Anterior-posterior Axis Formation Research Articles

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.

CRISPR/Cas9 and FLP-FRT mediated regulatory dissection of the BX-C of Drosophila melanogaster.

The homeotic genes or Hox define the anterior-posterior (AP) body axis formation in bilaterians and are often present on the chromosome in an order collinear to their function across the AP axis. However, there are many cases wherein the Hox are not collinear, but their expression pattern is conserved across the AP axis. The expression pattern of Hox is attributed to the cis-regulatory modules (CRMs) consisting of enhancers, initiators, or repressor elements that regulate the genes in a segment-specific manner. In the Drosophila melanogaster Hox complex, the bithorax complex (BX-C) and even the CRMs are organized in an order that is collinear to their function in the thoracic and abdominal segments. In the present study, the regulatorily inert regions were targeted using CRISPR/Cas9 to generate a series of transgenic lines with the insertion of FRT sequences. These FRT lines are repurposed to shuffle the CRMs associated with Abd-B to generate modular deletion, duplication, or inversion of multiple CRMs. The rearrangements yielded entirely novel phenotypes in the fly suggesting the requirement of such complex manipulations to address the significance of higher order arrangement of the CRMs. The functional map and the transgenic flies generated in this study are important resources to decipher the collective ability of multiple regulatory elements in the eukaryotic genome to function as complex modules.

Decoding anterior-posterior axis emergence among mouse, monkey, and human embryos.

Anterior-posterior axis formation regulated by the distal visceral endoderm (DVE) and anterior visceral endoderm (AVE) is essential for peri-implantation embryogenesis. However, the principles of the origin and specialization of DVE and AVE remain elusive. Here, with single-cell transcriptome analysis and pseudotime prediction, we show that DVE and AVE independently originate from the specialized primary endoderm in mouse blastocysts. Along distinct developmental paths, these two lineages, respectively, undergo four representative states with stage-specific transcriptional patterns around implantation. Further comparative analysis shows that AVE, but not DVE, is detected in human and non-human primate embryos, defining differences in polarity formation across species. Moreover, stem cell-assembled human blastoids lack DVE or AVE precursors, implying that additional induction of stem cells with DVE/AVE potential could promote the current embryo-like models and their post-implantation growth. Our work provides insight into understanding of embryonic polarity formation and early mammalian development.

Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom.

Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom.

Zbtb21 is required for the anterior-posterior patterning of neural tissue in the early Xenopus embryo

The vertebrate body is organized along the dorsal-ventral (DV) and anterior-posterior (AP) axes by the BMP and Wnt pathways, respectively. We previously reported that Xenopus Zbtb14 promotes dorsalization (neural induction) of ectoderm by inhibiting BMP signaling and also posteriorizes neural tissue by activating Wnt signaling, thereby coordinating the patterning of the DV and AP axes during early development. Although it has been reported that human ZBTB21 binds to ZBTB14 and is involved in gene expression in cultured mammalian cells, the function of Zbtb21 in early embryonic development remains unknown. Here, we show that Xenopus Zbtb21 plays an essential role in AP axis formation in the early Xenopus embryo. zbtb21 and zbtb14 are co-expressed in the dorsal region of embryos during gastrulation. Simultaneous overexpression of Zbtb21 and Zbtb14 in ectodermal explants enhances the neural-inducing activity of Zbtb14. Moreover, knockdown experiments showed that Zbtb21 is required for the formation of posterior neural tissue and the suppression of anterior neural development. Collectively, these results suggest that in cooperation with Zbtb14, Zbtb21 has a crucial function in AP patterning during early Xenopus embryogenesis.

The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation

SummaryThe Drosophila anterior-posterior axis is specified at mid-oogenesis when the Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarizes the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs, localize. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. Here we show that the myosin chaperone Unc-45 and non-muscle myosin II (MyoII) are required upstream of Par-1 in polarity establishment. Furthermore, the myosin regulatory light chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior that may increase cortical tension. Overexpression of MRLC-T21A that cannot be di-phosphorylated or treatment with the myosin light-chain kinase inhibitor ML-7 abolishes Par-1 localization, indicating that the posterior of MRLC di-phosphorylation is essential for both polarity establishment and maintenance. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with anterior-posterior axis formation in C. elegans, where MyoII also acts upstream of the PAR proteins to establish polarity, but to localize the anterior PAR proteins rather than Par-1.

