Endodermal Cell Fate Research Articles

ABCG2 Gene and ABCG2 Protein Expression in Colorectal Cancer-In Silico and Wet Analysis.

ABCG2 (ATP-binding cassette superfamily G member 2) is a cell membrane pump encoded by the ABCG2 gene. ABCG2 can protect cells against compounds initiating and/or intensifying neoplasia and is considered a marker of stem cells responsible for cancer growth, drug resistance and recurrence. Expression of the ABCG2 gene or its protein has been shown to be a negative prognostic factor in various malignancies. However, its prognostic significance in colorectal cancer remains unclear. Using publicly available data, ABCG2 was shown to be underexpressed in colon and rectum adenocarcinomas, with lower expression compared to both the adjacent nonmalignant lung tissues and non-tumour lung tissues of healthy individuals. This downregulation could result from the methylation level of some sites of the ABCG2 gene. This was connected with microsatellite instability, weight and age among patients with colon adenocarcinoma, and with tumour localization, population type and age of patients for rectum adenocarcinoma. No association was found between ABCG2 expression level and survival of colorectal cancer patients. In wet analysis of colorectal cancer samples, neither ABCG2 gene expression, analysed by RT-PCR, nor ABCG2 protein level, assessed by immunohistochemistry, was associated with any clinicopathological factors or overall survival. An ABCG2-centered protein-protein interaction network build by STRING showed proteins were found to be involved in leukotriene, organic anion and xenobiotic transport, endodermal cell fate specification, and histone methylation and ubiquitination. Hence, ABCG2 underexpression could be an indicator of the activity of certain signalling pathways or protein interactors essential for colorectal carcinogenesis.

An in vitro culture platform for studying the effect of collective cell migration on spatial self-organization within induced pluripotent stem cell colonies

BackgroundHuman induced pluripotent stem cells (hiPSCs) provide an in vitro system to identify the impact of cell behavior on the earliest stages of cell fate specification during human development. Here, we developed an hiPSC-based model to study the effect of collective cell migration in meso–endodermal lineage segregation and cell fate decisions through the control of space confinement using a detachable ring culture system.ResultsThe actomyosin organization of cells at the edge of undifferentiated colonies formed in a ring barrier differed from that of the cells in the center of the colony. In addition, even in the absence of exogenous supplements, ectoderm, mesoderm, endoderm, and extraembryonic cells differentiated following the induction of collective cell migration at the colony edge by removing the ring-barrier. However, when collective cell migration was inhibited by blocking E-cadherin function, this fate decision within an hiPSC colony was altered to an ectodermal fate. Furthermore, the induction of collective cell migration at the colony edge using an endodermal induction media enhanced endodermal differentiation efficiency in association with cadherin switching, which is involved in the epithelial-mesenchymal transition.ConclusionsOur findings suggest that collective cell migration can be an effective way to drive the segregation of mesoderm and endoderm lineages, and cell fate decisions of hiPSCs.

The RNA helicase DDX6 controls early mouse embryogenesis by repressing aberrant inhibition of BMP signaling through miRNA-mediated gene silencing.

The evolutionarily conserved RNA helicase DDX6 is a central player in post-transcriptional regulation, but its role during embryogenesis remains elusive. We here show that DDX6 enables proper cell lineage specification from pluripotent cells by analyzing Ddx6 knockout (KO) mouse embryos and employing an in vitro epiblast-like cell (EpiLC) induction system. Our study unveils that DDX6 is an important BMP signaling regulator. Deletion of Ddx6 causes the aberrant upregulation of the negative regulators of BMP signaling, which is accompanied by enhanced expression of Nodal and related genes. Ddx6 KO pluripotent cells acquire higher pluripotency with a strong inclination toward neural lineage commitment. During gastrulation, abnormally expanded Nodal and Eomes expression in the primitive streak likely promotes endoderm cell fate specification while inhibiting mesoderm differentiation. We also genetically dissected major DDX6 pathways by generating Dgcr8, Dcp2, and Eif4enif1 KO models in addition to Ddx6 KO. We found that the miRNA pathway mutant Dgcr8 KO phenocopies Ddx6 KO, indicating that DDX6 mostly works along with the miRNA pathway during early development, whereas its P-body-related functions are dispensable. Therefore, we conclude that DDX6 prevents aberrant upregulation of BMP signaling inhibitors by participating in miRNA-mediated gene silencing processes. Overall, this study delineates how DDX6 affects the development of the three primary germ layers during early mouse embryogenesis and the underlying mechanism of DDX6 function.

Identification of the Shared Gene Signatures and Biological Mechanism in Type 2 Diabetes and Pancreatic Cancer.

