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The vertebrate body forms by continuous generation of new tissue from progenitors at the posterior end of the embryo. The study of these axial progenitors has proved to be challenging in vivo largely because of the lack of unique molecular markers to identify them. Here, we elucidate the expression pattern of the transcription factor Nkx1-2 in the mouse embryo and show that it identifies axial progenitors throughout body axis elongation, including neuromesodermal progenitors and early neural and mesodermal progenitors. We create a tamoxifen-inducible Nkx1-2CreER T2 transgenic mouse and exploit the conditional nature of this line to uncover the lineage contributions of Nkx1-2 -expressing cells at specific stages. We show that early Nkx1-2 -expressing epiblast cells contribute to all three germ layers, mostly neuroectoderm and mesoderm, excluding notochord. Our data are consistent with the presence of some self-renewing axial progenitors that continue to generate neural and mesoderm tissues from the tail bud. This study identifies Nkx1-2 -expressing cells as the source of most trunk and tail tissues in the mouse and provides a useful tool to genetically label and manipulate axial progenitors in vivo ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Caudal somites are generated from a pool of progenitor cells located in the tailbud region. These progenitor cells form the presomitic mesoderm that gradually differentiates into somites under the action of the segmentation clock. The signals responsible for tailbud mesoderm progenitor pool maintenance during axial elongation are still elusive. Here, we show that Bmp signaling is sufficient to activate the entire mesoderm progenitor gene signature in primary cultures of caudal mesoderm cells. Bmp signaling acts through the key regulatory genes brachyury ( T ) and Nkx1-2 and contributes to the activation of several other regulators of the mesoderm progenitor gene network. In the absence of Bmp signaling, tailbud mesoderm progenitor cells acquire aberrant gene expression signatures of the heart, blood, muscle and skeletal embryonic lineages. Treatment of embryos with the Bmp inhibitor noggin confirmed the requirement for Bmp signaling for normal T expression and the prevention of abnormal lineage marker activation. Together, these results identify Bmp signaling as a non-cell-autonomous signal necessary for mesoderm progenitor cell homeostasis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Vertebrate body axis formation depends on a population of bipotential neuromesodermal cells along the posterior wall of the tailbud that make a germ layer decision after gastrulation to form spinal cord and mesoderm. Despite exhibiting germ layer plasticity, these cells never give rise to midline tissues of the notochord, floor plate and dorsal endoderm, raising the question of whether midline tissues also arise from basal posterior progenitors after gastrulation. We show in zebrafish that local posterior signals specify germ layer fate in two basal tailbud midline progenitor populations. Wnt signaling induces notochord within a population of notochord/floor plate bipotential cells through negative transcriptional regulation of sox2. Notch signaling, required for hypochord induction during gastrulation, continues to act in the tailbud to specify hypochord from a notochord/hypochord bipotential cell population. Our results lend strong support to a continuous allocation model of midline tissue formation in zebrafish, and provide an embryological basis for zebrafish and mouse bifurcated notochord phenotypes as well as the rare human congenital split notochord syndrome. We demonstrate developmental equivalency between the tailbud progenitor cell populations. Midline progenitors can be transfated from notochord to somite fate after gastrulation by ectopic expression of msgn1, a master regulator of paraxial mesoderm fate, or if transplanted into the bipotential progenitors that normally give rise to somites. Our results indicate that the entire non-epidermal posterior body is derived from discrete, basal tailbud cell populations. These cells remain receptive to extracellular cues after gastrulation and continue to make basic germ layer decisions., (© 2016. Published by The Company of Biologists Ltd.)

The outgrowth of the vertebrate tail is thought to involve the proliferation of regionalised stem/progenitor cell populations formed during gastrulation. To follow these populations over extended periods, we used cells from GFP-positive transgenic chick embryos as a source for donor tissue in grafting experiments. We determined that resident progenitor cell populations are localised in the chicken tail bud. One population, which is located in the chordoneural hinge (CNH), contributes descendants to the paraxial mesoderm, notochord and neural tube, and is serially transplantable between embryos. A second population of mesodermal progenitor cells is located in a separate dorsoposterior region of the tail bud, and a corresponding population is present in the mouse tail bud. Using heterotopic transplantations, we show that the fate of CNH cells depends on their environment within the tail bud. Furthermore, we show that the anteroposterior identity of tail bud progenitor cells can be reset by heterochronic transplantation to the node region of gastrula-stage chicken embryos.

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.

Elongation of the mouse anteroposterior axis depends on a small population of progenitors initially located in the primitive streak and later in the tail bud. Gene expression and lineage tracing have shown that there are many features common to these progenitor tissues throughout axial elongation. However, the identity and location of the progenitors is unclear. We show by lineage tracing that the descendants of 8.5 d.p.c. node and anterior primitive streak which remain in the tail bud are located in distinct territories: (1) ventral node descendants are located in the widened posterior end of the notochord; and (2) descendants of anterior streak are located in both the tail bud mesoderm, and in the posterior end of the neurectoderm. We show that cells from the posterior neurectoderm are fated to give rise to mesoderm even after posterior neuropore closure. The posterior end of the notochord, together with the ventral neurectoderm above it, is thus topologically equivalent to the chordoneural hinge region defined in Xenopus and chick. A stem cell model has been proposed for progenitors of two of the axial tissues, the myotome and spinal cord. Because it was possible that labelled cells in the tail bud represented stem cells, tail bud mesoderm and chordoneural hinge were grafted to 8.5 d.p.c. primitive streak to compare their developmental potency. This revealed that cells from the bulk of the tail bud mesoderm are disadvantaged in such heterochronic grafts from incorporating into the axis and even when they do so, they tend to contribute to short stretches of somites suggesting that tail bud mesoderm is restricted in potency. By contrast, cells from the chordoneural hinge of up to 12.5 d.p.c. embryos contribute efficiently to regions of the axis formed after grafting to 8.5 d.p.c. embryos, and also repopulate the tail bud. These cells were additionally capable of serial passage through three successive generations of embryos in culture without apparent loss of potency. This potential for self-renewal in chordoneural hinge cells strongly suggests that stem cells are located in this region.

