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The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture.In vivo, studies in theXenopussystem showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.SignificanceWnt/β-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylated-enriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes.Xenopusembryo assays revealed that TFG is requiredin vivofor canonical Wnt signaling.

Summary Lysosomes are the catabolic center of the cell. Limitations of many lysosomal tracers include low specificity and lack of reliable physiological readouts for changes in growth factor-regulated lysosomal activity. The imaging-based protocols described here provide insights at the cellular level to quantify functions essential to lysosomal biology, including β-glucosidase enzymatic cleavage, active Cathepsin D, and pH regulation in real time. These optimized protocols, applied in different cell types and pathophysiologic contexts, provide useful tools to study lysosome function in cultured living cells. For complete details on the use and execution of this protocol, please refer to Albrecht et al. (2020)., Graphical Abstract, Highlights • Protocol for visualizing and quantitively examining lysosomal catabolism • Details how organelle pH gradient shifts can be dynamically traced in living cells • Describes approaches to determine levels of active Cathepsin D in lysosomes • β-Glucosidase, a target in Gaucher disease, can be assessed by substrate cleavage, Lysosomes are the catabolic center of the cell. Limitations of many lysosomal tracers include low specificity and lack of reliable physiological readouts for changes in growth factor-regulated lysosomal activity. The imaging-based protocols described here provide insights at the cellular level to quantify functions essential to lysosomal biology, including β-glucosidase enzymatic cleavage, active Cathepsin D, and pH regulation in real time. These optimized protocols, applied in different cell types and pathophysiologic contexts, provide useful tools to study lysosome function in cultured living cells.

Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2(-/-) or Chd(-/-) mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2(-/-) mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2(-/-) embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2(-/-)vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.

The RNA-binding protein Bicaudal C is an important regulator of embryonic development in C. elegans, Drosophila and Xenopus. In mouse, bicaudal C (Bicc1) mutants are characterized by the formation of fluid-filled cysts in the kidney and by expansion of epithelial ducts in liver and pancreas. This phenotype is reminiscent of human forms of polycystic kidney disease (PKD). Here, we now provide data that Bicc1 functions by modulating the expression of polycystin 2 (Pkd2), a member of the transient receptor potential (TRP) superfamily. Molecular analyses demonstrate that Bicc1 acts as a post-transcriptional regulator upstream of Pkd2. It regulates the stability of Pkd2 mRNA and its translation efficiency. Bicc1 antagonized the repressive activity of the miR-17 microRNA family on the 3′UTR of Pkd2 mRNA. This was substantiated in Xenopus, in which the pronephric defects of bicc1 knockdowns were rescued by reducing miR-17 activity. At the cellular level, Bicc1 protein is localized to cytoplasmic foci that are positive for the P-body markers GW182 and HEDLs. Based on these data, we propose that the kidney phenotype in Bicc1−/− mutant mice is caused by dysregulation of a microRNA-based translational control mechanism.

Embryos and developing organs have the remarkable ability of self-regenerating after experimental manipulations. In the Xenopus blastula half-embryos can regenerate the missing part, producing identical twins. Studies on the molecular nature of Spemann's organizer have revealed that self-regulation results from the battle between two signaling centers under reciprocal transcriptional control. Long-range communication between the dorsal and ventral sides is mediated by the action of growth factor antagonists - such as the BMP antagonist Chordin - that regulate the flow of BMPs within the embryonic morphogenetic field. BMPs secreted by the dorsal Spemann organizer tissue are released by metalloproteinases of the Tolloid family, which cleave Chordin at a distance of where they were produced. The dorsal center secretes Chordin, Noggin, BMP2 and ADMP. The ventral center of the embryo secretes BMP4, BMP7, Sizzled, Crossveinless-2 and Tolloid-related. Crossveinless-2 binds Chordin/BMP complexes, facilitating their flow towards the ventral side, where BMPs are released by Tolloid allowing peak BMP signaling. Self-regulation occurs because transcription of ventral genes is induced by BMP while transcription of dorsal genes is repressed by BMP signals. This assures that for each action of Spemann's organizer there is a reaction in the ventral side of the embryo. Because both dorsal and ventral centers express proteins of similar biochemical activities, they can compensate for each other. A novel biochemical pathway of extracellular growth factor signaling regulation has emerged from these studies in Xenopus. This remarkable dorsal-ventral positional information network has been conserved in evolution and is ancestral to all bilateral animals.

