Evo-devo: variations on ancestral themes.

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.

The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.

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.

Integrating positional information at the level of Smad1/5/8.

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.

Activity-induced protocadherin arcadlin regulates dendritic spine number by triggering N-cadherin endocytosis via TAO2beta and p38 MAP kinases.

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.

Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos.

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.

Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta.

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.

Asymmetric mitosis: Unequal segregation of proteins destined for degradation.

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-beta-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.

Protocol for Probing Regulated Lysosomal Activity and Function in Living Cells.

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 ß-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).

Development of the vertebral morphogenetic field in the mouse: interactions between Crossveinless-2 and Twisted Gastrulation.

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.

Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.

We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.

Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center is required for brain formation in Xenopus laevis embryos.

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.

Mesodermal patterning by a gradient of the vertebrate homeobox gene goosecoid.

Amphibian mesoderm arises from the marginal zone of the early gastrula and generates various tissues such as notochord, muscle, kidney, and blood. Small changes (twofold) in the amount of microinjected messenger RNA encoding the goosecoid (gsc) homeodomain protein resulted in marked changes in the differentiation of mesoderm in Xenopus laevis. At least three thresholds were observed, which were sufficient to specify four mesodermal cell states. Endogenous gsc messenger RNA was expressed in the marginal zone in a graded fashion that is congruent with a role for this gene in dorso-ventral patterning of mesoderm at the early gastrula stage.

Organizer-specific homeobox genes in Xenopus laevis embryos.

The dorsal blastopore lip of the early Xenopus laevis gastrula can organize a complete secondary body axis when transplanted to another embryo. A search for potential gene regulatory components specifically expressed in the organizer was undertaken that resulted in the identification of four types of complementary DNAs from homeobox-containing genes that fulfill this criterion. The most abundant of these encodes a DNA-binding specificity similar to that of the Drosophila melanogaster anterior morphogen bicoid. The other three are also homologous to developmentally significant Drosophila genes. These four genes may participate in the regulation of the developmental potential of the organizer.

Vertebrate homeodomain proteins: families of region-specific transcription factors.

Vertebrate homeodomain proteins are transcription factors whose genes can be isolated via a conserved DNA-binding domain called the homeobox. We review recent studies suggesting that one function of these genes is the early subdivision of the embryo along the antero-posterior axis into 'fields' of cells with different developmental potential.

Neural plate patterning by secreted signals.

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.

Vertebrate axis formation.

Molecular understanding of axis formation has recently taken a great leap forward with the identification and functional characterization of regulatory genes that appear to act at the top of the hierarchy leading to positional specification in the vertebrate. Analysis of these genes, which encode peptide growth factors and their receptors as well as transcription factors, is disclosing principles of early cell fate specification that are common to all vertebrates.

The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos.

BACKGROUND: One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary. RESULTS: Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM. CONCLUSIONS: Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that establishes the segmental boundaries between posterior and anterior cells in adjacent segments.

Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus.

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.

Systems biology of embryonic morphogens.

A network of secreted proteins that interact with each other in the extracellular space regulates embryonic morphogenesis. Mathematical modeling offers an excellent opportunity to understand how morphogens signal and self-regenerate pattern.