In Caenorhabditis elegans, Wnt signaling pathways are important in controlling cell polarity and cell migrations. In the embryo, a novel Wnt pathway functions through a (beta)-catenin homolog, WRM-1, to downregulate the levels of POP-1/Tcf in the posterior daughter of the EMS blastomere. The level of POP-1 is also lower in the posterior daughters of many anteroposterior asymmetric cell divisions during development. I have found that this is the case for of a pair of postembryonic blast cells in the tail. In wild-type animals, the level of POP-1 is lower in the posterior daughters of the two T cells, TL and TR. Furthermore, in lin-44/Wnt mutants, in which the polarities of the T cell divisions are frequently reversed, the level of POP-1 is frequently lower in the anterior daughters of the T cells. I have used a novel RNA-mediated interference technique to interfere specifically with pop-1 zygotic function and have determined that pop-1 is required for wild-type T cell polarity. Surprisingly, none of the three C. elegans (beta)-catenin homologs appeared to function with POP-1 to control T cell polarity. Wnt signaling by EGL-20/Wnt controls the migration of the descendants of the QL neuroblast by regulating the expression the Hox gene mab-5. Interfering with pop-1 zygotic function caused defects in the migration of the QL descendants that mimicked the defects in egl-20/Wnt mutants and blocked the expression of mab-5. This suggests that POP-1 functions in the canonical Wnt pathway to control QL descendant migration and in novel Wnt pathways to control EMS and T cell polarities.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.

In order to understand the developmental mechanisms of posterior body formation in the zebrafish, a fate map of the zebrafish tailbud was generated along with a detailed analysis of tailbud cell movements. The fate map of the zebrafish tailbud shows that it contains tissue-restricted domains and is not a homogeneous blastema. Furthermore, time-lapse analysis shows that some cell movements and behaviors in the tailbud are similar to those seen during gastrulation, while others are unique to the posterior body. The extension of axial mesoderm and the continuation of ingression throughout zebrafish tail development suggests the continuation of processes initiated during gastrulation. Unique properties of zebrafish posterior body development include the bilateral distribution of tailbud cell progeny and the exhibition of different forms of ingression within specific tailbud domains. The ingression of cells in the anterior tailbud only gives rise to paraxial mesoderm, at the exclusion of axial mesoderm. Cells of the posterior tailbud undergo subduction, a novel form of ingression resulting in the restriction of this tailbud domain to paraxial mesodermal fates. The intermixing of spinal cord and muscle precursor cells, as well as evidence for pluripotent cells within the tailbud, suggest that complex inductive mechanisms accompany these cell movements throughout tail elongation. Rates of cell proliferation in the tailbud were examined and found to be relatively low at the tip of the tail indicating that tail elongation is not due to growth at its posterior end. However, higher rates of cell proliferation in the dorsomedial region of the tail may contribute to the preferential posterior movement of cells in this tailbud region and to the general extension of the tail. Understanding the cellular movements, cell fates and gene expression patterns in the tailbud will help to determine the nature of this important aspect of vertebrate development.

We have tested directly the hypothesis that the endogenous electrical field in the chick embryo plays a causal role in development. Conductive implants, which shunt currents out of the embryo and thus alter the internal field, were placed under the dorsal skin at the mid-trunk level of stage 11-15 embryos. Currents leaving the posterior intestinal portal (p.i.p.) of these embryos were reduced by an average of 30%. Control embryos receiving non-conductive implants showed no change in p.i.p. currents. In the group receiving current shunts, 92% of the embryos exhibited some developmental abnormality. Only 11% of the control group displayed defects. The most common defect in the experimental group (81%) was in tail development. Tail defects ranged from complete absence to the formation of a normal length, but morphologically abnormal tail. Internally, tail structures (neural tube, notochord and somites) were frequently absent or aberrantly formed. In 33% of the experimental embryos, the notochord continued lengthening in the absence of any other tail development. This led to the formation of ourenteric outgrowths from the hindgut. Defects in limb bud and head development were also found in experimentally treated embryos, but at a much lower frequency than tail defects. The abnormalities observed in experimental embryos were very similar to those produced naturally in rumpless mutant chicks. A vibrating probe analysis of these mutants (from both dominant and recessive strains) showed that currents leaving the p.i.p. were significantly lower in phenotypically abnormal mutants than in wild-type and phenotypically normal mutant embryos from both strains. There was no apparent correlation between the average transepithelial potential (TEP) of these mutants and the development of tail abnormalities. The possible role of endogenous electrical fields in chick tail development is discussed.