CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 µg/L, 1 µg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Crosstalk between Thyroid Hormone and Corticosteroid Signaling Targets Cell Proliferation in Xenopus tropicalis Tadpole Liver.

Thyroid hormones (TH) and glucocorticoids (GC) are involved in numerous developmental and physiological processes. The effects of individual hormones are well documented, but little is known about the joint actions of the two hormones. To decipher the crosstalk between these two hormonal pathways, we conducted a transcriptional analysis of genes regulated by TH, GC, or both hormones together in liver of Xenopus tropicalis tadpoles using RNA-Seq. Among the differentially expressed genes (DE), 70.5% were regulated by TH only, 0.87% by GC only, and 15% by crosstalk between the two hormones. Gene ontology analysis of the crosstalk-regulated genes identified terms referring to DNA replication, DNA repair, and cell-cycle regulation. Biological network analysis identified groups of genes targeted by the hormonal crosstalk and corroborated the gene ontology analysis. Specifically, we found two groups of functionally linked genes (chains) mainly composed of crosstalk-regulated hubs (highly interactive genes), and a large subnetwork centred around the crosstalk-regulated genes psmb6 and cdc7. Most of the genes in the chains are involved in cell-cycle regulation, as are psmb6 and cdc7, which regulate the G2/M transition. Thus, the biological action of these two hormonal pathways acting together in the liver targets cell-cycle regulation.

Are paedomorphs actual larvae?

Amphibians display very diverse life cycles and development can be direct, where it occurs in ovo and a juvenile hatches directly, or biphasic, where an aquatic larva hatches and later undergoes metamorphosis followed by sexual maturation. In both cases, metamorphosis, corresponds to the post embryonic transition (PETr). A third strategy, only found in Urodeles, is more complex as larvae reach sexual maturity before metamorphosis, which can become accessory. The resulting paedomorphs retain their larval characters and keep their aquatic habitat. Does it mean that paedomorphs do not undergo PETr? Recent work using high throughput technologies coupled to system biology and developmental endocrinology revisited this question and provided novel datasets indicating that a paedomorph's "larval" tissue undergoes a proper developmental transition. Together with historical data, we propose that this transition is a marker of the PETr, which would be distinct from metamorphosis. This implies that (a) complex life cycles would result from the uncoupling of PETr and metamorphosis, and (b) biphasic life cycles would be a special cases where they occur simultaneously.

DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain.

Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the methylcytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period.

ERGO: Breaking Down the Wall between Human Health and Environmental Testing of Endocrine Disrupters.

ERGO (EndocRine Guideline Optimization) is the acronym of a European Union-funded research and innovation action, that aims to break down the wall between mammalian and non-mammalian vertebrate regulatory testing of endocrine disruptors (EDs), by identifying, developing and aligning thyroid-related biomarkers and endpoints (B/E) for the linkage of effects between vertebrate classes. To achieve this, an adverse outcome pathway (AOP) network covering various modes of thyroid hormone disruption (THD) in multiple vertebrate classes will be developed. The AOP development will be based on existing and new data from in vitro and in vivo experiments with fish, amphibians and mammals, using a battery of different THDs. This will provide the scientifically plausible and evidence-based foundation for the selection of B/E and assays in lower vertebrates, predictive of human health outcomes. These assays will be prioritized for validation at OECD (Organization for Economic Cooperation and Development) level. ERGO will re-think ED testing strategies from in silico methods to in vivo testing and develop, optimize and validate existing in vivo and early life-stage OECD guidelines, as well as new in vitro protocols for THD. This strategy will reduce requirements for animal testing by preventing duplication of testing in mammals and non-mammalian vertebrates and increase the screening capacity to enable more chemicals to be tested for ED properties.

A novel stress hormone response gene in tadpoles of Xenopus tropicalis.

Previous work identified a transcribed locus, Str. 34945, induced by the frog stress hormone corticosterone (CORT) in Xenopus tropicalis tails. Because thyroid hormone had no influence on its expression, Str. 34945 was dubbed the first "CORT-only" gene known from tadpoles. Here, we examine the genomic annotation for this transcript, hormone specificity, time course of induction, tissue distribution, and developmental expression profile. The location of Str. 34945 on the X. tropicalis genome lies between the genes ush1g (Usher syndrome 1G) and fads6 (fatty acid desaturase 6). A blast search showed that it maps to the same region on the X. laevis genome, but no hits were found in the human genome. Using RNA-seq data and conventional reverse transcriptase PCR and sequencing, we show that Str. 34945 is part of the 3' untranslated region of ush1g. We find that CORT but not aldosterone or thyroid hormone treatment induces Str. 34945 in tadpole tails and that expression of Str. 34945 achieves maximal expression within 12-24 h of CORT treatment. Among tissues, Str. 34945 is induced to the highest degree in tail, with lesser induction in lungs, liver, and heart, and no induction in the brain or kidney. During natural metamorphosis, Str. 34945 expression in tails peaks at metamorphic climax. The role of ush1g in metamorphosis is not understood, but the specificity of its hormone response and its expression in tail make ush1g valuable as a marker of CORT-response gene induction independent of thyroid hormone.

