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The mammalian gut is the largest endocrine organ. Dozens of hormones secreted by enteroendocrine cells regulate a variety of physiological functions of the gut but also of the pancreas and brain. Here, we examined the role of the helix-loop-helix transcription factor ID2 during the differentiation of intestinal stem cells along the enteroendocrine lineage.To assess the functions of ID2 in the adult mouse small intestine, we used single-cell RNA sequencing, genetically modified mice, and organoid assays.We found that in the adult intestinal epithelium Id2 is predominantly expressed in enterochromaffin and peptidergic enteroendocrine cells. Consistently, the loss of Id2 leads to the reduction of Chromogranin A-positive enteroendocrine cells. In contrast, the numbers of tuft cells are increased in Id2 mutant small intestine. Moreover, ablation of Id2 elevates the numbers of SerotoninID2 plays an important role in cell fate decisions in the adult small intestine. First, ID2 is essential for establishing a differentiation gradient for enterochromaffin and X-cells along the anterior-posterior axis of the gut. Next, ID2 is necessary for the differentiation of N-cells thus ensuring a differentiation gradient along the crypt-villi axis. Finally, ID2 suppresses the commitment of secretory intestinal epithelial progenitors towards tuft cell lineage and thus controls host immune response to commensal and parasitic microbiota.

The gastrointestinal tract is a functionally and anatomically segmented organ that is colonized by microbial communities from birth. While the genetics of mouse gut development is increasingly understood, how nutritional factors and the commensal gut microbiota act in concert to shape tissue organization and morphology of this rapidly renewing organ remains enigmatic. Here, we provide an overview of embryonic mouse gut development, with a focus on the intestinal vasculature and the enteric nervous system. We review how nutrition and the gut microbiota affect the adaptation of cellular and morphologic properties of the intestine, and how these processes are interconnected with innate immunity. Furthermore, we discuss how nutritional and microbial factors impact the renewal and differentiation of the epithelial lineage, influence the adaptation of capillary networks organized in villus structures, and shape the enteric nervous system and the intestinal smooth muscle layers. Intriguingly, the anatomy of the gut shows remarkable flexibility to nutritional and microbial challenges in the adult organism.

The adult intestinal stem cells (ISCs) are transcriptionally heterogeneous. As the mechanisms governing their developmental specification are still poorly understood, whether this heterogeneity reflects an early determination of distinct cellular sub-types with potentially distinct physiological functions remains an open question. We investigate the cellular heterogeneity within the mouse embryonic midgut epithelium at the molecular and functional levels. Cell fate mapping analysis revealed that multiple early embryonic epithelial progenitors give rise to Lgr5+ ISCs. The origin of the molecularly distinct early precursors along the anterior-posterior axis defines the transcriptional signature of embryonic Lgr5+ ISC progenitors. We further show that the early epithelial progenitors have different capacity to generate Lgr5+ ISC progenitors and Axin2+ early precursors display the highest potential.

The identity of embryonic gastric epithelial progenitors is unknown. We used single-cell RNA sequencing, genetic lineage tracing and organoid assays to assess whether Axin2 and Lgr5 expressing cells are gastric progenitors in the developing mouse stomach. We show that Axin2+ cells represent a transient population of embryonic epithelial cells in the forestomach. Lgr5+ cells generate both glandular corpus and squamous forestomach organoids ex vivo. Only Lgr5+ progenitors give rise to zymogenic cells in culture. Modulating the activity of the WNT, BMP and Notch pathways in vivo and ex vivo, we found that WNTs are essential for the maintenance of Lgr5+ epithelial cells. Notch prevents differentiation of the embryonic epithelial cells along all secretory lineages and hence ensures their maintenance. While WNTs promote differentiation of the embryonic progenitors along zymogenic cell lineage, BMPs enhance their differentiation along the parietal lineage. In contrast, WNTs and BMPs are required to suppress differentiation of embryonic gastric epithelium along pit cell lineage. Thus, coordinated action of the WNT, BMP and Notch pathways controls cell fate determination in the embryonic gastric epithelium.

A controlled organ homeostasis is essential to sustain the integrity of any living organism. This homeostasis relies on stem cells, which maintain themselves and give rise to the various types of differentiated cells. It is well established that the differentiation of pluripotent embryonic stem cells (ESCs) depends on major changes in transcriptional programs. Covalent modifications of DNA, RNA and proteins are instrumental in setting up the genetic programs associated with cell fates. Another important mechanism for regulation of gene expression is protein localisation and stability. In addition to ubiquitination, SUMOylation and phosphorylation, methylation of proteins is an important determinant of their properties.