Mitotic cell division ensures that two daughter somatic cells inherit identical genetic material. Previous work has shown that signaling by the Smad1 transcription factor is terminated by polyubiquitinylation and proteasomal degradation after essential phosphorylations by MAPK and glycogen synthase kinase 3 (GSK3). Here, we show that, unexpectedly, proteins specifically targeted for proteasomal degradation are inherited preferentially by one mitotic daughter during somatic cell division. Experiments with dividing human embryonic stem cells and other mammalian cultured cell lines demonstrated that in many supposedly equal mitoses the segregation of proteins destined for degradation (Smad1 phosphorylated by MAPK and GSK3, phospho-β-catenin, and total polyubiquitinylated proteins) was asymmetric. Transport of pSmad1 targeted for degradation to the centrosome required functional microtubules. In vivo , an antibody specific for Mad phosphorylated by MAPK showed that this antigen was associated preferentially with one of the two centrosomes in Drosophila embryos at cellular blastoderm stage. We propose that this remarkable cellular property may be explained by the asymmetric inheritance of peripheral centrosomal proteins when centrioles separate and migrate to opposite poles of the cell, so that one mitotic daughter remains pristine. We conclude that many mitotic divisions are unequal, unlike what was previously thought.

The blastula Chordin- and Noggin-expressing (BCNE) center located in the dorsal animal region of the Xenopus blastula embryo contains both prospective anterior neuroectoderm and Spemann organizer precursor cells. Here we show that, contrary to previous reports, the canonical Wnt target homeobox genes, Double knockdown of these genes using antisense morpholinos in Xenopus laevis blocked head formation, reduced the expression of the other BCNE center genes, upregulated Bmp4 expression, and nullified hyperdorsalization by lithium chloride. Moreover, gain- and loss-of-function experiments showed that Siamois and Twin expression is repressed by the vegetal transcription factor VegT. We propose that VegT expression causes maternal beta-Catenin signals to restrict Siamois and Twin expression to the BCNE region. A two-step inhibition of BMP signals by Siamois and Twin-- first by transcriptional repression of Bmp4 and then by activation of the expression of the BMP inhibitors Chordin and Noggin--in the BCNE center is required for head formation.

Most animals evolved from a common ancestor, Urbilateria, which already had in place the developmental genetic networks for shaping body plans. Comparative genomics has revealed rather unexpectedly that many of the genes present in bilaterian animal ancestors were lost by individual phyla during evolution. Reconstruction of the archetypal developmental genomic tool-kit present in Urbilateria will help to elucidate the contribution of gene loss and developmental constraints to the evolution of animal body plans.

Synaptic activity induces changes in the number of dendritic spines. Here, we report a pathway of regulated endocytosis triggered by arcadlin, a protocadherin induced by electroconvulsive and other excitatory stimuli in hippocampal neurons. The homophilic binding of extracellular arcadlin domains activates TAO2beta, a splice variant of the thousand and one amino acid protein kinase 2, cloned here by virtue of its binding to the arcadlin intracellular domain. TAO2beta is a MAPKKK that activates the MEK3 MAPKK, which phosphorylates the p38 MAPK. Activation of p38 feeds-back on TAO2beta, phosphorylating a key serine required for triggering endocytosis of N-cadherin at the synapse. Arcadlin knockout increases the number of dendritic spines, and the phenotype is rescued by siRNA knockdown of N-cadherin. This pathway of regulated endocytosis of N-cadherin via protocadherin/TAO2beta/MEK3/p38 provides a molecular mechanism for transducing neuronal activity into changes in synaptic morphologies.

SummaryHere we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl ┤ Xlr ┤ Chd ┤ BMP → P-Smad1 → Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10−8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.

SummaryEmbryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.

Xenopus embryonic ectodermal cells dissociated for three or more hours differentiate into neural tissue instead of adopting their normal epidermal fate. This default type of neural induction occurs in the absence of Spemann's organizer signals and is thought to be caused by the dilution of endogenous BMPs into the culture medium. Unexpectedly, we observed that BMP ligands continue to signal in dissociated cells. Instead, cell dissociation induces a sustained activation of the Ras/MAPK pathway, which causes the phosphorylation of Smad1 at sites that inhibit the activity of this transcription factor. It is this activation of Ras/MAPK that is required for neuralization in dissociated ectoderm.

