Cell Fate Patterning Research Articles

Logic programming to predict cell fate patterns and retrodict genotypes in organogenesis.

Caenorhabditis elegans vulval development is a paradigm system for understanding cell differentiation in the process of organogenesis. Through temporal and spatial controls, the fate pattern of six cells is determined by the competition of the LET-23 and the Notch signalling pathways. Modelling cell fate determination in vulval development using state-based models, coupled with formal analysis techniques, has been established as a powerful approach in predicting the outcome of combinations of mutations. However, computing the outcomes of complex and highly concurrent models can become prohibitive. Here, we show how logic programs derived from state machines describing the differentiation of C. elegans vulval precursor cells can increase the speed of prediction by four orders of magnitude relative to previous approaches. Moreover, this increase in speed allows us to infer, or 'retrodict', compatible genomes from cell fate patterns. We exploit this technique to predict highly variable cell fate patterns resulting from dig-1 reduced-function mutations and let-23 mosaics. In addition to the new insights offered, we propose our technique as a platform for aiding the design and analysis of experimental data.

Cryptic genetic variation uncovers evolution of environmentally sensitive parameters in Caenorhabditis vulval development.

Understanding the robustness of developmental systems requires insights into the sensitivity of underlying molecular and cellular parameters to perturbations, and how such sensitivity evolves. We address these issues using vulval cell fate determination--a reproducible and robust patterning process regulated by a cross-talk of EGF-Ras-MAPK and Delta-Notch pathways. Although the final vulval cell fate pattern is identical in all Caenorhabditis species, the patterning process underlies extensive cryptic genetic variation between and within species. Here, we tested whether this cryptic genetic variation translates into variation in developmental sensitivity to environmental perturbations. We disrupted vulval patterning using thermal perturbations to quantify and compare environmental sensitivity of different system parameters between distinct genotypes of C. elegans and C. briggsae. Thermal perturbations globally debuffered vulval development, triggering diverse pattering variants, whose frequency and spectra were strongly species- and genotype-dependent. This condition-dependent variation indicates that environmental sensitivity of different system properties, such as vulval competence or vulval induction, is subject to evolutionary change. High temperature induced a genotype-specific decrease of secondary fate induction and corresponding Notch pathway activity in the C. elegans N2 strain; in contrast, hypoinduction of the primary cell fate was never observed. Vulval precursor cells therefore differ in temperature sensitivity and such cell-specific sensitivity shows evolutionary variation. We further compared spectra of temperature-induced vulval variants to the ones induced by mutation accumulation in the same genotypes. In response to either perturbation, we observed similar genotype-dependence of variant production, allowing identification of distinct system features most sensitive to both mutation and environment. Taken together, we show how sensitivity of system parameters regulating Caenorhabditis vulval development depends on subtle interactions between perturbations and genetic background. Our results imply that cryptic genetic variation may reflect evolutionary variation in developmental robustness, therefore potentially contributing to the maintenance of phenotypic precision when facing perturbations.

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.

Control of neural stem cell self-renewal and differentiation in Drosophila.

The neural stem cells of Drosophila, called neuroblasts, have the ability to self-renew and at the same time produce many different types of neurons and glial cells. In the central brain and ventral ganglia, neuroblasts are specified and delaminate from the neuroectoderm during embryonic development under the control of proneural and neurogenic genes. In contrast, in the optic lobes, neuroepithelial cells are transformed into neuroblasts postembryonically by a spatial wave of proneural gene expression. Central brain and ventral nerve cord neuroblasts manifest a short embryonic proliferation period followed by a stage of quiescence and then undergo a prolonged postembryonic proliferation period during which most of the differentiated neurons of the adult CNS are generated. While most neuroblasts belong to a type I class that produces neuronal lineages through non-self-renewing ganglion mother cells, a small subset of type II neuroblasts generates exceptionally large neuronal lineages through self-renewing intermediate progenitor cells that have a transit amplifying function. All neuroblasts in the CNS generate their neural progeny through an asymmetric cell division mode in which the interplay of apical complex and basal complex molecules in the mitotically active progenitor results in the segregation of cell fate determinants into the smaller more differentiated daughter cell. Defects in this molecular control of asymmetric cell division in neuroblasts can result in brain tumor formation. Proliferating neuroblast lineages in the developing CNS utilize transcription factor cascades as a generic mechanism for temporal patterning and birth order-dependent determination of differential neural cell fate. This contributes to the generation of a remarkable diversity of cell types in the developing CNS from a surprisingly small set of neural stem cell-like precursors.