Epigenetic mechanisms, including chromatin structure, chromatin dynamics and histone modifications play an important role for maintenance and differentiation of pluripotent embryonic stem cells. However, little is known about the molecular mechanisms of adult stem cell specification and differentiation. Here, we used intestinal stem cells (ISCs) as a model system to reveal the epigenetic changes coordinating gene expression programs during these processes. We found that two distinct epigenetic mechanisms participate in establishing the transcriptional program promoting ISC specification from embryonic progenitors. A large number of adult ISC signature genes are targets of repressive DNA methylation in embryonic intestinal epithelial progenitors. On the other hand, genes essential for embryonic development acquire H3K27me3 and are silenced during ISC specification. We also show that the repression of ISC signature genes as well as the activation of enterocyte specific genes is accompanied by a global loss of H2A.Z during ISCs differentiation. Our results reveal that, already during ISC specification, an extensive remodeling of chromatin both at promoters and distal regulatory elements organizes transcriptional landscapes operating in differentiated enterocytes, thus explaining similar chromatin modification patterns in the adult gut epithelium.

The adult intestinal stem cells (ISCs), their hierarchies, mechanisms of maintenance and differentiation have been extensively studied. However, when and how ISCs are established during embryogenesis remains unknown. We show here that the transcription regulator Id2 controls the specification of embryonic Lgr5 + progenitors in the developing murine small intestine. Cell fate mapping analysis revealed that Lgr5 + progenitors emerge at E13.5 in wild‐type embryos and differ from the rest on the intestinal epithelium by a characteristic ISC signature. In the absence of Id2, the intestinal epithelium differentiates into Lgr5 + cells already at E9.5. Furthermore, the size of the Lgr5 + cell pool is significantly increased. We show that Id2 restricts the activity of the Wnt signalling pathway at early stages and prevents precocious differentiation of the embryonic intestinal epithelium. Id2‐deficient embryonic epithelial cells cultured ex vivo strongly activate Wnt target genes as well as markers of neoplastic transformation and form fast growing undifferentiated spheroids. Furthermore, adult ISCs from Id2‐deficient mice display a distinct transcriptional signature, supporting an essential role for Id2 in the correct specification of ISCs.

Hox genes encode transcription factors defining cellular identities along the major and secondary body axes. Their coordinated expression in both space and time is critical for embryonic patterning. Accordingly, Hox genes transcription is tightly controlled at multiple levels, and involves an intricate combination of local and long-range cis-regulatory elements. Recent studies revealed that in addition to transcription factors, dynamic patterns of histone marks and higher-order chromatin structure are important determinants of Hox gene regulation. Furthermore, the emerging picture suggests an involvement of various species of non-coding RNA in targeting activating and repressive complexes to Hox clusters. I review these recent developments and discuss their relevance to the control of Hox gene expression in vivo, as well as to our understanding of transcriptional regulatory mechanisms.

The vertebrate body plan is characterized by an increased complexity relative to that of all other chordates and large-scale gene amplifications have been associated with key morphological innovations leading to their remarkable evolutionary success. Here, we use compound full Hox clusters deletions to investigate how Hox genes duplications may have contributed to the emergence of vertebrate-specific innovations. We show that the combined deletion of HoxA and HoxB leads to an atavistic heart phenotype, suggesting that the ancestral HoxA/B cluster was co-opted to help in diversifying the complex organ in vertebrates. Other phenotypic effects observed seem to illustrate the resurgence of ancestral (plesiomorphic) features. This indicates that the duplications of Hox clusters were associated with the recruitment or formation of novel cis-regulatory controls, which were key to the evolution of many vertebrate features and hence to the evolutionary radiation of this group.

The four R-spondin secreted ligands (RSPO1–RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1–3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers. Independently of the LGR4/5/6 receptors, RSPO2 acts as a direct antagonistic ligand to RNF43 and ZNRF3 during embryogenesis, and specifies the position and number of limbs that an embryo should form.

Dynamic changes in gene expression are tightly controlled during development, as a single totipotent zygote gives rise to distinct cell lineages. The establishment and maintenance of these diverse transcriptional programs rely on changes of chromatin state, mainly through histone modifications. Polycomb and Trithorax complexes participate in setting apart active and inactive genes by respectively repressing and activating key developmental regulators in different cell types. Over the last decade, our understanding of the biochemical mechanisms underlying their activities has greatly improved, but the signals targeting these proteins to specific regions of the genome are still poorly understood, particularly in vertebrates. Recent findings highlight the highly dynamic activities of Polycomb and Trithorax complexes in vivo. Surprisingly, although their role in transcriptional regulation is deeply conserved during evolution, the time sequence in which they act seems to vary across species. Birth Defects Research (Part A), 2011. © 2011 Wiley-Liss, Inc.