Frogs model man: In vivo thyroid hormone signaling during development.

Thyroid hormone (TH) signaling comprises TH transport across cell membranes, metabolism by deiodinases, and molecular mechanisms of gene regulation. Proper TH signaling is essential for normal perinatal development, most notably for neurogenesis and fetal growth. Knowledge of perinatal TH endocrinology needs improvement to provide better treatments for premature infants and endocrine diseases during gestation and to counteract effects of endocrine disrupting chemicals. Studies in amphibians have provided major insights to understand in vivo mechanisms of TH signaling. The frog model boasts dramatic TH-dependent changes directly observable in free-living tadpoles with precise and easy experimental control of the TH response at developmental stages comparable to fetal stages in mammals. The hormones, their receptors, molecular mechanisms, and developmental roles of TH signaling are conserved to a high degree in humans and amphibians, such that with respect to developmental TH signaling "frogs are just little people that hop." The frog model is exceptionally illustrative of fundamental molecular mechanisms of in vivo TH action involving TH receptors, transcriptional cofactors, and chromatin remodeling. This review highlights the current need, recent successes, and future prospects using amphibians as a model to elucidate molecular mechanisms and functional roles of TH signaling during post-embryonic development.

Mechanisms of thyroid hormone receptor action during development: lessons from amphibian studies.

BACKGROUND: Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. However, the in vivo mechanism of TR action remains poorly explored. SCOPE OF REVIEW: Frog metamorphosis is controlled by TH and mimics the postembryonic period in mammals when high levels of TH are also required. We review here some of the findings on the developmental functions of TH and TR and the associated mechanisms obtained from this model system. MAJOR CONCLUSION: A dual function model for TR in Anuran development was proposed over a decade ago. That is, unliganded TR recruits corepressors to TH response genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes. Subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model. Specifically, it has been shown that unliganded TR recruits histone deacetylase containing corepressor complexes during larval stages to control metamorphic timing, while liganded TR recruits multiple histone modifying and chromatin remodeling coactivator complexes during metamorphosis. These complexes can alter chromatin structure via nucleosome position alterations or eviction and histone modifications to contribute to the recruitment of transcriptional machinery and gene activation. GENERAL SIGNIFICANCE: The molecular mechanisms of TR action in vivo as revealed from studies on amphibian metamorphosis are very likely applicable to mammalian development as well. These findings provide a new perspective for understanding the diverse effects of TH in normal physiology and diseases caused by TH dysfunction. This article is part of a Special Issue entitled Thyroid hormone signalling.

Overlapping action of T3 and T4 during Xenopus laevis development.

Thyroid hormones are involved in many biological processes such as neurogenesis, metabolism, and development. However, compounds called endocrine disruptors can alter thyroid hormone signaling and induce unwanted effects on human and ecosystems health. Regulatory tests have been developed to detect these compounds but need to be significantly improved by proposing novel endpoints and key events. The Xenopus Eleutheroembryonic Thyroid Assay (XETA, OECD test guideline no. 248) is one such test. It is based on Xenopus laevis tadpoles, a particularly sensitive model system for studying the physiology and disruption of thyroid hormone signaling: amphibian metamorphosis is a spectacular (thus easy to monitor) life cycle transition governed by thyroid hormones. With a long-term objective of providing novel molecular markers under XETA settings, we propose first to describe the differential effects of thyroid hormones on gene expression, which, surprisingly, are not known. After thyroid hormones exposure (T3 or T4), whole tadpole RNAs were subjected to transcriptomic analysis. By using standard approaches coupled to system biology, we found similar effects of the two thyroid hormones. They impact the cell cycle and promote the expression of genes involves in cell proliferation. At the level of the whole tadpole, the immune system is also a prime target of thyroid hormone action.

Lithium and endocrine disruption: A concern for human health?