Reconstitution of Human Peri-Implantation Embryogenesis With Integrated Stem Cell-Based Embryo Models

Studies of cultured human embryos have provided insights into human peri-implantation embryogenesis but are limited by ethical and technical constraints. Here we assembled a 3D integrated embryo model (IEM) using naive human embryonic stem cells (hESCs) and hESC-derived proliferating trophoblasts. By comparing phenotypic cell fate and comprehensive single-cell transcriptome of IEMs with 3D-cultured human embryos, we reveal that IEMs with embryonic and extra-embryonic cell types mimic developmental landmarks and 3D morphological features of natural human post­­­ ­­-implantation embryos, including pluripotent state transitions, epithelial-mesenchymal transition, anterior-posterior axis formation, amniotic cavity, yolk sac, bilaminar embryonic disc, extra-embryonic mesenchyme, primitive streak and primordial germ cells. Using IEMs, we unravel composition and gene expression patterns of embryonic lineages, and that trophoblasts secret WNT, BMP and TGFβ/Activin signals to instruct post-implantation embryogenesis . Our findings show that IEMs provide a scalable and tractable model for decoding regulatory mechanisms of human peri-implantation embryogenesis.

The <i>Drosophila</i> Anterior-Posterior Axis is Polarized by Asymmetric Myosin Activation

The Drosophila anterior-posterior (AP) axis is specified at mid-oogenesis when Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarises the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs localise. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. Here we show that the myosin chaperone, Unc-45 and Non-Muscle Myosin II (MyoII) are required in the germ line upstream of Par-1 in polarity establishment. Furthermore, the Myosin regulatory Light Chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior and increased cortical tension. Over-expression of MRLC-T21A that cannot be di-phosphorylated or acute treatment with the Myosin light chain kinase inhibitor ML-7 abolish Par-1 localisation, indicating that posterior of MRLC di-phosphorylation is essential for polarity. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with AP axis formation in C. elegans where MyoII is also required to establish polarity, but functions to localize the anterior PAR proteins rather than PAR-1.

Intracellular Communication among Morphogen Signaling Pathways during Vertebrate Body Plan Formation.

During embryonic development in vertebrates, morphogens play an important role in cell fate determination and morphogenesis. Bone morphogenetic proteins (BMPs) belonging to the transforming growth factor-β (TGF-β) family control the dorsal–ventral (DV) patterning of embryos, whereas other morphogens such as fibroblast growth factor (FGF), Wnt family members, and retinoic acid (RA) regulate the formation of the anterior–posterior (AP) axis. Activation of morphogen signaling results in changes in the expression of target genes including transcription factors that direct cell fate along the body axes. To ensure the correct establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated by a fine-tuning of morphogen signaling. In this review, we focus on the interplay of various intracellular regulatory mechanisms and discuss how communication among morphogen signaling pathways modulates body axis formation in vertebrate embryos.

Differentially and Co-expressed Genes in Embryo, Germ-Line and Somatic Tissues of Tribolium castaneum.

Transcriptomic studies of Tribolium castaneum have led to significant advances in our understanding of co-regulation and differential expression of genes in development. However, previously used microarray approaches have covered only a subset of known genes. The aim of this study was to investigate gene expression patterns of beetle embryo, germ-line and somatic tissues. We identified 12,302 expressed genes and determined differentially expressed up and down-regulated genes among all samples. For example, 1624 and 3639 genes were differentially increased in expression greater than or equal to twofold change (FDR < 0.01) in testis vs. ovary (virgin female) and ovary vs. embryo (0-5 hr), respectively. Of these, many developmental, somatic and germ-line differentially expressed genes were identified. Furthermore, many maternally deposited transcripts were identified, whose expression either decreased rapidly or persisted during embryogenesis. Genes with the largest change in expression were predominantly decreased during early embryogenesis as compared to ovary or were increased in testis compared to embryo. We also identify zygotic genes induced after fertilization. The genome wide variation in transcript regulation in maternal and zygotic genes could provide additional information on how the anterior posterior axis formation is established in Tribolium embryos as compared to Drosophila. Together, our data will facilitate studies of comparative developmental biology as well as help identify candidate genes for identifying cis-elements to drive transgenic constructs.