Twisted gastrulation (Tsg) is a secreted protein that regulates Bmp signaling in the extracellular space through its direct interaction with Bmp/Dpp and Chordin (Chd)/Short gastrulation (Sog). The ternary complex of Tsg/Chd/Bmp is cleaved by the metalloprotease Tolloid (Tld)/Xolloid (Xld). Studies in Drosophila, Xenopus and zebrafish suggest that Tsg can act both as an anti-Bmp and as a pro-Bmp. We have analyzed Tsg loss-of-function in the mouse. Tsg homozygous mutants are viable but of smaller size and display mild vertebral abnormalities and osteoporosis. We provide evidence that Tsg interacts genetically with Bmp4 . When only one copy of Bmp4 is present, a requirement of Tsg for embryonic development is revealed. Tsg -/- ;Bmp4 +/- compound mutants die at birth and display holoprosencephaly, first branchial arch and eye defects. The results show that Tsg functions to promote Bmp4 signaling during mouse head development.

The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. We present the phenotype produced by the targeted inactivation of the chordin gene in mouse. Chordin homozygous mutant mice show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally,displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis.

A growing body of work indicates that neural induction may be initiated prior to the establishment of the gastrula mesodermal organizer. Here, we examine neural induction in Xenopus embryos in which mesoderm induction has been blocked by Cerberus-short, a reagent that specifically inhibits Nodal-related (Xnr) signals. We find that extensive neural structures with cyclopic eyes and brain tissue are formed despite the absence of mesoderm. This neural induction correlates with the expression of chordin and other BMP inhibitors-such as noggin, follistatin, and Xnr3-at the blastula stage, and requires beta-Catenin signaling. Activation of the beta-Catenin pathway by mRNA microinjections or by treatment with LiCl leads to differentiation of neurons, as well as neural crest, in ectodermal explants. Xnr signals are required for the maintenance, but not for the initiation, of BMP antagonist expression. Recent work has demonstrated a role for beta-Catenin signaling in neural induction mediated by the transcriptional down-regulation of BMP-4 expression. The present results suggest an additional function for beta-Catenin, the early activation of expression of secreted BMP antagonists, such as Chordin, in a preorganizer region in the dorsal side of the Xenopus blastula.

Cysteine-rich repeats (CRs) of the type described in Chordin constitute conserved domains present in an expanding family of secreted molecules. These motifs were shown to mediate directly the antagonism of BMP signaling by Chordin and play a major role during development. Here we report the cloning and expression pattern of neuralin-1, a new member of the chordin family. The mouse cDNA was cloned by homology with a human genomic sequence encoding putative CRs. In the human genome, neuralin-1 transcripts are encoded by 8 exons that span a region of at least 80 kilobases located on chromosome Xq22.1-23. Neuralin-1 is a 333 amino acid protein containing three CRs, two of them highly similar to the Chordin CRs that bind BMP. Like chordin, neuralin-1 is able to induce secondary axes after mRNA injection in Xenopus embryos. Interestingly, during late gastrulation, neuralin-1 and chordin present distinct and complementary expression patterns in the mouse: neuralin-1 expression starts in the neural plate at mid-gastrulation, whereas chordin expression at that stage is restricted to the node and midline mesendoderm. Later on, neuralin-1 expression becomes restricted to discrete regions of the central nervous system and to derivatives of the neural crest cells. During organogenesis, neuralin-1 presents a broad expression pattern in many tissues such as dorsal root ganglia, gut, condensing cartilages of the skeleton and developing hair follicles.

To determine the spectrum of secreted proteins that are present in the extracellular space of early Xenopus embryos, a direct secretion screen was performed. Surprisingly, 24% of previously identified bona fide secretory proteins corresponded to four secreted Wnt antagonists of the same family: frzb-1, sizzled, sfrp-2 and crescent. sfrp-2 and crescent are novel components of the growing cocktail of growth factor antagonists secreted by Spemann organizer cells in Xenopus. Crescent is first expressed at blastula, defining a deep endodermal region that may be homologous to the avian hypoblast. Unlike other members of this family of inhibitors, microinjection of crescent mRNA causes the development of cyclopic embryos, even though the amount of anterior neural tissue is normal. In crescent-injected embryos, studies with specific markers indicate that morphogenetic movements of the anterior midline are abnormal, resulting in a more posterior location of prechordal plate and ventral forebrain markers with respect to the developing eye field. The results are discussed in light of recent findings in zebrafish and Xenopus that suggest that Wnt signaling through non-canonical (non-beta-catenin dependent) pathways plays a pivotal role in the regulation of morphogenetic movements.

