ZSWIM4 regulates embryonic patterning and BMP signaling by promoting nuclear Smad1 degradation.

The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning. Zinc Finger SWIM-Type Containing 4 (zswim4) is expressed in the Spemann-Mangold organizer at the onset of Xenopus gastrulation and is then enriched in the developing neuroectoderm at the mid-gastrula stages. Knockdown or knockout of zswim4 causes ventralization. Overexpression of zswim4 decreases, whereas knockdown of zswim4 increases the expression levels of ventrolateral mesoderm marker genes. Mechanistically, ZSWIM4 attenuates the BMP signal by reducing the protein stability of SMAD1 in the nucleus. Stable isotope labeling by amino acids in cell culture (SILAC) identifies Elongin B (ELOB) and Elongin C (ELOC) as the interaction partners of ZSWIM4. Accordingly, ZSWIM4 forms a complex with the Cul2-RING ubiquitin ligase and ELOB and ELOC, promoting the ubiquitination and degradation of SMAD1 in the nucleus. Our study identifies a novel mechanism that restricts BMP signaling in the nucleus.

Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus.

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F1Fo ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when ß-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling.

Mink1 regulates spemann organizer cell fate in the xenopus gastrula via Hmga2.

Congenital Heart Disease (CHD) is the most common birth defect and leading cause of infant mortality, yet molecular mechanisms explaining CHD remain mostly unknown. Sequencing studies are identifying CHD candidate genes at a brisk rate including MINK1, a serine/threonine kinase. However, a plausible molecular mechanism connecting CHD and MINK1 is unknown. Here, we reveal that mink1 is required for proper heart development due to its role in left-right patterning. Mink1 regulates canonical Wnt signaling to define the cell fates of the Spemann Organizer and the Left-Right Organizer, a ciliated structure that breaks bilateral symmetry in the vertebrate embryo. To identify Mink1 targets, we applied an unbiased proteomics approach and identified the high mobility group architectural transcription factor, Hmga2. We report that Hmga2 is necessary and sufficient for regulating Spemann's Organizer. Indeed, we demonstrate that Hmga2 can induce Spemann Organizer cell fates even when ß-catenin, a critical effector of the Wnt signaling pathway, is depleted. In summary, we discover a transcription factor, Hmga2, downstream of Mink1 that is critical for the regulation of Spemann's Organizer, as well as the LRO, defining a plausible mechanism for CHD.

The embryonic node behaves as an instructive stem cell niche for axial elongation.

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

Targeted search for scaling genes reveals matrixmetalloproteinase 3 as a scaler of the dorsal-ventral pattern in Xenopus laevis embryos.

How embryos scale patterning according to size is still not fully understood. Through in silico screening and analysis of reaction-diffusion systems that could be responsible for scaling, we predicted the existence of genes whose expression is sensitive to embryo size and which regulate the scaling of embryonic patterning. To find these scalers, we identified genes with strongly altered expression in half-size Xenopus laevis embryos compared with full-size siblings at the gastrula stage. Among found genes, we investigated the role of matrix metalloproteinase-3 (mmp3), which was most strongly downregulated in half-size embryos. We show that Mmp3 scales dorsal-ventral patterning by degrading the slowly diffusing embryonic inducers Noggin1 and Noggin2 but preventing cleavage of the more rapidly diffusing inducer Chordin via degradation of a Tolloid-type proteinase. In addition to unraveling the mechanism underlying the scaling of dorsal-ventral patterning, this work provides proof of principal for scalers identification in embryos of other species.

Lysosomal degradation of the maternal dorsal determinant Hwa safeguards dorsal body axis formation.

The Spemann and Mangold Organizer (SMO) is of fundamental importance for dorsal ventral body axis formation during vertebrate embryogenesis. Maternal Huluwa (Hwa) has been identified as the dorsal determinant that is both necessary and sufficient for SMO formation. However, it remains unclear how Hwa is regulated. Here, we report that the E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) is essential for restricting the spatial activity of Hwa and therefore correct SMO formation in Xenopus laevis. ZNRF3 interacts with and ubiquitinates Hwa, thereby regulating its lysosomal trafficking and protein stability. Perturbation of ZNRF3 leads to the accumulation of Hwa and induction of an ectopic axis in embryos. Ectopic expression of ZNRF3 promotes Hwa degradation and dampens the axis-inducing activity of Hwa. Thus, our findings identify a substrate of ZNRF3, but also highlight the importance of the regulation of Hwa temporospatial activity in body axis formation in vertebrate embryos.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear ß-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.

