Embryonic timing, axial stem cells, chromatin dynamics, and the Hox clock.

Collinear regulation of Hox genes in space and time has been an outstanding question ever since the initial work of Ed Lewis in 1978. Here we discuss recent advances in our understanding of this phenomenon in relation to novel concepts associated with large-scale regulation and chromatin structure during the development of both axial and limb patterns. We further discuss how this sequential transcriptional activation marks embryonic stem cell-like axial progenitors in mammals and, consequently, how a temporal genetic system is further translated into spatial coordinates via the fate of these progenitors. In this context, we argue the benefit and necessity of implementing this unique mechanism as well as the difficulty in evolving an alternative strategy to deliver this critical positional information.

Polarized regulatory landscape and Wnt responsiveness underlie Hox activation in embryos.

Sequential 3'-to-5' activation of the Hox gene clusters in early embryos is a most fascinating issue in developmental biology. Neither the trigger nor the regulatory elements involved in the transcriptional initiation of the 3'-most Hox genes have been unraveled in any organism. We demonstrate that a series of enhancers, some of which are Wnt-dependent, is located within a HoxA 3' subtopologically associated domain (subTAD). This subTAD forms the structural basis for multiple layers of 3'-polarized features, including DNA accessibility and enhancer activation. Deletion of the cassette of Wnt-dependent enhancers proves its crucial role in initial transcription of HoxA at the 3' side of the cluster.

At the base of colinear Hox gene expression: cis-features and trans-factors orchestrating the initial phase of Hox cluster activation.

Hox genes are crucial players in the generation and pattering of the vertebrate trunk and posterior body during embryogenesis. Their initial expression takes place shortly after the establishment of the primitive streak, in the posterior-most part of the mouse embryo and is a determinant step for setting up the definitive Hox expression boundaries along the antero-posterior body axis. The developmental signals and epigenetic mechanisms underlying this early activation remained unsolved until recently. The development of novel embryo-derived model systems, combined with methods that examine chromatin status and chromosome conformation, led to deeper understanding of the process of Hox activation in the early embryo. Here we summarize how the early Hox cis-regulatory landscape becomes active upon receiving the appropriate developmental signal, and we discuss the importance of the local topological segmentation of the HoxA cluster during early Hox activation.

On the shoulders of Hubrecht: From embryos to stem cells.

One hundred years of the Hubrecht Institute were celebrated in May 2016 with the organization of a one-day symposium "From embryos to stem cells" on the Uithof Campus, Utrecht, the Netherlands. Nine distinguished speakers were invited. They all represent a research branch originating from the passion of Institute founder, Ambrosius Hubrecht, for embryology:, regulation of gene expression, genome structure and function, embryonic and adult stem cells, nuclear reprogramming, and understanding cancer and other diseases using model organisms. The centennial symposium not only retraced the history of the Institute and of modern developmental biology, but was also a tribute to basic research. From there, avenues to therapeutics are being developed and implemented. The symposium was organized, introduced and chaired by Jeroen den Hertog and Alexander van Oudenaarden, the present Directors of the Institute, who also stand on Hubrecht's shoulders.

Cdx is crucial for the timing mechanism driving colinear Hox activation and defines a trunk segment in the Hox cluster topology.

Cdx and Hox transcription factors are important regulators of axial patterning and are required for tissue generation along the vertebrate body axis. Cdx genes have been demonstrated to act upstream of Hox genes in midgestation embryos. Here, we investigate the role of Cdx transcription factors in the gradual colinear activation of the Hox clusters. We found that Hox temporally colinear expression is severely affected in epiblast stem cells derived from Cdx null embryos. We demonstrate that after initiation of 3' Hox gene transcription, Cdx activity is crucial for H3K27ac deposition and for accessibility of cis-regulatory elements around the central - or 'trunk' - Hox genes. We thereby identify a Cdx-responsive segment of HoxA, immediately 5' to the recently defined regulatory domain orchestrating initial transcription of the first Hox gene. We propose that this partition of HoxA into a Wnt-driven 3' part and the newly found Cdx-dependent middle segment of the cluster, forms a structural fundament of Hox colinearity of expression. Subsequently to initial Wnt-induced activation of 3' Hox genes, Cdx transcription factors would act as crucial effectors for activating central Hox genes, until the last gene of the cluster arrests the process.