Axial structures (neural tube/notochord) and surface ectoderm activate myogenesis in the mouse embryo; their action can be reproduced, at least in part, by several molecules such as Sonic hedgehog and Wnts. Recently, soluble Wnt antagonists have been identified. Among those examined only Frzb1 was found to be expressed in the presomitic mesoderm and newly formed somites and thus its possible role in regulating myogenesis was investigated in detail. When presomitic mesoderm or newly formed somites were cultured with axial structures and surface ectoderm on a feeder layer of C3H10T1/2 cells expressing Frzb1, myogenesis was abolished or severely reduced in presomitic mesoderm and the three most recently formed somites. In contrast, no effect was observed on more mature somites. Inhibition of myogenesis did not appear to be associated with increased cell death since the final number of cells in the explants grown in the presence of Frzb1 was only slightly reduced in comparison with controls. In order to examine the possible function of Frzb1 in vivo, we developed a method based on the overexpression of the soluble antagonist by transient transfection of WOP cells with a Frzb1 expression vector and injection of transfected cells into the placenta of pregnant females before the onset of maternofoetal circulation. Frzb1, secreted by WOP cells, accumulated in the embryo and caused a marked reduction in size of caudal structures. Myogenesis was strongly reduced and, in the most severe cases, abolished. This was not due to a generalized toxic effect since only several genes downstream of the Wnt signaling pathway such as En1, Noggin and Myf5 were downregulated; in contrast, Pax3 and Mox1 expression levels were not affected even in embryos exhibiting the most severe phenotypes. Taken together, these results suggest that Wnt signals may act by regulating both myogenic commitment and expansion of committed cells in the mouse mesoderm.

Objective: To study pressure-volume (P/V) curves over a wide pressure and volume range in pigs.¶Design: Dynamic and static P/V curves (Pdyn/V and Pst/V) and compliance of the respiratory system were studied. The effects of recruitment, positive end-expiratory pressure (PEEP) and body position were analysed.¶Setting: Research animal laboratory.¶Materials: Seven anaesthetised, paralysed and ventilated healthy pigs of 21 kg.¶Measurements: P/V curves up to a pressure of about 40 cmH2O were recorded with a computer-controlled ventilator. Pst/V curves were obtained with the static occlusion method and Pdyn/V curves during an insufflation at a low, constant flow rate.¶Results: Pdyn/V recording showed a complex pattern. During the insufflation compliance increased, fell, increased and fell again. A 2nd ¶Pdyn/V recording immediately following the 1st one was displaced towards higher volumes and showed only one maximum of compliance. The difference between the two curves reflected: (1) lung collapse during a period of 5 min of ventilation at zero end-expiratory pressure (ZEEP) following a recruitment manoeuvre, (2) recruitment during the measurement of the 1st Pdyn/V curve. These observations were similar in the supine and in the left lateral position. After ventilation at PEEP, 4 cmH2O, the signs of collapse and recruitment were reduced. It was confirmed that PEEP offers a partial protection against collapse. Pst/V curves showed higher volumes and higher compliance values compared to Pdyn/V curves. This reflects the influence of viscoelastance on Pdyn/V curves.¶Conclusion: The study demonstrates a particularly strong tendency to lung collapse in pigs.

The head inducer Cerberus is a multivalent extracellular inhibitor Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants.

Zebrafish paraxial protocadherin (papc) encodes a transmembrane cell adhesion molecule (PAPC) expressed in trunk mesoderm undergoing morphogenesis. Microinjection studies with a dominant-negative secreted construct suggest that papc is required for proper dorsal convergence movements during gastrulation. Genetic studies show that papc is a close downstream target of spadetail, gene encoding a transcription factor required for mesodermal morphogenetic movements. Further, we show that the floating head homeobox gene is required in axial mesoderm to repress the expression of both spadetail and papc, promoting notochord and blocking differentiation of paraxial mesoderm. The PAPC structural cell-surface protein may provide a link between regulatory transcription factors and the actual cell biological behaviors that execute morphogenesis during gastrulation.