Dact-4 is a Xenopus laevis Spemann organizer gene related to the Dapper/Frodo antagonist of ß-catenin family of proteins.

Dact/Dapper/Frodo members belong to an evolutionarily conserved family of Dishevelled-binding proteins present in mammals, birds, amphibians and fishes that are involved in the regulation of Wnt and TGF-ß signaling. In addition to the three established genes (Dact1-3) that compose the Dact family, a fourth paralogue group of related proteins has been recently identified and named Dact-4. Interestingly, Dact-4 is the most rapidly evolving gene of the entire family, as it displays very low homology with other Dact proteins and has lost key conserved domains. Dact-4 is not present in mammals, but weakly conserved homologs were found in reptiles and fishes. Recent RNAseq from our group identified new genes specifically expressed in the Xenopus laevis Spemann organizer. Among these, LOC100170590 mRNA encoded a protein sharing weak homology with a coelacanth Dact-like protein member. Here, by analyzing protein phylogeny and synteny, we show that this organizer gene corresponds to Dact-4. We report that Dact-4 is expressed in the Xenopus blastula pre-organizer region in addition to the gastrula organizer, as well as in placodes, eyes, neural tube, presomitic mesoderm and pronephros. Dact-4-Flag microinjection experiments suggest it is a nucleocytoplasmic protein, as are the other Dact paralogues.

Mcl1 protein levels and Caspase-7 executioner protease control axial organizer cells survival.

BACKGROUND: Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS: We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS: Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.

The tumor suppressor PTPRK promotes ZNRF3 internalization and is required for Wnt inhibition in the Spemann organizer.

A hallmark of Spemann organizer function is its expression of Wnt antagonists that regulate axial embryonic patterning. Here we identify the tumor suppressor Protein tyrosine phosphatase receptor-type kappa (PTPRK), as a Wnt inhibitor in human cancer cells and in the Spemann organizer of Xenopus embryos. We show that PTPRK acts via the transmembrane E3 ubiquitin ligase ZNRF3, a negative regulator of Wnt signaling promoting Wnt receptor degradation, which is also expressed in the organizer. Deficiency of Xenopus Ptprk increases Wnt signaling, leading to reduced expression of Spemann organizer effector genes and inducing head and axial defects. We identify a '4Y' endocytic signal in ZNRF3, which PTPRK maintains unphosphorylated to promote Wnt receptor depletion. Our discovery of PTPRK as a negative regulator of Wnt receptor turnover provides a rationale for its tumor suppressive function and reveals that in PTPRK-RSPO3 recurrent cancer fusions both fusion partners, in fact, encode ZNRF3 regulators.

Matrix-filled microcavities in the emerging avian left-right organizer.

BACKGROUND: Hensen node of the amniote embryo plays a central role in multiple developmental processes, especially in induction and formation of axial organs. In the chick, it is asymmetrical in shape and has recently been considered to represent the left-right organizer. As mechanisms of breaking the initial left-right symmetry of the embryo are still ill-understood, analyzing the node's microarchitecture may provide insights into functional links between symmetry breaking and asymmetric morphology. RESULTS: In the course of a light- and electron-microscopic study addressing this issue we discovered novel intercellular matrix-filled cavities in the node of the chick during gastrulation and during early neurulation stages; measuring up to 45 µm, they are surrounded by densely packed cells and filled with nanoscale fibrils, which immunostaining suggests to consist of the basement membrane-related proteins fibronectin and perlecan. The cavities emerge immediately prior to node formation in the epiblast layer adjacent to the tip of the primitive streak and later, with emerging node asymmetry, they are predominantly located in the right part of the node. Almost identical morphological features of microcavities were found in the duck node. CONCLUSIONS: We address these cavities as "nodal microcavities" and propose their content to be involved in the function of the avian node by mediating morphogen signaling and storage.

Organization of Embryonic Morphogenesis via Mechanical Information.