Meis transcription factor maintains the neurogenic ectoderm and regulates the anterior-posterior patterning in embryos of a sea urchin, Hemicentrotus pulcherrimus

Precise body axis formation is an essential step in the development of multicellular organisms, for most of which the molecular gradient and/or specifically biased localization of cell-fate determinants in eggs play important roles. In sea urchins, however, any biased proteins and mRNAs have not yet been identified in the egg except for vegetal cortex molecules, suggesting that sea urchin development is mostly regulated by uniformly distributed maternal molecules with contributions to axis formation that are not well characterized. Here, we describe that the maternal Meis transcription factor regulates anterior-posterior axis formation through maintenance of the most anterior territory in embryos of a sea urchin, Hemicentrotus pulcherrimus. Loss-of-function experiments revealed that Meis is intrinsically required for maintenance of the anterior neuroectoderm specifier foxQ2 after hatching and, consequently, the morphant lost anterior neuroectoderm characteristics. In addition, the expression patterns of univin and VEGF, the lateral ectoderm markers, and the mesenchyme-cell pattern shifted toward the anterior side in Meis morphants more than they did in control embryos, indicating that Meis contributes to the precise anteroposterior patterning by regulating the anterior neuroectodermal fate.

Haploinsufficiency of the Chromatin Remodeler BPTF Causes Syndromic Developmental and Speech Delay, Postnatal Microcephaly, and Dysmorphic Features

Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight denovo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes.

Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm

The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation.

Conserved and divergent expression patterns of markers of axial development in eutherian mammals.

Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.

AVE protein expression and visceral endoderm cell behavior during anterior–posterior axis formation in mouse embryos: Asymmetry in OTX2 and DKK1 expression

The initial landmark of anterior–posterior (A–P) axis formation in mouse embryos is the distal visceral endoderm, DVE, which expresses a series of anterior genes at embryonic day 5.5 (E5.5). Subsequently, DVE cells move to the future anterior region, generating anterior visceral endoderm (AVE). Questions remain regarding how the DVE is formed and how the direction of the movement is determined. This study compares the detailed expression patterns of OTX2, HHEX, CER1, LEFTY1 and DKK1 by immunohistology and live imaging at E4.5-E6.5. At E6.5, the AVE is subdivided into four domains: most anterior (OTX2, HHEX, CER1-low/DKK1-high), anterior (OTX2, HHEX, CER1-high/DKK1-low), main (OTX2, HHEX, CER1, LEFTY1-high) and antero-lateral and posterior (OTX2, HHEX-low). The study demonstrates how this pattern is established. AVE protein expression in the DVE occurs de novo at E5.25-E5.5. Neither HHEX, LEFTY1 nor CER1 expression is asymmetric. In contrast, OTX2 expression is tilted on the future posterior side with the DKK1 expression at its proximal domain; the DVE cells move in the opposite direction of the tilt.

A histological evaluation of development and axis formation in freshwater fish ectoparasite Argulus bengalensis Ramakrishna, 1951 (Crustacea: Branchiura).

The present investigation was carried out to underscore the developmental events of a crustacean ectoparasite of fish, Argulus bengalensis. Serial histological sections of the embryo were made at lateral, sagittal and longitudinal planes to explain its cleavage, gastrulation and axis specification. The centrolecithal egg of A. bengalensis underwent meroblastic superficial cleavage. The cleavage initiated at the future dorsal side of the egg within 5 h to 5 h and 30 min of incubation. Consequently, a small mass of energids appeared superficially at the future dorsal side within 6 h. Later, energids were found at the future ventral and lateral sides. A syncytial blastoderm was formed around the centrally placed yolk material which was transformed into a cellular blastoderm within 30 h of incubation. In the blastoderm, two cell masses were formed at the dorsal and ventral part which initially extended towards each other and later spread out though future anterior-posterior direction. The pressure exerted by the cell flow displaced the entire yolk material at the future postero-ventral side. At the time of egg laying, a prototype of the embryonic axes is determined. The substratum side of the egg formed the dorsal part, whilst the side facing water turned to the ventral part. The broader end of the egg formed the anterior side and the narrow end formed the posterior side of the embryo. The anterior-posterior axis formation was initiated within 72 to 96 h of incubation when the blastodermal cells displaced the yolk material at the future posterior end. Within 120 h of incubation, the germ layers of the embryo were determined. The study reveals that the cleavage pattern of A. bengalensis shows close similarities with that of the Malacostraca amongst the crustaceans and dipteran and hymenopteran amongst the insects.