Nuclear receptors are ligand-modulated transcription factors that respond to steroids, retinoids, and thyroid hormones to control development and body physiology. Orphan nuclear receptors, which lack identified ligands, provide a unique, and largely untapped, resource to discover new principles of physiologic homeostasis. We describe the isolation and characterization of the vertebrate orphan receptor, BXR, which heterodimerizes with RXR and binds high-affinity DNA sites composed of a variant thyroid hormone response element. A bioactivity-guided screen of embryonic extracts revealed that BXR is activatable by low-molecular-weight molecules with spectral patterns distinct from known nuclear receptor ligands. Mass spectrometry and 1 H NMR analysis identified alkyl esters of amino and hydroxy benzoic acids as potent, stereoselective activators. In vitro cofactor association studies, along with competable binding of radiolabeled compounds, establish these molecules as bona fide ligands. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway.

Spemann's organizer has potent neural inducing and mesoderm dorsalizing activities in the Xenopus gastrula. A third activity, the organizer's ability to induce a secondary gut, has been difficult to analyze experimentally due to the lack of early gene markers. Here we introduce endodermin, a pan-endodermal gene marker, and use it to demonstrate that chordin (Chd), a protein secreted by the organizer region, is able to induce endodermal differentiation in Xenopus. The ability of chd, as well as that of noggin, to induce endoderm in animal cap explants is repressed by the ventralizing factor BMP-4. When FGF signaling is blocked by a dominant-negative FGF receptor in chd-injected animal caps, neural induction is inhibited and most of the explant is induced to become endoderm. The results suggest that proteins secreted by the organizer, acting together with known peptide growth factors, regulate differentiation of the endodermal germ layer.

The availability of molecular markers now permits the analysis of the common elements of vertebrate gastrulation. While gastrulation appears to be very diverse in the verte brates, by analyzing a head-organizer marker, goosecoid, and a marker common to all forming mesoderm, Brachyury, we attempt to identify homologous structures and equivalent stages in Xenopus, zebrafish, chick and mouse gastrulation. Using a tail-organizer marker, Xnot-2, we also discuss how the late stages of gastrulation lead to the formation of the postanal tail, a structure characteris tic of the chordates.

The patterning of the CNS relies on the interaction of multiple signaling molecules such as Sonic Hedgehog, Wnts, and BMPs and their antagonists Chordin and Noggin. The identification of the secreted molecule Tiarin (Tsuda et al., 2002, this issue of Neuron), produced by the nonneural ectoderm at border of the anterior and lateral neural plate, now introduces a novel signaling pathway participating in CNS development.

Three lines of evidence suggest that tail formation in Xenopus is a direct continuation of events initiated during gastrulation. First, the expression of two gene markers, Xbra and Xnot2, can be followed from the blastopore lip into distinct cell populations of the developing tailbud. Second, the tip of the tail retains Spemann’s tail organizer activity until late stages of development. Third, lineage studies with the tracer DiI indicate that the cells of the late blastopore are fated to form specific tissues of the tailbud, and that intercalation of dorsal cells continues during tail elongation. In particular, the fate map shows that the tip of the tail is a direct descendant of the late dorsal blastopore lip. Thus, the tailbud is not an undifferentiated blastema as previously thought, but rather consists of distinct cell populations which arise during gastrulation.

IN 1941 caspersson, Landstrom-Hyden and Aquilonius1 demonstrated that the chromidial substance contained in the pancreatic exocrine cells and in other serous secreting elements had a typical absorption spectrum in the ultra-violet region with a peak in 2600 A. As this substance showed a negative Feulgen reaction, they concluded that it is a nucleoprotein with a high content of nucleotides of ribonucleic type. In the apical part of the cell containing the granules, they found absorption curves of a protein type, but with a concentration much lower than in the basal zone ; they explain this difference as due to the dilution of the secretion.

Publisher Summary This chapter focuses on the integration of three different pathways, bone morphogenetic protein (BMP), RTK, and Wnt, at the level of a single transcription factor, Smad1, in the control of the differentiation of the ectoderm into neural or epidermal fates. It also discusses other situations in which Smads are utilized as a platform for the integration of different signaling pathways. During embryonic development, there are multiple situations in which the fibroblast growth factors (FGF)/mitogen-activated protein kinase (MAPK) pathway cross-talks with BMP signals. The MAPK-dependent phosphorylations of Smad1 are required for the inhibitory effects of BMP/Smads on androgen receptor function, and the integration of MAPK signals on Smad1 has a critical role in the androgen regulation of prostate cancer progression. An analogous mechanism of signal integration has been found for the transcription factors Smad2 and Smad3, which transduce signals of transforming growth factor β (TGFβ). The TGFβ growth factor pathway is a very important tumor suppressor in mammals. In Xenopus , the TGFβ molecule activin induces the expression of mesodermal genes in ectodermal cells, but this ability is lost after a certain point of development. This loss of competence correlated with the inability of Smad2 to accumulate in the nucleus, and mutation of conserved MAPK phosphorylation sites (SP) at the linker region of Smad2 prolongs the competence of cells to respond to activin by inducing mesodermal genes. TGFβs are also potent inhibitors of cell proliferation, and have crucial roles in the control of tumorigenesis. Certain types of cancer are resistant to the growth-inhibitory effects of TGFβ. In transformed cell lines with activated oncogenic Ras/MAPK signals, Smad2/3 protein displays elevated phosphorylation at their MAPK sites, which inhibits TGFβ-induced nuclear accumulation.