Embryonic organizers establish gradients of diffusible signaling molecules to pattern the surrounding cells. Here, we elucidate an additional mechanism of embryonic organizers that is a secondary consequence of morphogen signaling. Using pharmacological and localized transgenic perturbations, 4D imaging of the zebrafish embryo, systematic analysis of cell motion, and computational modeling, we find that the vertebrate tail organizer orchestrates morphogenesis over distances beyond the range of morphogen signaling. The organizer regulates the rate and coherence of cell motion in the elongating embryo using mechanical information that is transmitted via relay between neighboring cells. This mechanism is similar to a pressure front in granular media and other jammed systems, but in the embryo the mechanical information emerges from self-propelled cell movement and not force transfer between cells. The propagation likely relies upon local biochemical signaling that affects cell contractility, cell adhesion, and/or cell polarity but is independent of transcription and translation.

Barhl2 maintains T cell factors as repressors and thereby switches off the Wnt/ß-Catenin response driving Spemann organizer formation.

A hallmark of Wnt/ß-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in ß-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/ß-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/ß-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.

Zebrafish znfl1s regulate left-right asymmetry patterning through controlling the expression of fgfr1a.

Proper left-right (LR) axis establishment is critical for organogenesis in vertebrates. Previously, we reported that zinc finger transcription factors zinc finger transcription factor 1 (znfl1s) are expressed in the tailbud and axial mesoderm in zebrafish. However, a role of znfl1s in LR axis development has not been demonstrated. Here, we discovered that the knockdown of znfl1s using morpholino (MO) in whole embryos or dorsal forerunner cells (DFCs) interrupted LR asymmetry and normal development of the heart, liver, and pancreas. Whole-embryo knockdown of znfl1s by MO or clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) resulted in the absent expression of nodal gene spaw and Nodal signaling-related genes lft1, lft2, and pitx2c in the left lateral plate mesoderm (LPM), and Spaw, Lft1, Lft2, and Pitx2c play important roles in LR axis development in zebrafish. However, specific knockdown of znfl1s in DFCs resulted in random expression of spaw, lft1, lft2, and pitx2c. Knockdown of znfl1s led to abnormal cilia formation by the downregulation of fgfr1a and foxj1a expression. The expression of spaw, lft1, lft2, and pitx2c was partially rescued by the overexpression of fgfr1a mRNA in znfl1s morphants. Taken together, our results suggest that znfl1s regulate laterality development in zebrafish embryos through controlling the expression of fgfr1a.

On the Enigma of the Human Neurenteric Canal.

Existence and biomedical relevance of the neurenteric canal, a transient midline structure during early neurulation in the human embryo, have been controversially discussed for more than a century by embryologists and clinicians alike. In this study, the authors address the long-standing enigma by high-resolution histology and three-dimensional reconstruction using new and historic histological sections of 5 human 17- to 21-day-old embryos and of 2 marmoset monkey embryos of the species Callithrix jacchus at corresponding stages. The neurenteric canal presents itself as the classical vertical connection between the amniotic cavity and the yolk sac cavity and is lined (a) craniolaterally by a horseshoe-shaped "hinge of involuting notochordal cells" within Hensen's node and (b) caudally by the receding primitive streak epiblast dorsally and by notochordal plate epithelium ventrally, the latter of which covered the (longitudinal) notochordal canal on its ventral side at the preceding stage. Furthermore, asymmetric parachordal nodal expression in Callithrix and morphological asymmetries within the nodes of the other specimens suggest an early non-cilium-dependent left-right symmetry breaking mode previously postulated for other mammals. We conclude that structure and position of the mammalian neurenteric canal support the notion of its homology with the reptilian blastopore as a whole and with a dorsal segment of the blastopore in amphibia. These new features of the neurenteric canal may further clarify the aetiology of foetal malformations such as junctional neurulation defects, neuroendodermal cysts, and the split notochord syndrome.

WDR5 regulates left-right patterning via chromatin-dependent and -independent functions.