Simplet/Fam53b is required for Wnt signal transduction by regulating β-catenin nuclear localization

Canonical β-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of β-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating β-catenin nuclear localization are poorly understood. We show that Simplet/Fam53B (Smp) is required for Wnt signaling by positively regulating β-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two β-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of β-catenin. Conversely, overexpression of smp increases β-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the β-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for β-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating β-catenin nuclear localization: the protein colocalizes with β-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces β-catenin nuclear localization and transcriptional activity; human SMP mediates β-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human β-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of β-catenin-dependent Wnt signal transduction.

Simplet/Fam53b is required for Wnt signal transduction by regulating β-catenin nuclear localization.

Canonical β-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of β-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating β-catenin nuclear localization are poorly understood. We show that Simplet/Fam53B (Smp) is required for Wnt signaling by positively regulating β-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two β-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of β-catenin. Conversely, overexpression of smp increases β-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the β-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for β-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating β-catenin nuclear localization: the protein colocalizes with β-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces β-catenin nuclear localization and transcriptional activity; human SMP mediates β-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human β-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of β-catenin-dependent Wnt signal transduction.

Bmp Suppression in Mangrove Killifish Embryos Causes a Split in the Body Axis

Bone morphogenetic proteins (Bmp) are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV) axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL) movements, and anterior-posterior (AP) axis formation in embryos of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM) produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.

Nuclearization of β-catenin in ectodermal precursors confers organizer-like ability to induce endomesoderm and pattern a pluteus larva.

BackgroundIn many bilaterians, asymmetric activation of canonical Wnt (cWnt) signaling at the posterior pole is critical for anterior-posterior (AP) body axis formation. In 16-cell stage sea urchins, nuclearization of β-catenin in micromeres activates a gene regulatory network that defines body axes and induces endomesoderm. Transplanting micromeres to the animal pole of a host embryo induces ectopic endomesoderm in the mesomeres (ectoderm precursors) whereas inhibiting cWnt signaling blocks their endomesoderm-inducing activity and the micromeres become ectoderm-like. We have tested whether ectopic activation of cWnt signaling in mesomeres is sufficient to impart the cells with organizer-like abilities, allowing them to pattern normal embryonic body axes when recombined with a field of mesomeres.ResultsFertilized eggs were microinjected with constitutively active Xenopus β-catenin (actβ-cat) mRNA and allowed to develop until the 16-cell stage. Two mesomeres from injected embryos were then recombined with isolated animal halves (AH) from uninjected 16-cell stage embryos. Control chimeras produced animalized phenotypes (hollow balls of ectoderm) and rarely formed skeletogenic mesoderm (SM)-derived spicules, endoderm or pigment cells, a type of non-skeletogenic mesoderm (NSM). In contrast, over half of the 0.5 pg/pL actβ-cat mesomere/AH chimeras formed a partial or complete gut (exhibiting AP polarity), contained mesenchyme-like cells similar to SM, and produced pigment cells. At three days, chimeras formed plutei with normal embryonic body axes. When fates of the actβ-cat mRNA-injected mesomeres were tracked, we found that injected mesomeres formed mesenchyme-like and pigment cells, but endoderm was induced. Higher concentrations of actβ-cat mRNA were less likely to induce endoderm or pigment cells, but had similar mesenchyme-like cell production to 0.5 pg/pL actβ-cat mesomere/AH chimeras.ConclusionsOur results show that nuclear β-catenin is sufficient to endow naïve cells with the ability to act as an organizing center and that β-catenin has both cell-autonomous and non-autonomous effects on cell fate specification in a concentration-dependent manner. These results are consistent with the hypothesis that a shift in the site of early cWnt signaling in cleaving embryos could have modified polarity of the main body axes during metazoan evolution.