SummaryThe evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a “regulatory archipelago,” provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.PaperFlick

In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.

SummaryCCCTC-binding factor (CTCF) is a nuclear zinc-finger protein that displays insulating activity in a variety of biological assays. For example, CTCF-binding sites have been suggested to isolate Hox gene clusters from neighboring transcriptional interference. We investigated this issue during limb development, where Hoxd genes must remain isolated from long-range effects to allow essential regulation within independent sub-groups. We used conditional Ctcf inactivation in incipient forelimbs and show that the overall pattern of Hoxd gene expression remains unchanged. Transcriptome analysis using tiling arrays covering chromosomes 2 and X confirmed the weak effect of CTCF depletion on global gene regulation. However, Ctcf deletion caused massive apoptosis, leading to a nearly complete loss of limb structure at a later stage. We conclude that, at least in this physiological context, rather than being an insulator, CTCF is required for cell survival via the direct transcriptional regulation of target genes critical for cellular homeostasis.

Temporal and spatial control of Hox gene expression is essential for correct patterning of many animals. In both Drosophila and vertebrates, Polycomb and Trithorax group complexes control the maintenance of Hox gene expression in appropriate domains. In vertebrates, dynamic changes in chromatin modifications are also observed during the sequential activation of Hox genes in the embryo, suggesting that progressive epigenetic modifications could regulate collinear gene activation.

Hox genes are critical regulators of embryonic development in bilaterian animals. They exhibit a unique mode of transcriptional regulation where the position of the genes along the chromosome corresponds to the time and place of their expression during development. The sequential temporal activation of these genes in the primitive streak helps determining their subsequent pattern of expression along the anterior-posterior axis of the embryo, yet the precise correspondence between these two collinear processes is not fully understood. In addition, vertebrate Hox genes evolved similar modes of regulation along secondary body axes, such as the developing limbs. We review the current understanding of the mechanisms operating during activation, maintenance and silencing of Hox gene expression in these various contexts, and discuss the evolutionary significance of their genomic organization.

By conditional gene ablation in mice, we found that β-catenin, an essential downstream effector of canonical Wnt signaling, is a key regulator of formation of the apical ectodermal ridge (AER) and of the dorsal–ventral axis of the limbs. By generation of compound mutants, we also show that β-catenin acts downstream of the BMP receptor IA in AER induction, but upstream or parallel in dorsal–ventral patterning. Thus, AER formation and dorsal–ventral patterningof limbs are tightly controlled by an intricate interplay between Wnt/β-catenin and BMP receptor signaling.

Hox genes encode transcription factors essential for patterning the anterior to posterior animal body axis. In vertebrates, these genes are arranged in clusters. During development, Hox genes are activated in a time sequence that follows their physical order within the cluster. To elucidate the mechanism of ‘temporal collinearity’ we looked at the in vivo dynamics of chromatin state during successive Hox gene activation in the mouse embryo. We show that chromatin marks are important parameters in the temporal regulation of the Hox gene family as judged by the dynamic progression of transcription-competent modifications: increases in activation marks correspond to decreases in repressive marks. Furthermore, using a mouse where a Hox cluster was split into two pieces, we document the necessity to maintain a clustered organization to properly implement this temporal process.

SummaryThe emergence of limb-driven locomotor behaviors was a key event in the evolution of vertebrates and fostered the transition from aquatic to terrestrial life. We show that the generation of limb-projecting lateral motor column (LMC) neurons in mice relies on a transcriptional autoregulatory module initiated via transient activity of multiple genes within the HoxA and HoxC clusters. Repression of this module at thoracic levels restricts expression of LMC determinants, thus dictating LMC position relative to the limbs. This suppression is mediated by a key regulatory domain that is specifically found in the Hoxc9 proteins of appendage-bearing vertebrates. The profile of Hoxc9 expression inversely correlates with LMC position in land vertebrates and likely accounts for the absence of LMC neurons in limbless species such as snakes. Thus, modulation of both Hoxc9 protein function and Hoxc9 gene expression likely contributed to evolutionary transitions between undulatory and ambulatory motor circuit connectivity programs.PaperClip

Hox Clocks Homeobox or Hox genes are historically significant for demonstrating evolutionary conservation and homology in developmentally important genes. In addition, in many species, the Hox genes are fundamental to the organization of the embryo. Modifications in the regulation of their colinear, temporal activation may represent a way of altering their expression patterns and to elaborate body plans during evolution. Soshnikova and Duboule (p. 1320 ) now report highly dynamic modifications of chromatin marks along with progressive Hox gene activation during axial extension in the mouse. This work supports the proposal that the “ Hox clock” may be controlled, at least in part, by epigenetic mechanisms.