In a search for nuclear hormone receptors expressed in early development we found that Xenopus laevis eggs contain mRNAs from two retinoic acid receptor genes (xRAR alpha and xRAR gamma) and two retinoid "X" receptor genes (xRXR alpha and xRXR gamma). We also show that RXRs are members of a family of at least three genes, thus expanding the number of genes encoding retinoic acid-responsive transcription factors to six. With the exception of xRXR gamma, these maternal mRNAs are degraded before gastrulation. The RXRs isolated are differentially activated by retinoic acid and by 3,4-didehydroretinoic acid. Considered together, these four receptors provide a molecular basis for the pleiotropic effects of retinoic acid on early development, and their pattern of expression suggests a role for retinoic acid at the earliest stages of embryonic determination.

BMPs pattern the dorsal–ventral axis of vertebrate embryos. Smad1/5/8 transduces the BMP signal, and receives phosphorylation inputs from both MAPK and GSK3. Phosphorylation of Smad1 by MAPK and GSK3 result in its polyubiquitination and transport to the centrosome where it is degraded by the proteasome. These linker phosphorylations inhibit BMP/Smad1signaling by shortening its duration. Wnt, which negatively regulates GSK3 activity, prolongs the BMP/Smad1 signal. Remarkably, linker-phosphorylated Smad1 has been shown to be inherited asymmetrically during cell division. Drosophila contains a single Smad1/5/8 homologue, Mad, and is stabilized by phosphorylation-resistant mutations at GSK3 sites, causing Wingless-like effects. We summarize here the significance of linker-phosphorylated Smad1/Mad in relation to signal intensity and duration, and how this integrates the Wnt and BMP pathways during cell differentiation.

We have injected XIHbox 6 mRNA together with the lineage tracer colloidal gold into individual dorso-anterior blastomeres of the 32-cell stage Xenopus embryo and analyzed their cell fate during embryogenesis. While the developing tadpoles appeared entirely normal, the fate of the progeny of the injected blastomere was altered. In the brain injected cells failed to differentiate terminally, as indicated by a loss of labeled cranial nerves. Differentiation of spinal nerves remained unaffected. Fate change in the CNS occurred at about the time of normal XIHbox 6 protein expression. In addition, progeny of injected blastomeres gained head epidermal fate and lost anterior notochord fate as a result of altered cell migrations during gastrulation. The results show that a homeodomain protein is capable of altering cell fate in a position-specific and cell-autonomous manner in Xenopus embryos. The experimental approach used here should be applicable to other molecules specifying cell fate.

We review here old experiments that defined the existence of morphogenetic gradient fields in vertebrate embryos. The rather abstract idea of cell fields of organforming potential has become less popular among modern developmental and molecular biologists. Results obtained with antibodies directed against homeodomain proteins suggest that gradient fields may indeed be visualized at the level of individual regulatory molecules in vertebrate embryos.

The intensity of the BMP signal is determined by cell surface receptors that phosphorylate Smad1/5/8 at the C-terminus. In addition to this BMP-activated phosphorylation, recent studies have shown that sequential phosphorylations by MAPK and GSK3 kinases can negatively regulate the activity of the pSmad1Cter signal. These phosphorylations in the linker region cause Smad1 to be transported to the centrosomal region, polyubiquitinylated and degraded by the proteasomal machinery. In Xenopus embryos, Wnt signals, which regulate GSK3, induce ectoderm to adopt an epidermal fate, and this Wnt effect requires an active BMP-Smad1/5/8 signaling pathway. These findings have profound implications for understanding how dorsal-ventral and anterior-posterior patterning are seamlessly integrated in the early embryonic morphogenetic field.