Lithium is a key medication for the treatment of psychiatric disorders and is also used in various industrial applications (including battery production and recycling). Here, we review published data on the endocrine-disrupting potential of lithium, with a particular focus on the thyroid hormone system. To this end, we used PubMed and Scopus databases to search for, select and review primary research addressing human and animal health endpoints during or after lithium exposure at non-teratogenic doses. Given the key role of thyroid hormones in neurodevelopmental processes, we focused at studies of the neural effects of developmental exposure to lithium in humans and animals. Our results show that lithium meets the World Health Organization's definition of a thyroid hormone system disruptor - particularly when used at therapeutic doses. When combined with knowledge of adverse outcome pathways linking molecular initiating events targeting thyroid function and neurodevelopmental outcomes, the neurodevelopmental data reported in animal experiments prompt us to suggest that lithium influences neurodevelopment. However, we cannot rule out the involvement of additional modes of action (i.e. unrelated to the thyroid hormone system) in the described neural effects. Given the increasing use of lithium salts in new technologies, attention must be paid to this emerging pollutant - particularly with regard to its potential effects at environmental doses on the thyroid hormone system and potential consequences on the developing nervous system.

Xenopus tropicalis osteoblast-specific open chromatin regions reveal promoters and enhancers involved in human skeletal phenotypes and shed light on early vertebrate evolution.

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Evolution of the vertebrate bone matrix: an expression analysis of the network forming collagen paralogues in amphibian osteoblasts.

The emergence of vertebrates is closely associated to the evolution of mineralized bone tissue. However, the molecular basis underlying the origin and subsequent diversification of the skeletal mineralized matrix is still poorly understood. One efficient way to tackle this issue is to compare the expression, between vertebrate species, of osteoblastic genes coding for bone matrix proteins. In this work, we have focused on the evolution of the network forming collagen family which contains the Col8a1, Col8a2, and Col10a1 genes. Both phylogeny and synteny reveal that these three paralogues are vertebrate-specific and derive from two independent duplications in the vertebrate lineage. To shed light on the evolution of this family, we have analyzed the osteoblastic expression of the network forming collagens in endochondral and intramembraneous skeletal elements of the amphibian Xenopus tropicalis. Remarkably, we find that amphibian osteoblasts express Col10a1, a gene strongly expressed in osteoblasts in actinopterygians but not in amniotes. In addition, while Col8a1 is known to be robustly expressed in mammalian osteoblasts, the expression levels of its amphibian orthologue are dramatically reduced. Our work reveals that while a skeletal expression of network forming collagen members is widespread throughout vertebrates, osteoblasts from divergent vertebrate lineages express different combinations of network forming collagen paralogues.

Tetrabromobisphenol A effects on differentiating mouse embryonic stem cells reveals unexpected impact on immune system.

Tetrabromobisphenol A (TBBPA) is a potent flame retardant used in numerous appliances and a major pollutant in households and ecosystems. In vertebrates, it was shown to affect neurodevelopment, the hypothalamic-pituitary-gonadal axis and thyroid signaling, but its toxicity and modes of actions are still a matter of debate. The molecular phenotype resulting from exposure to TBBPA is only poorly described, especially at the level of transcriptome reprogramming, which further limits our understanding of its molecular toxicity. In this work, we combined functional genomics and system biology to provide a system-wide description of the transcriptomic alterations induced by TBBPA acting on differentiating mESCs, and provide potential new toxicity markers. We found that TBBPA-induced transcriptome reprogramming affect a large collection of genes loosely connected within the network of biological pathways, indicating widespread interferences on biological processes. We also found two hotspots of action: at the level of neuronal differentiation markers, and surprisingly, at the level of immune system functions, which has been largely overlooked until now. This effect is particularly strong, as terminal differentiation markers of both myeloid and lymphoid lineages are strongly reduced: the membrane T cell receptor (Cd79a, Cd79b), interleukin seven receptor (Il7r), macrophages cytokine receptor (Csf1r), monocyte chemokine receptor (Ccr2). Also, the high affinity IgE receptor (Fcer1g), a key mediator of allergic reactions, is strongly induced. Thus, the molecular imbalance induce by TBBPA may be stronger than initially realized.

Thyroid and Corticosteroid Signaling in Amphibian Metamorphosis.