Congenital heart disease (CHD) is a major cause of infant mortality and morbidity, yet the genetic causes and mechanisms remain opaque. In a patient with CHD and heterotaxy, a disorder of left-right (LR) patterning, a de novo mutation was identified in the chromatin modifier gene WDR5 WDR5 acts as a scaffolding protein in the H3K4 methyltransferase complex, but a role in LR patterning is unknown. Here, we show that Wdr5 depletion leads to LR patterning defects in Xenopus via its role in ciliogenesis. Unexpectedly, we find a dual role for WDR5 in LR patterning. First, WDR5 is expressed in the nuclei of monociliated cells of the LR organizer (LRO) and regulates foxj1 expression. LR defects in wdr5 morphants can be partially rescued with the addition of foxj1 Second, WDR5 localizes to the bases of cilia. Using a mutant form of WDR5, we demonstrate that WDR5 also has an H3K4-independent role in LR patterning. Guided by the patient phenotype, we identify multiple roles for WDR5 in LR patterning, providing plausible mechanisms for its role in ciliopathies like heterotaxy and CHD.

Self-organization of a human organizer by combined Wnt and Nodal signalling.

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

ADHFe1: a novel enzyme involved in retinoic acid-dependent Hox activation.

Retinoic acid (RA) signaling is a central pathway regulating anterior-posterior patterning of the embryo through its targets, the Hox genes. RA is produced by two sequential oxidations from vitamin A (retinol) and this biosynthesis has to be regulated temporally, spatially and quantitatively. Mining Xenopus embryonic expression databases identified a novel component of the RA metabolic network, ADHFe1. Using Xenopus laevis embryos as our experimental system we determined the temporal and spatial pattern of AdhFe1 expression. Gain- and loss-of-function of ADHFe1 were induced to study its function and the regulation of the AdhFe1 gene by RA was studied. Expression analysis localized the ADHFe1 protein to the late Spemann's organizer, the trunk organizer. Subsequently, ADHFe1 can be detected in the prechordal mesoderm, the notochord and the lateral plate mesoderm. Manipulation of ADHFe1 levels affects the normal Hox gene expression. The effects of ADHFe1 manipulation can by rescued by increasing the levels of RA or its biosynthesis. Expression of the AdhFe1 gene is regulated by RA itself. ADHFe1 is an enzyme active already during gastrula stages and the protein is still present during neurula stages. ADHFe1 regulates the expression of the Hox genes during the early patterning of the trunk. The effect of ADHFe1 on Hox expression is mediated through regulation of RA levels. ADHFe1 probably reduces retinaldehyde to retinol thereby restricting the availability of retinaldehyde, the substrate needed by retinaldehyde dehydrogenases to produce RA making it a novel regulator of RA concentrations in the embryo and RA homeostasis.

A novel role of the organizer gene Goosecoid as an inhibitor of Wnt/PCP-mediated convergent extension in Xenopus and mouse.

Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.

Genome-wide analysis of dorsal and ventral transcriptomes of the Xenopus laevis gastrula.

RNA sequencing has allowed high-throughput screening of differential gene expression in many tissues and organisms. Xenopus laevis is a classical embryological and cell-free extract model system, but its genomic sequence had been lacking due to difficulties arising from allotetraploidy. There is currently much excitement surrounding the release of the completed X. laevis genome (version 9.1) by the Joint Genome Institute (JGI), which provides a platform for genome-wide studies. Here we present a deep RNA-seq dataset of transcripts expressed in dorsal and ventral lips of the early Xenopus gastrula embryo using the new genomic information, which was further annotated by blast searches against the human proteome. Overall, our findings confirm previous results from differential screenings using other methods that uncovered classical dorsal genes such as Chordin, Noggin and Cerberus, as well as ventral genes such as Sizzled, Ventx, Wnt8 and Bambi. Complete transcriptome-wide tables of mRNAs suitable for data mining are presented, which include many novel dorsal- and ventral-specific genes. RNA-seq was very quantitative and reproducible, and allowed us to define dorsal and ventral signatures useful for gene set expression analyses (GSEA). As an example of a new gene, we present here data on an organizer-specific secreted protein tyrosine kinase known as Pkdcc (protein kinase domain containing, cytoplasmic) or Vlk (vertebrate lonesome kinase). Overexpression experiments indicate that Pkdcc can act as a negative regulator of Wnt/ ß-catenin signaling independently of its kinase activity. We conclude that RNA-Seq in combination with the X. laevis complete genome now available provides a powerful tool for unraveling cell-cell signaling pathways during embryonic induction.