The combination of DNA cloning in bacterial plasmids and DNA injection into frog oocyte nuclei permits a novel type of genetic analysis in which the function of defined sequences of DNA may be more readily and precisely investigated than before. Purified segments of chromosomal DNA containing 5S genes of Xenopus have been injected into the nuclei of Xenopus oocytes and the products of these genes recognized as labelled 5S RNA. Since short linear molecules of DNA are degraded in oocytes, the function of particular regions of DNA near a 5S gene is best determined by excising these regions and joining them to other kinds of DNA. This is achieved most simply by inserting regions of 5S DNA into a plasmid. Our results with alpha-amanitin show that the activity of a 5S DNA promoter inserted into a plasmid can be detected independently of the plasmid's own promoters, using DNA injection into oocytes. The observation that plasmids containing variant 5S genes result in the synthesis of abnormal 5S RNA molecules illustrates the possibility of mutanting recombinant plasmids and then testing the biological effect of these mutations by DNA injection.

Homeoproteins are functionally involved in pattern formation. Recently, homeoproteins have been shown to be distributed in a graded fashion in developing limb buds. Here we examine the expression of homeoproteins in chicken feather development by immunocytochemical localization. We find that XlHbox 1 antigen is present in cell nuclei and is distributed in a gradient in the mesoderm of developing feather buds, with strongest expression in the anterior-proximal region. The gradient is most obvious in feather buds from the mid-trunk level. Feather buds from the scapular level express very high levels of XlHbox 1 and feather buds from the caudal region express no XlHbox 1, suggesting that a broad gradient along the body axis is superimposed on a smaller gradient within each individual feather bud. Feather ectoderm also expresses XlHbox 1 antigen but without an obvious graded pattern. Another homeoprotein, Hox 5.2, is also expressed in developing feather buds in a graded way, and its distribution pattern is partially complementary to that of XlHbox 1. These observations suggest that homeoproteins may be involved in setting up the anteroposterior polarity of cell fields at different levels, first for the body axis, then for the limb axis and finally for the feather axis.

What is the nature of positional information during embryogenesis? By using Xenopus homeo box genes as anteroposterior (A-P) markers, we confirm the findings of others that mesoderm-inducing growth factors and retinoic acid (RA) can provide positional information along the axis of the body. Xenopus tissue culture-mesoderm-inducing factor (XTC-MIF) selectively activates an anteriorly expressed homeo box gene (XlHbox 1), while basic fibroblast growth factor (bFGF) activates selectively a posteriorly expressed homeo box gene (XlHbox 6). RA activates expression of the posterior gene XlHbox 6, but not of XlHbox 1. This activation, however, requires exposure to growth factors. The data suggest that growth factors and RA may cooperate with each other to provide positional information in vertebrates.

Homeobox gene XlHbox 1 is expressed in a mesodermal gradient in vertebrate forelimbs with maximal expression anteriorly and proximally and may encode positional values. In chick wing buds, anterior cells can be reprogrammed to form posterior structures by grafts of polarizing region tissue and by beads soaked in retinoic acid (RA), which is a good candidate for an endogenous morphogen. Applications of RA anteriorly or at the bud apex, treatments which produce duplicated digits or truncations respectively, substantially increase the extent of mesodermal XlHbox 1 expression. Polarizing region grafts that also produce additional digits lead to a moderate increase. The effects of RA application and the behaviour of transplanted tissue show that only anterior cells are competent to express XlHbox 1 and that expression is cell autonomous. Ectodermal expression in wing buds is enhanced by RA but not by polarizing region grafts and ectoderm/mesoderm recombinations show that the mesoderm is irreversibly affected. The changes in mesodermal expression do not fit the predictions of the simple model that XlHbox 1 encodes anterior positional values but are correlated with a series of novel malformations of the shoulder girdle which, in normal wing buds, is derived from cells expressing XlHbox 1.