Competition in Notch Signaling with Cis Enriches Cell Fate Decisions

Notch signaling is involved in cell fate choices during the embryonic development of Metazoa. Commonly, Notch signaling arises from the binding of the Notch receptor to its ligands in adjacent cells driving cell-to-cell communication. Yet, cell-autonomous control of Notch signaling through both ligand-dependent and ligand-independent mechanisms is known to occur as well. Examples include Notch signaling arising in the absence of ligand binding, and cis-inhibition of Notch signaling by titration of the Notch receptor upon binding to its ligands within a single cell. Increasing experimental evidences support that the binding of the Notch receptor with its ligands within a cell (cis-interactions) can also trigger a cell-autonomous Notch signal (cis-signaling), whose potential effects on cell fate decisions and patterning remain poorly understood. To address this question, herein we mathematically and computationally investigate the cell states arising from the combination of cis-signaling with additional Notch signaling sources, which are either cell-autonomous or involve cell-to-cell communication. Our study shows that cis-signaling can switch from driving cis-activation to effectively perform cis-inhibition and identifies under which conditions this switch occurs. This switch relies on the competition between Notch signaling sources, which share the same receptor but differ in their signaling efficiency. We propose that the role of cis-interactions and their signaling on fine-grained patterning and cell fate decisions is dependent on whether they drive cis-inhibition or cis-activation, which could be controlled during development. Specifically, cis-inhibition and not cis-activation facilitates patterning and enriches it by modulating the ratio of cells in the high-ligand expression state, by enabling additional periodic patterns like stripes and by allowing localized patterning highly sensitive to the precursor state and cell-autonomous bistability. Our study exemplifies the complexity of regulations when multiple signaling sources share the same receptor and provides the tools for their characterization.

Mechanobiology and Developmental Control

Morphogenesis is the remarkable process by which cells self-assemble into complex tissues and organs that exhibit specialized form and function during embryological development. Many of the genes and chemical cues that mediate tissue and organ formation have been identified; however, these signals alone are not sufficient to explain how tissues and organs are constructed that exhibit their unique material properties and three-dimensional forms. Here, we review work that has revealed the central role that physical forces and extracellular matrix mechanics play in the control of cell fate switching, pattern formation, and tissue development in the embryo and how these same mechanical signals contribute to tissue homeostasis and developmental control throughout adult life.

An organizing activity is required for head patterning and cell fate specification in the polychaete annelid Capitella teleta: New insights into cell–cell signaling in Lophotrochozoa

Many lophotrochozoans (i.e., molluscs, annelids, nemerteans, and polyclad flatworms) display a well-conserved early developmental program called spiral cleavage that contrasts with the high diversity of adult body forms present in this group. Due to this stereotypical development, each cell can be uniquely identified and its lineage history known following intracellular injection of lineage tracers. Cell deletion experiments performed mainly in molluscs have demonstrated that one or two cells associated with the endomesodermal lineage represent an embryonic organizer of subsequent development and are causally involved in cell fate and body patterning. Utilizing the published fate map of the spiral-cleaving annelid Capitella teleta, we used infrared laser cell deletions to dissect the role of individual cells on the patterning of the larval body. Thirteen uniquely identifiable individual blastomeres and two double cell combination deletions were studied to assess larval phenotypes by scoring multiple morphological structures and cell type-specific molecular markers differentially expressed along the antero-posterior and dorso-ventral axes. Surprisingly, our results show that in C. teleta, the cellular identity of the “organizing cell” and the timing of the organizing activity are different from that of other spiralians. In C. teleta, the ectodermal primary somatoblast, 2d, is the key cell responsible for organizing activity during early embryonic development, and is necessary for bilateral symmetry and dorso-ventral axis organization of the head as well as neural, foregut and mesoderm tissue formation. Furthermore, we show that the ERK/MAPK signaling pathway does not appear to be involved in organizing activity in C. teleta. This contrasts with data from molluscs and the molecular mechanism suggested for another polychaete, Hydroides elegans, highlighting likely molecular level variation among spiralian embryos. These results reinforce the idea that an embryonic organizing activity is present across spiralians. Our data also emphasize the developmental variation within lophotrochozoans, and may ultimately provide insight into the role of developmental processes in the evolution of diverse body forms in metazoans.