Evolutionarily conserved requirement of Cdx for post-occipital tissue emergence.

Mouse Cdx genes are involved in axial patterning and partial Cdx mutants exhibit posterior embryonic defects. We found that mouse embryos in which all three Cdx genes are inactivated fail to generate any axial tissue beyond the cephalic and occipital primordia. Anterior axial tissues are laid down and well patterned in Cdx null embryos, and a 3' Hox gene is initially transcribed and expressed in the hindbrain normally. Axial elongation stops abruptly at the post-occipital level in the absence of Cdx, as the posterior growth zone loses its progenitor activity. Exogenous Fgf8 rescues the posterior truncation of Cdx mutants, and the spectrum of defects of Cdx null embryos matches that resulting from loss of posterior Fgfr1 signaling. Our data argue for a main function of Cdx in enforcing trunk emergence beyond the Cdx-independent cephalo-occipital region, and for a downstream role of Fgfr1 signaling in this function. Cdx requirement for the post-head section of the axis is ancestral as it takes place in arthropods as well.

Cdx2 determines the fate of postnatal intestinal endoderm.

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.

Region-specific regulation of posterior axial elongation during vertebrate embryogenesis.

BACKGROUND: The vertebrate body axis extends sequentially from the posterior tip of the embryo, fueled by the gastrulation process at the primitive streak and its continuation within the tailbud. Anterior structures are generated early, and subsequent nascent tissues emerge from the posterior growth zone and continue to elongate the axis until its completion. The underlying processes have been shown to be disrupted in mouse mutants, some of which were described more than half a century ago. RESULTS: Important progress in elucidating the cellular and genetic events involved in body axis elongation has recently been made on several fronts. Evidence for the residence of self-renewing progenitors, some of which are bipotential for neurectoderm and mesoderm, has been obtained by embryo-grafting techniques and by clonal analyses in the mouse embryo. Transcription factors of several families including homeodomain proteins have proven instrumental for regulating the axial progenitor niche in the growth zone. A complex genetic network linking these transcription factors and signaling molecules is being unraveled that underlies the phenomenon of tissue lengthening from the axial stem cells. The concomitant events of cell fate decision among descendants of these progenitors begin to be better understood at the levels of molecular genetics and cell behavior. CONCLUSIONS: The emerging picture indicates that the ontogenesis of the successive body regions is regulated according to different rules. In addition, parameters controlling vertebrate axial length during evolution have emerged from comparative experimental studies. It is on these issues that this review will focus, mainly addressing the study of axial extension in the mouse embryo with some comparison with studies in chick and zebrafish, aiming at unveiling the recent progress, and pointing at still unanswered questions for a thorough understanding of the process of embryonic axis elongation.

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone.

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

Cdx2 contributes to the expansion of the early primordial germ cell population in the mouse.

Cdx gene products regulate the extent of axial elongation from the posterior growth zone. These transcription factors sustain the emergence of trunk and tail tissues by providing a suitable niche in the axial progenitor zone, via regulation of Wnt signaling. Cdx genes are expressed in and along the complete primitive streak including its posterior part wherefrom the extraembryonic mesoderm of the allantois emerges. Cdx genes are required for the full development of the allantois and its derivatives in the placental labyrinth. The mouse germ cell lineage also originates from the proximo-posterior epiblast of the primitive streak, and is established within the extraembryonic mesoderm that generates the allantois. We asked whether the expression of Cdx genes around the newly specified PGCs is necessary for the maintenance and expansion of this population, as it is for the allantois and axial progenitors. We observed a significantly lower number of PGCs in Cdx2(null) embryos than in controls. We found that Wnt3a loss of function decreases the PGC population to the same extent as Cdx2 inactivation. Moreover, exogenous Wnt3a corrects the lower PGC number in Cdx2(null) posterior embryonic tissues cultured in vitro. Cdx2 is not expressed in PGCs themselves, and we propose that the expression of Cdx2 in posterior extraembryonic tissues contributes to the proper niche of the germ cell progenitors by stimulating canonical Wnt signaling. Since PGC residence within the posterior growth zone is a mouse-specific feature, our data suggest that mouse PGCs opportunistically became dependent on the axial progenitor niche.