Crossveinless-2 (Cv2), Twisted Gastrulation (Tsg) and Chordin (Chd) are components of an extracellular biochemical pathway that regulates Bone Morphogenetic Protein (BMP) activity during dorso-ventral patterning of Drosophila and Xenopus embryos, the formation of the fly wing, and mouse skeletogenesis. Because the nature of their genetic interactions remained untested in the mouse, we generated a null allele for Cv2 which was crossed to Tsg and Chd mutants to obtain Cv2; Tsg and Cv2; Chd compound mutants. We found that Cv2 is essential for skeletogenesis as its mutation caused the loss of multiple bone structures and posterior homeotic transformation of the last thoracic vertebra. During early vertebral development, Smad1 phosphorylation in the intervertebral region was decreased in the Cv2 mutant, even though CV2 protein is normally located in the future vertebral bodies. Because Cv2 mutation affects BMP signaling at a distance, this suggested that CV2 is involved in the localization of the BMP morphogenetic signal. Cv2 and Chd mutations did not interact significantly. However, mutation of Tsg was epistatic to all CV2 phenotypes. We propose a model in which CV2 and Tsg participate in the generation of a BMP signaling morphogenetic field during vertebral formation in which CV2 serves to concentrate diffusible Tsg/BMP4 complexes in the vertebral body cartilage.

We present a loss-of-function study using antisense morpholino (MO) reagents for the organizer-specific gene Goosecoid (Gsc) and the ventral genes Vent1 and Vent2. Unlike in the mouse Gsc is required in Xenopus for mesodermal patterning during gastrulation, causing phenotypes ranging from reduction of head structures-including cyclopia and holoprosencephaly-to expansion of ventral tissues in MO-injected embryos. The overexpression effects of Gsc mRNA require the expression of the BMP antagonist Chordin, a downstream target of Gsc. Combined Vent1 and Vent2 MOs strongly dorsalized the embryo. Unexpectedly, simultaneous depletion of all three genes led to a rescue of almost normal development in a variety of embryological assays. Thus, the phenotypic effects of depleting Gsc or Vent1/2 are caused by the transcriptional upregulation of their opposing counterparts. A principal function of Gsc and Vent1/2 homeobox genes might be to mediate a self-adjusting mechanism that restores the basic body plan when deviations from the norm occur, rather than generating individual cell types. The results may shed light on the molecular mechanisms of genetic redundancy.

In Xenopus, two signaling systems, maternal beta-Catenin and Nodal-related, are required for induction of the Spemann organizer and establishment of the body plan. By screening cDNA macroarrays for genes activated by these two signaling pathways, we identified Xenopus xBtg-x, a novel member of the Btg/Tob gene family of antiproliferative proteins. We show that xBtg-x is expressed in the dorsal mesendoderm (Spemann organizer tissue) of gastrula stage embryos and that its expression is regulated by both beta-Catenin and Nodal-related signals. Microinjection of synthetic xBtg-x mRNA into Xenopus embryos induced axis duplication and completely rescued the ventralizing effects of UV irradiation through the activation of the canonical Wnt/beta-Catenin signaling pathway. Interestingly, xBtg-x stimulated beta-Catenin-dependent transcription without affecting the stability of beta-Catenin protein. These data suggest that xBtg-x is a novel component of the Wnt/beta-Catenin signaling pathway regulating early embryonic patterning.

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early beta-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator beta-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula beta-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.

Embryology is a discipline that has been traditionally strongly influenced by its history. Its most important experiment was carried out by Hans Spemann and Hilde Mangold in Freiburg, Germany, in 1924. An English translation of their epoch-making paper, by Viktor Hamburger, is available in the Festschrift celebrating the 75th anniversary of this work (Spemann and Mangold 1924; de Robertis and Arechaga 2001). By transplanting the dorsal lip into the ventral side of a salamander gastrula of a different species, they found that the graft could induce a secondary neural plate and a twinned body axis. The dorsal lip was able to induce central nervous system (CNS), dorsal mesoderm (in particular somites and pronephros) and a secondary gut cavity. Since the fate of grafted cells could be followed by their different pigmentation, they were able to demonstate that the dorsal blastopore lip had very powerful inductive properties on neighboring cells. This experiment led to the realization that vertebrate cell differentiation progresses through a series of successive cell-cell inductions. For this well-known work Spemann received the Nobel Prize of Medicine in 1935 (Spemann 1938; Sander and Faessler 2001). When cloning became practical, it became possible to identify the molecules responsible for this remarkable inducing activity. In this chapter we review the molecular exploration of the gene products that execute the phenomenon of the Spemann organizer.

Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells. In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin. Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases. We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins. Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut. By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin. Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus. The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.

Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-beta binding domains. Here we show that CTGF directly binds BMP4 and TGF-beta 1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-beta 1. These results show that CTGF inhibits BMP and activates TGF-beta signals by direct binding in the extracellular space.