ERK1/2‐RSK Regulation of Cell Fate in Human Mammary Ductal Development

Understanding the initiation and progression of breast cancer requires knowledge of the normal development of the mammary ductal network. We have developed a platform based on primary human breast tissue grown in a highly‐defined, 3D mammary explant system, which faithfully recapitulates the normal morphogenesis of human mammary ducts. This system allows for the study of signaling cues that regulate normal development. Strikingly, although different HER1 ligands all permit ductal development, they generate different distinct patterns of cell fate. The intensity of signaling through the ERK1/2‐RSK signaling pathways alters cell fate decisions by progenitor populations within the duct. EGF causes an expansion of basal cells whereas amphiregulin enables normal lineage specification. In EGF conditions inhibition of RSK restores normal ductal development. These findings are relevant to triple negative breast cancer (TNBC) in which increased RSK signaling is observed, and thus RSK may play a role in the inappropriate expansion of the basal population that occurs in some types of TNBC. Identifying the cell‐autonomous and extrinsic cues that regulate ductal development will aid in identifying novel drug targets for breast cancer.

DNMT1 represses p53 to maintain progenitor cell survival during pancreatic organogenesis

In the developing pancreas, self-renewal of progenitors and patterning of cell fates are coordinated to ensure the correct size and cellular makeup of the organ. How this coordination is achieved, however, is not clear. We report that deletion of DNA methyltransferase 1 (Dnmt1) in pancreatic progenitors results in agenesis of the pancreas due to apoptosis of progenitor cells. We show that DNMT1 is bound to the p53 regulatory region and that loss of Dnmt1 results in derepression of the p53 locus. Haploinsufficiency of p53 rescues progenitor cell survival and cellular makeup of the Dnmt1-deleted pancreas.

Retention of Stem Cell Plasticity in Avian Primitive Streak Cells and the Effects of Local Microenvironment

Primitive streak (PS) is the first structure occurring in embryonic gastrulation, in which the epiblast cells undergo the epithelial-mesenchymal transition to become the loose mesoderm cells subsequently. Because the mesoderm cells departing from different portions of PS are blessed with disparate migration trajectory and differentiation fate, one question is when the cell fate is determinated. To understand whether the cell fate and cell migration pattern will be alternated along with the microenvironment transformation, the traditional transplantation technology was used to replace the anterior PS cells in HH4 host embryo using posterior PS tissue labeled by green fluorescent protein (GFP) in the same stage donor embryo, and then, we tracked the migration trajectory of the GFP-positive cells with fluorescence stereomicroscope after incubation, and eventually verified the cell contribution from the transplants with in situ hybridization and immunocytochemistry. The same experimental strategy applied for posterior PS site replacement in host embryo. We found that the transplanted posterior PS cells to anterior part of streak followed the anterior PS cell migration pattern rather than kept its posterior streak cell migration trajectory, and so did vice versa. In addition, the transplants were involved in the contribution to the subsequent organogenesis as the local PS tissues affirmed by specific expression of myocardial or hematopoietic markers. Therefore, our data strongly suggest that the PS cells still keep stem cell plasticity during gastrulation and the eventual cell fate will depend on the spatial gene expression within local microenvironment along with development.

Control of cell-fate plasticity and maintenance of multipotency by DAF-16/FoxO in quiescent Caenorhabditis elegans

The Caenorhabditis elegans vulval precursor cells (VPCs) offer a paradigm for investigating how multipotency of progenitor cells is maintained during periods of quiescence. The VPCs are born in the first larval stage. When hermaphrodites are grown under favorable conditions, the EGF-mediated "inductive" signal and the LIN-12/Notch-mediated "lateral" signal confer a precise spatial pattern of distinct vulval cell fates in the third larval stage, a day after hatching. Under adverse conditions, hermaphrodites undergo a prolonged quiescent period as dauer larvae, which can endure for several months with progenitor cells such as VPCs in developmental arrest. If favorable conditions ensue, larvae recover and resume development as postdauer third stage larvae, with the same VPC spatial-patterning events as in continuously developing third stage larvae. Here, we identify several consequences of dauer life history for VPC specification. In wild-type dauers, VPCs undergo a phenomenon reminiscent of natural direct reprogramming to maintain or reestablish multipotency; they acquire an active block to signal transduction by EGF receptor and LIN-12/Notch and have a different mechanism for regulating transcription of the lateral signal. Furthermore, DAF-16/FoxO, a target of insulin/insulin-like growth factor signaling, is required to promote VPC fate plasticity during dauer and for normal vulval patterning after passage through dauer, suggesting that DAF-16/FoxO coordinates environment and life history with plasticity of cell fate.