Cdx mutant axial progenitor cells are rescued by grafting to a wild type environment.

Cdx transcription factors are required for axial extension. Cdx genes are expressed in the posterior growth zone, a region that supplies new cells for axial elongation. Cdx2(+/-)Cdx4(-/-) (Cdx2/4) mutant embryos show abnormalities in axis elongation from E8.5, culminating in axial truncation at E10.5. These data raised the possibility that the long-term axial progenitors of Cdx mutants are intrinsically impaired in their ability to contribute to posterior growth. We investigated whether we could identify cell-autonomous defects of the axial progenitor cells by grafting mutant cells into a wild type growth zone environment. We compared the contribution of GFP labeled mutant and wild type progenitors grafted to unlabeled wild type recipients subsequently cultured over the period during which Cdx2/4 defects emerge. Descendants of grafted cells were scored for their contribution to differentiated tissues in the elongating axis and to the posterior growth zone. No difference between the contribution of descendants from wild type and mutant grafted progenitors was detected, indicating that rescue of the Cdx mutant progenitors by the wild type recipient growth zone is provided non-cell autonomously. Recently, we showed that premature axial termination of Cdx mutants can be partly rescued by stimulating canonical Wnt signaling in the posterior growth zone. Taken together with the data shown here, this suggests that Cdx genes function to maintain a signaling-dependent niche for the posterior axial progenitors.

Hox genes and regional patterning of the vertebrate body plan.

Several decades have passed since the discovery of Hox genes in the fruit fly Drosophila melanogaster. Their unique ability to regulate morphologies along the anteroposterior (AP) axis (Lewis, 1978) earned them well-deserved attention as important regulators of embryonic development. Phenotypes due to loss- and gain-of-function mutations in mouse Hox genes have revealed that the spatio-temporally controlled expression of these genes is critical for the correct morphogenesis of embryonic axial structures. Here, we review recent novel insight into the modalities of Hox protein function in imparting specific identity to anatomical regions of the vertebral column, and in controlling the emergence of these tissues concomitantly with providing them with axial identity. The control of these functions must have been intimately linked to the shaping of the body plan during evolution.

Ancestral and recently recruited global control of the Hox genes in development.

Genes from the Hox family are involved in the common task of providing nascent embryonic tissues with their positional identity. They are organised in clusters in most species. Mouse Hox genes are regulated in part by gene-proximal regulatory elements, but owe several of their essential properties to the use of global regulatory elements located outside the complexes. The clustered Hox genes in that sense behave as a single large locus. Genomic and sequence data from different animal species suggest that a concerted regulation of the Hox clusters, inherently coupled to their patterning properties, originated early during evolution and pre-figured the temporal colinearity of expression of vertebrate Hox genes. In addition, vertebrates have recruited novel global mechanisms to control the expression of linear subsets of Hox genes in specific embryonic structures. Several of such novel global regulatory circuits have recently been characterised at the molecular genetic level in the mouse.

Loss of Hoxb8 alters spinal dorsal laminae and sensory responses in mice.