In multicellular organisms, development is based in part on the integration of communication systems. Two neuroendocrine axes, the hypothalamic-pituitary-thyroid and the hypothalamic-pituitary-adrenal/interrenal axes, are central players in orchestrating body morphogenesis. In all vertebrates, the hypothalamic-pituitary-thyroid axis controls thyroid hormone production and release, whereas the hypothalamic-pituitary-adrenal/interrenal axis regulates the production and release of corticosteroids. One of the most salient effects of thyroid hormones and corticosteroids in post-embryonic developmental processes is their critical role in metamorphosis in anuran amphibians. Metamorphosis involves modifications to the morphological and biochemical characteristics of all larval tissues to enable the transition from one life stage to the next life stage that coincides with an ecological niche switch. This transition in amphibians is an example of a widespread phenomenon among vertebrates, where thyroid hormones and corticosteroids coordinate a post-embryonic developmental transition. The review addresses the functions and interactions of thyroid hormone and corticosteroid signaling in amphibian development (metamorphosis) as well as the developmental roles of these two pathways in vertebrate evolution.

Transcriptome and Methylome Analysis Reveal Complex Cross-Talks between Thyroid Hormone and Glucocorticoid Signaling at Xenopus Metamorphosis.

BACKGROUND: Most work in endocrinology focus on the action of a single hormone, and very little on the cross-talks between two hormones. Here we characterize the nature of interactions between thyroid hormone and glucocorticoid signaling during Xenopus tropicalis metamorphosis. METHODS: We used functional genomics to derive genome wide profiles of methylated DNA and measured changes of gene expression after hormonal treatments of a highly responsive tissue, tailfin. Clustering classified the data into four types of biological responses, and biological networks were modeled by system biology. RESULTS: We found that gene expression is mostly regulated by either T3 or CORT, or their additive effect when they both regulate the same genes. A small but non-negligible fraction of genes (12%) displayed non-trivial regulations indicative of complex interactions between the signaling pathways. Strikingly, DNA methylation changes display the opposite and are dominated by cross-talks. CONCLUSION: Cross-talks between thyroid hormones and glucocorticoids are more complex than initially envisioned and are not limited to the simple addition of their individual effects, a statement that can be summarized with the pseudo-equation: TH ∙ GC > TH + GC. DNA methylation changes are highly dynamic and buffered from genome expression.

The invention of aldosterone, how the past resurfaces in pediatric endocrinology.

Sodium and water homeostasis are drastically modified at birth, in mammals, by the transition from aquatic life to terrestrial life. Accumulating evidence during the past ten years underscores the central role for the mineralocorticoid signaling pathway, in the fine regulation of this equilibrium, at this critical period of development. Interestingly, regarding evolution, while the mineralocorticoid receptor is expressed in fish, the appearance of its related ligand, aldosterone, coincides with terrestrial life, as it is first detected in lungfish and amphibian. Thus, aldosterone is likely one of the main hormones regulating the transition from an aquatic environment to an air environment. This review will focus on the different actors of the mineralocorticoid signaling pathway from aldosterone secretion in the adrenal gland, to mineralocorticoid receptor expression in the kidney, summarizing their regulation and roles throughout fetal and neonatal development, in the light of evolution.

Two families of Xenopus tropicalis skeletal genes display well-conserved expression patterns with mammals in spite of their highly divergent regulatory regions.

The origin of bone and cartilage, and their subsequent diversification in specific vertebrate lineages, is intimately linked to the precise transcriptional control of genes involved in matrix mineralization. It is not yet clear, however, to which extent the osteoblasts, osteocytes, and chondrocytes of each of the major vertebrate groups express similar sets of genes. In this study we have focused on the evolution of two independent families of genes that code for extracellular matrix components of the skeleton and that include secreted protein, acidic, cysteine-rich (SPARC), bone sialoprotein (BSP) and dentin matrix protein 1 (DMP1) paralogues, and the osteocalcin (OC) and matrix gla protein (MGP) paralogues. Analyzing developing Xenopus tropicalis skeletal elements, we show that the expression patterns of these genes are well conserved with mammals. The fact that only a few osteoblasts express DMP1, while only some osteocytes express SPARC and BSP, reveals a significant degree of molecular heterogeneity for these two populations of X. tropicalis cells, similarly to what has been described in mouse. Although the cis-regulatory modules (CRM) of the mammalian OC, DMP1, and BSP orthologs have been functionally characterized, we found no evidence of sequence similarity between these regions and the X. tropicalis genome. Furthermore, these regulatory elements evolve rapidly, as they are only poorly conserved between human and rodents. Therefore, the SPARC/DMP1/BSP and the OC/MGP families provide a good paradigm to study how transcriptional output can be maintained in skeletal cells despite extensive sequence divergence of CRM.