GO terms, protein interaction data, ORFeome support, and improved connectivity to other biomedical and bioinformatic resources.

Novel vectors for functional interrogation of Xenopus ORFeome coding sequences.

The current Xenopus ORFeome contains ~10,250 validated, full-length cDNA sequences without stop codons from Xenopus laevis and ~3,970 from Xenopus tropicalis cloned into Gateway-compatible entry vectors. To increase the utility of the ORFeome, we have constructed the Gateway-compatible destination vectors pDXTP and pDXTR, which in combination can control the spatial and temporal expression of any open reading frame (ORF). pDXTP receives a promoter/enhancer of interest, which controls the spatial expression of a doxycycline-inducible transcription factor rtTA. pDXTR receives an ORF of interest, which is controlled by a tetracycline response element enabling temporal control of ORF expression via rtTA activation by simple addition of doxycycline to the rearing water at any desired time point. These vectors can be integrated into the genome via well-established microinjection-based SceI, tol2, or phi-C31 transgenesis procedures and contain fluorescence reporters to confirm transgene integration. Cell-autonomous verification of ORF expression occurs via red nuclear fluorescence due to an mCherry-histone H2B fusion protein that is cleaved from the ORF during translation. Function of all essential features of pDXTP and pDXTR has been experimentally validated. pDXTP and pDXTR provide flexible molecular cloning and transgenesis options to accomplish tissue-specific inducible control of ORF expression in transgenic Xenopus.

Timing is everything: Reiterative Wnt, BMP and RA signaling regulate developmental competence during endoderm organogenesis.

A small number of signaling pathways are used repeatedly during organogenesis, and they can have drastically different effects on the same population of cells depending on the embryonic stage. How cellular competence changes over developmental time is not well understood. Here we used Xenopus, mouse, and human pluripotent stem cells to investigate how the temporal sequence of Wnt, BMP, and retinoic acid (RA) signals regulates endoderm developmental competence and organ induction, focusing on respiratory fate. While Nkx2-1+ lung fate is not induced until late somitogenesis stages, here we show that lung competence is restricted by the gastrula stage as a result of Wnt and BMP-dependent anterior-posterior (A-P) patterning. These early Wnt and BMP signals make posterior endoderm refractory to subsequent RA/Wnt/BMP-dependent lung induction. We further mapped how RA modulates the response to Wnt and BMP in a temporal specific manner. In the gastrula RA promotes posterior identity, however in early somite stages of development RA regulates respiratory versus pharyngeal potential in anterior endoderm and midgut versus hindgut potential in posterior endoderm. Together our data suggest a dynamic and conserved response of vertebrate endoderm during organogenesis, wherein early Wnt/BMP/RA impacts how cells respond to later Wnt/BMP/RA signals, illustrating how reiterative combinatorial signaling can regulate both developmental competence and subsequent fate specification.

FOXF1 transcription factor promotes lung morphogenesis by inducing cellular proliferation in fetal lung mesenchyme.

Organogenesis is regulated by mesenchymal-epithelial signaling events that induce expression of cell-type specific transcription factors critical for cellular proliferation, differentiation and appropriate tissue patterning. While mesenchymal transcription factors play a key role in mesenchymal-epithelial interactions, transcriptional networks in septum transversum and splanchnic mesenchyme remain poorly characterized. Forkhead Box F1 (FOXF1) transcription factor is expressed in mesenchymal cell lineages; however, its role in organogenesis remains uncharacterized due to early embryonic lethality of Foxf1-/- mice. In the present study, we generated mesenchyme-specific Foxf1 knockout mice (Dermo1-Cre Foxf1-/-) and demonstrated that FOXF1 is required for development of respiratory, cardiovascular and gastrointestinal organ systems. Deletion of Foxf1 from mesenchyme caused embryonic lethality in the middle of gestation due to multiple developmental defects in the heart, lung, liver and esophagus. Deletion of Foxf1 inhibited mesenchyme proliferation and delayed branching lung morphogenesis. Gene expression profiling of micro-dissected distal lung mesenchyme and ChIP sequencing of fetal lung tissue identified multiple target genes activated by FOXF1, including Wnt2, Wnt11, Wnt5A and Hoxb7. FOXF1 decreased expression of the Wnt inhibitor Wif1 through direct transcriptional repression. Furthermore, using a global Foxf1 knockout mouse line (Foxf1-/-) we demonstrated that FOXF1-deficiency disrupts the formation of the lung bud in foregut tissue explants. Finally, deletion of Foxf1 from smooth muscle cell lineage (smMHC-Cre Foxf1-/-) caused hyper-extension of esophagus and trachea, loss of tracheal and esophageal muscle, mispatterning of esophageal epithelium and decreased proliferation of smooth muscle cells. Altogether, FOXF1 promotes lung morphogenesis by regulating mesenchymal-epithelial signaling and stimulating cellular proliferation in fetal lung mesenchyme.

Xenopus genomic data and browser resources.

The two species of Xenopus most commonly used in biomedical research are the diploid Xenopus (Silurana) tropicalis and the tetraploid Xenopus laevis. The X. tropicalis genome sequence has been available since 2010 and this year the X. laevis, genome from two distinct genetic backgrounds has been published. Multiple genome assemblies available for both species and transcriptomic and epigenetic data sets are growing rapidly, all of which are available from a variety of web resources. This review describes the contents of these resources, how to locate and download genomic data, and also how to view and manipulate these data on various public genome browsers, with an emphasis on Xenbase, the Xenopus model organism database.

Osr1 functions downstream of Hedgehog pathway to regulate foregut development.

During early fetal development, paracrine Hedgehog (HH) ligands secreted from the foregut epithelium activate Gli transcription factors in the surrounding mesenchyme to coordinate formation of the respiratory system, digestive track and the cardiovascular network. Although disruptions to this process can lead to devastating congenital defects, the underlying mechanisms and downstream targets, are poorly understood. We show that the zinc finger transcription factor Osr1 is a novel HH target as Osr1 expression in the foregut mesenchyme depends on HH signaling and the effector of HH pathway Gli3 binds to a conserved genomic loci near Osr1 promoter region. Molecular analysis of mouse germline Osr1 mutants reveals multiple functions of Osr1 during foregut development. Osr1 mutants exhibit fewer lung progenitors in the ventral foregut. Osr is then required for the proper branching of the primary lung buds, with mutants exhibiting miss-located lung lobes. Finally, Osr1 is essential for proper mesenchymal differentiation including pulmonary arteries, esophageal and tracheal smooth muscle as well as tracheal cartilage rings. Tissue specific conditional knockouts in combination with lineage tracing indicate that Osr1 is required cell autonomously in the foregut mesenchyme. We conclude that Osr1 is a novel downstream target of HH pathway, required for lung specification, branching morphogenesis and foregut mesenchymal differentiation.