Although Hox gene expression has been linked to motoneuron identity, a role of these genes in development of the spinal sensory system remained undocumented. Hoxb genes are expressed at high levels in the dorsal horn of the spinal cord. Hoxb8 null mutants manifest a striking phenotype of excessive grooming and hairless lesions on the lower back. Applying local anesthesia underneath the hairless skin suppressed excessive grooming, indicating that this behavior depends on peripheral nerve activity. Functional ablation of mouse Hoxb8 also leads to attenuated response to nociceptive and thermal stimuli. Although spinal ganglia were normal, a lower postmitotic neural count was found in the dorsalmost laminae at lumbar levels around birth, leading to a smaller dorsal horn and a correspondingly narrowed projection field of nociceptive and thermoceptive afferents. The distribution of the dorsal neuronal cell types that we assayed, including neurons expressing the itch-specific gastrin-releasing peptide receptor, was disorganized in the lumbar region of the mutant. BrdU labeling experiments and gene-expression studies at stages around the birth of these neurons suggest that loss of Hoxb8 starts impairing development of the upper laminae of the lumbar spinal cord at approximately embryonic day (E)15.5. Because none of the neuronal markers used was unexpressed in the adult dorsal horn, absence of Hoxb8 does not impair neuronal differentiation. The data therefore suggest that a lower number of neurons in the upper spinal laminae and neuronal disorganization in the dorsal horn underlie the sensory defects including the excessive grooming of the Hoxb8 mutant.

Hoxb8-Cre mice: A tool for brain-sparing conditional gene deletion.

The spinal cord is the first site of temporal and spatial integration of nociceptive signals in the pain pathway. Neuroplastic changes occurring at this site contribute critically to various chronic pain syndromes. Gene targeting in mice has generated important insights into these processes. However, the analysis of constitutive (global) gene-deficient mice is often hampered by confounding effects arising from supraspinal sites. Here, we describe a novel Cre mouse line that expresses the Cre recombinase under the transcriptional control of the Hoxb8 gene. Within the neural axis of these mice, Hoxb8-Cre expression is found in spinal cord neurons and glial cells, and in virtually all neurons of the dorsal root ganglia, but spares the brain apart from a few cells in the spinal trigeminal nucleus. The Hoxb8-Cre mouse line should be a valuable new tool for the in vivo analysis of peripheral and spinal gene functions in pain pathways.

Colinearity loops out.

Modulation of chromatin structure has long been proposed to underlie the colinear regulation of Hox genes during animal development. In a recent paper, Chambeyron and Bickmore explore this possibility in retinoic acid-induced ES cells. They show that, while chromatin remodeling confers transcriptional competence to the gene cluster, subsequent sequential extrusion of genes from their chromosome territory may determine their coordinated expression in time.

The Cdx2 homeobox gene suppresses intestinal tumorigenesis through non-cell-autonomous mechanisms.

Developmental genes contribute to cancer, as reported for the homeobox gene Cdx2 playing a tumor suppressor role in the gut. In this study, we show that human colon cancers exhibiting the highest reduction in CDX2 expression belong to the serrated subtype with the worst evolution. In mice, mosaic knockout of Cdx2 in the adult intestinal epithelium induces the formation of imperfect gastric-type metaplastic lesions. The metaplastic knockout cells do not spontaneously become tumorigenic. However, they induce profound modifications of the microenvironment that facilitate the tumorigenic evolution of adjacent Cdx2-intact tumor-prone cells at the surface of the lesions through NF-κB activation, induction of inducible nitric oxide synthase, and stochastic loss of function of Apc This study presents a novel paradigm in that metaplastic cells, generally considered as precancerous, can induce tumorigenesis from neighboring nonmetaplastic cells without themselves becoming cancerous. It unveils the novel property of non-cell-autonomous tumor suppressor gene for the Cdx2 gene in the gut.

Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems?

When, where and how is the head-tail axis of the embryo set up during development? These are such fundamental and intensely studied questions that one might expect them to have been answered long ago. Not so; we still understand very little about the cellular or molecular mechanisms that lead to the orderly arrangement of body elements along the head-tail axis in vertebrates. In this paper, we outline some of the major outstanding problems and controversies and try to identify some reasons why it has been so difficult to resolve this important issue.

Fine-tuning and autoregulation of the intestinal determinant and tumor suppressor homeobox gene CDX2 by alternative splicing.

On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases.

Birth and upgrowth of the Hox topological domains during evolution.

The recently discovered chromatin compartments called topologically associating domains (TADs) are essential for the three-dimensional organization of regulatory interactions driving gene expression. A new study documents the emergence of a TAD flanking the amphioxus Hox cluster, prefiguring the vertebrate anterior Hox TAD and preceding the appearance of the concurring posterior Hox TAD.