Robustness and Epistasis in the C. elegans Vulval Signaling Network Revealed by Pathway Dosage Modulation

Biological systems may perform reproducibly to generate invariant outcomes, despite external or internal noise. One example is the C. elegans vulva, in which the final cell fate pattern is remarkably robust. Although this system has been extensively studied and the molecular network underlying cell fate specification is well understood, very little is known in quantitative terms. Here, through pathway dosage modulation and single molecule fluorescence in situ hybridization, we show that the system can tolerate a 4-fold variation in genetic dose of the upstream signaling molecule LIN-3/epidermal growth factor (EGF) without phenotypic change in cell fate pattern. Furthermore, through tissue-specific dosage perturbations of the EGF and Notch pathways, we determine the first-appearing patterning errors. Finally, by combining different doses of both pathways, we explore how quantitative pathway interactions influence system behavior. Our results highlight the feasibility and significance of launching experimental studies of robustness and quantitative network analysis in genetically tractable, multicellular eukaryotes.

Mitochondrial GCD1 Dysfunction Reveals Reciprocal Cell-to-Cell Signaling during the Maturation of Arabidopsis Female Gametes

Cell-to-cell communication in embryo sacs is thought to regulate the development of female gametes in flowering plants, but the details remain poorly understood. Here, we report a mitochondrial protein, GAMETE CELL DEFECTIVE 1 (GCD1), enriched in gametophytes that is essential for final maturation of female gametes. Using Arabidopsis gcd1 mutants, we found that final maturation of the egg and central cells is not required for double fertilization but is necessary for embryogenesis initiation and endosperm development. Furthermore, nonautonomous effects, observed when GCD1 or AAC2 function is disrupted, suggest that mitochondrial function influences reciprocal signaling between central and egg cells to regulate maturation of the partner (egg or central) cell. Our findings confirm that cell-to-cell communication is important in functional maturation of female gametic cells and suggest that both egg and central cells sense and transmit their mitochondrial metabolic status as animportant cue that regulates the coordination of gamete maturation.

PTEN Negatively Regulates MAPK Signaling during Caenorhabditis elegans Vulval Development

Vulval development in Caenorhabditis elegans serves as an excellent model to examine the crosstalk between different conserved signaling pathways that are deregulated in human cancer. The concerted action of the RAS/MAPK, NOTCH, and WNT pathways determines an invariant pattern of cell fates in three vulval precursor cells. We have discovered a novel form of crosstalk between components of the Insulin and the RAS/MAPK pathways. The insulin receptor DAF-2 stimulates, while DAF-18 PTEN inhibits, RAS/MAPK signaling in the vulval precursor cells. Surprisingly, the inhibitory activity of DAF-18 PTEN on the RAS/MAPK pathway is partially independent of its PIP3 lipid phosphatase activity and does not involve further downstream components of the insulin pathway, such as AKT and DAF-16 FOXO. Genetic and biochemical analyses indicate that DAF-18 negatively regulates vulval induction by inhibiting MAPK activation. Thus, mutations in the PTEN tumor suppressor gene may result in the simultaneous hyper-activation of two oncogenic signaling pathways.

Caenorhabditis elegans vulval cell fate patterning

The spatial patterning of three cell fates in a row of competent cells is exemplified by vulva development in the nematode Caenorhabditis elegans. The intercellular signaling network that underlies fate specification is well understood, yet quantitative aspects remain to be elucidated. Quantitative models of the network allow us to test the effect of parameter variation on the cell fate pattern output. Among the parameter sets that allow us to reach the wild-type pattern, two general developmental patterning mechanisms of the three fates can be found: sequential inductions and morphogen-based induction, the former being more robust to parameter variation. Experimentally, the vulval cell fate pattern is robust to stochastic and environmental challenges, and minor variants can be detected. The exception is the fate of the anterior cell, P3.p, which is sensitive to stochastic variation and spontaneous mutation, and is also evolving the fastest. Other vulval precursor cell fates can be affected by mutation, yet little natural variation can be found, suggesting stabilizing selection. Despite this fate pattern conservation, different Caenorhabditis species respond differently to perturbations of the system. In the quantitative models, different parameter sets can reconstitute their response to perturbation, suggesting that network variation among Caenorhabditis species may be quantitative. Network rewiring likely occurred at longer evolutionary scales.

Signaling mechanisms controlling cell fate and embryonic patterning.

During development, signaling pathways specify cell fates by activating transcriptional programs in response to extracellular signals. Extensive studies in the past 30 years have revealed that surprisingly few pathways exist to regulate developmental programs and that dysregulation of these can lead to human diseases, including cancer. Although these pathways use distinct signaling components and signaling strategies, a number of common themes have emerged regarding their organization and regulation in time and space. Examples from Drosophila, such as Notch, Hedgehog, Wingless/WNT, BMP (bone morphogenetic proteins), EGF (epidermal growth factor), and FGF (fibroblast growth factor) signaling, illustrate their abilities to act either at a short range or over a long distance, and in some instances to generate morphogen gradients that pattern fields of cells in a concentration-dependent manner. They also show how feedback loops and transcriptional cascades are part of the logic of developmental regulation.

302 Dynamic Regulation of Intestinal Crypt Base Columnar Stem Cells by Notch Signaling

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

303 High Altitude Journeys and Flights are Associated With the Increased Risk of Flares in IBD Patients

Background/Aim: The intestinal epithelium is a rapidly renewed tissue, requiring continual replenishment by intestinal stem cells (ISCs) in order to sustain life. The crypt base columnar cell (CBC) is an actively dividing ISC population marked by the expression of Lgr5, Ascl2, and Olfm4. Our recent studies show that Notch signaling regulates epithelial cell homeostasis by directing differentiated cell fate and promoting stem cell maintenance. In this study we examined Notch regulation of CBCs by characterizing Olfm4 expression and function. Methods: Gamma-secretase inhibitors (GSIs) were used to block Notch signaling in C57BL/ 6 mice (30μmol/kg dibenzazepine; DBZ) or in the human colon cancer cell line LS174T (40μM DAPT). Tissue was analyzed at various time points for progenitor and differentiated marker expression and epithelial proliferation by histological staining and qRT-PCR. LS174T cells were used to investigate the mechanism of Notch regulation of Olfm4 expression. Genetically engineered Olfm4-deficient mouse intestine was studied to examine the function of this CBC marker for intestinal epithelial cell homeostasis. Results: Chronic Notch inhibition for 6 days resulted in marked secretory cell hyperplasia, decreased cellular proliferation, and a striking decrease in Olfm4 mRNA, showing that Notch is critical for many different aspects of cellular renewal. Surprisingly, acute block of Notch with a single dose of DBZ was sufficient to cause a rapid and transient loss of Olfm4, in addition to a delayed, yet sustained surge in secretory cells and cellular proliferation. This surge in proliferation was in stark contrast to the loss of proliferation observed with chronic DBZ treatment. The acute DBZ model revealed exquisite Olfm4 regulation: mice treated with one dose showed a significant decrease in Olfm4 mRNA within 12 hours, but recovery to baseline within 2 days. Similarly, LS174T colon cancer cells showed decreased Olfm4 mRNA 4 hours after DAPT administration. To test whether changes in Olfm4 expression may mediate Notch effects in the intestine we examinedOlfm4-/mice. This analysis showed normal proliferation, cell fate, and CBC marker gene expression patterns, demonstrating that Olfm4 is not required for CBC function. However, we observed expression of other Olfactomedin family members in intestine, suggesting functional redundancy.Conclusions:Olfm4 is dynamically regulated by Notch signaling, with rapid loss of transcripts observed upon acute Notch inhibition. Thus this CBC marker is a sensitive read out of Notch activity in the intestine. Olfm4-/mice did not exhibit an intestinal phenotype, although related family members may play a compensatory role, suggesting compound mouse mutants may be needed to understand the function of Notch regulation of Olfm4 in the CBC.

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.

Relationship between brassinosteroids and genes controlling stomatal production in the Arabidopsis hypocotyl

Stomata are excellent model systems for examining the mechanisms that regulate cell fate determination and pattern formation. It has recently been demonstrated that brassinosteroids control stomatal development by regulating both the MAPK kinase kinase YODA and the basic helix-loop-helix transcriptional factor SPEECHLESS. Here, we show that these plant regulators positively regulate stomatal formation in the hypocotyl and also accelerate their development. Hormone tests, reporter gene studies and mutant analyses revealed that brassinosteroids act upstream of the transcriptional factors CAPRICE and GLABRA2. These plant regulators control an earlier stage of stomatal production than those regulated by the membrane receptor TOO MANY MOUTHS. This work highlights differences in the genetic control of stomatal development between cotyledons or leaves and hypocotyls.