BackgroundThe relationship between pancreatic cancer (PC) and type 2 diabetes mellitus (T2DM) has long been widely recognized, but the interaction mechanisms are still unknown. This study was aimed to investigate the shared gene signatures and molecular processes between PC and T2DM.MethodsThe Gene Expression Omnibus (GEO) database was used to retrieve the RNA sequence and patient information of PC and T2DM. Weighted gene co-expression network analysis (WGCNA) was performed to discover a co-expression network associated with PC and T2DM. Enrichment analysis of shared genes present in PC and T2DM was performed by ClueGO software. These results were validated in the other four cohorts based on differential gene analysis. The predictive significance of S100A6 in PC was evaluated using univariate and multivariate Cox analyses, as well as Kaplan–Meier plots. The biological process of S100A6 enrichment in PC was detected using Gene Set Enrichment Analysis (GSEA). The involvement of S100A6 in the tumor immune microenvironment (TIME) was assessed by CIBERSORT. In vitro assays were used to further confirm the function of S100A6 in PC.ResultsWGCNA recognized three major modules for T2DM and two major modules for PC. There were 44 shared genes identified for PC and T2DM, and Gene Ontology (GO) analysis showed that regulation of endodermal cell fate specification was primarily enriched. In addition, a key shared gene S100A6 was derived in the validation tests. S100A6 was shown to be highly expressed in PC compared to non-tumor tissues. PC patients with high S100A6 expression had worse overall survival (OS) than those with low expression. GSEA revealed that S100A6 is involved in cancer-related pathways and glycometabolism-related pathways. There is a strong relationship between S100A6 and TIME. In vitro functional assays showed that S100A6 helped to induce the PC cells’ proliferation and migration. We also proposed a diagram of common mechanisms of PC and T2DM.ConclusionsThis study firstly revealed that the regulation of endodermal cell fate specification may be common pathogenesis of PC and T2DM and identified S100A6 as a possible biomarker and therapeutic target for PC and T2DM patients.

Abstract PO-008: Multi-omic Profiling of primary pancreatic adenocarcinomas obtained from the APACT adjuvant trial of nab-paclitaxel + gemcitabine vs gemcitabine

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a difficult disease to treat, with few therapies available that target specific patient subgroups. Translational studies in pancreatic cancer can be technically challenging due to biopsy specimen characteristics including low tumor cellularity and dense fibrotic stroma. Resected primary pancreatic tumors obtained during the phase 3 APACT clinical trial (NCT01964430 adjuvant nab-paclitaxel + gemcitabine versus gemcitabine monotherapy n = 866) are a unique resource for characterizing molecular and immune subtypes among PDAC tumors and their association with treatment outcomes in the adjuvant chemotherapy setting. Tumor-infiltrating immune cells were assessed in serial sections of 453 tumors by dual-chromogen immuno-histochemical (IHC) staining (CD4 CD8 CD20 CD163 CMAF CD56 FoxP3 PD-1 PD-L1). Image alignment, segmentation, and spatial localization of stained cells relative to a pan-cytokeratin staining-based tumor epithelial mask was performed using a commercial analysis platform. Expression profiles were obtained for 515 macrodissected tumor biopsy regions by RNA-seq. Transcriptional subtypes were assigned based on schema previously reported by Moffit and Bailey, and molecular pathway correlates were characterized using gene set enrichment analysis. Based on immunochemical staining, higher intratumoral CD8+ or lower CD163+ cell densities were associated with modestly longer disease-free or overall survival in patients treated with nab-paclitaxel plus gemcitabine. The combination of both high CD8+ and low CD163+ cell density was notably associated with longer overall survival compared to other subjects treated with the combination regimen (mOS>55months versus 36 months HR=0.46 p=<0.01) Transcriptional subtyping of tumors, based on schema such as those previously reported by Moffit et al or Bailey et al, identified groups defined by anticipated signatures. Classical/progenitor subtypes differentially expressed genes involved in oxidative respiration and endodermal cell fate, while basal/squamous subtypes showed high expression of signatures for hypoxia, mesenchymal transformation, and TGFb signaling. Longer overall survival in subjects treated with nab-paclitaxel plus gemcitabine as compared to gemcitabine monotherapy was seen principally in subjects with tumors of Moffit classical (HR=0.59 P=0.01) and Bailey progenitor (HR=0.42 P<0.01) subtypes. The observations highlighted provide insight into primary PDAC tumor characteristics that are associated with treatment-specific outcomes to nab-paclitaxel based adjuvant chemotherapy. Future work will combine data described here with mutational and genetic studies in progress as well as with other public, proprietary, or commercial data sources. In combination, we anticipate that these data may be useful in identifying patient subsets and molecular mechanisms that might be targeted by novel combination therapies for improved treatment of pancreatic cancer. Citation Format: Thomas Lila, Andrew Biankin, Andrew Browne, David J. Reiss, Brian Lu, Daniel Pierce, Alexander Ratushny, Kao-tai Tsai, Sneh Lata, Sitharthan Kamalakaran, Tomas Babak, Brian Fox, Sam Danziger, Konstantinos Mavrommatis, Matthew W. B. Trotter, David Chang, Fadi Towfic. Multi-omic Profiling of primary pancreatic adenocarcinomas obtained from the APACT adjuvant trial of nab-paclitaxel + gemcitabine vs gemcitabine [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-008.

Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumor fate.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.

Dnmt1 is required for proximal-distal patterning of the lung endoderm and for restraining alveolar type 2 cell fate

Lung endoderm development occurs through a series of finely coordinated transcriptional processes that are regulated by epigenetic mechanisms. However, the role of DNA methylation in regulating lung endoderm development remains poorly understood. We demonstrate that DNA methyltransferase 1 (Dnmt1) is required for early branching morphogenesis of the lungs and for restraining epithelial fate specification. Loss of Dnmt1 leads to an early branching defect, a loss of epithelial polarity and proximal endodermal cell differentiation, and an expansion of the distal endoderm compartment. Dnmt1 deficiency also disrupts epithelial-mesenchymal crosstalk and leads to precocious distal endodermal cell differentiation with premature expression of alveolar type 2 cell restricted genes. These data reveal an important requirement for Dnmt1 mediated DNA methylation in early lung development to promote proper branching morphogenesis, maintain proximal endodermal cell fate, and suppress premature activation of the distal epithelial fate.

XEN and the Art of Stem Cell Maintenance: Molecular Mechanisms Maintaining Cell Fate and Self-Renewal in Extraembryonic Endoderm Stem (XEN) Cell Lines.

The extraembryonic endoderm is one of the first cell types specified during mammalian development. This extraembryonic lineage is known to play multiple important roles throughout mammalian development, including guiding axial patterning and inducing formation of the first blood cells during embryogenesis. Moreover, recent studies have uncovered striking conservation between mouse and human embryos during the stages when extraembryonic endoderm cells are first specified, in terms of both gene expression and morphology. Therefore, mouse embryos serve as an excellent model for understanding the pathways that maintain extraembryonic endoderm cell fate. In addition, self-renewing multipotent stem cell lines, called XEN cells, have been derived from the extraembryonic endoderm of mouse embryos. Mouse XEN cell lines provide an additional tool for understanding the basic mechanisms that contribute to maintaining lineage potential, a resource for identifying how extraembryonic ectoderm specifies fetal cell types, and serve as a paradigm for efforts to establish human equivalents. Given the potential conservation of essential extraembryonic endoderm roles, human XEN cells would provide a considerable advance. However, XEN cell lines have not yet been successfully derived from human embryos. Given the potential utility of human XEN cell lines, this chapter focuses on reviewing the mechanisms known to govern the stem cell properties of mouse XEN, in hopes of facilitating new ways to establish human XEN cell lines.

Reconstructed cell fate-regulatory programs in stem cells reveal hierarchies and key factors of neurogenesis.

Cell lineages, which shape the body architecture and specify cell functions, derive from the integration of a plethora of cell intrinsic and extrinsic signals. These signals trigger a multiplicity of decisions at several levels to modulate the activity of dynamic gene regulatory networks (GRNs), which ensure both general and cell-specific functions within a given lineage, thereby establishing cell fates. Significant knowledge about these events and the involved key drivers comes from homogeneous cell differentiation models. Even a single chemical trigger, such as the morphogen all-trans retinoic acid (RA), can induce the complex network of gene-regulatory decisions that matures a stem/precursor cell to a particular step within a given lineage. Here we have dissected the GRNs involved in the RA-induced neuronal or endodermal cell fate specification by integrating dynamic RXRA binding, chromatin accessibility, epigenetic promoter epigenetic status, and the transcriptional activity inferred from RNA polymerase II mapping and transcription profiling. Our data reveal how RA induces a network of transcription factors (TFs), which direct the temporal organization of cognate GRNs, thereby driving neuronal/endodermal cell fate specification. Modeling signal transduction propagation using the reconstructed GRNs indicated critical TFs for neuronal cell fate specification, which were confirmed by CRISPR/Cas9-mediated genome editing. Overall, this study demonstrates that a systems view of cell fate specification combined with computational signal transduction models provides the necessary insight in cellular plasticity for cell fate engineering. The present integrated approach can be used to monitor the in vitro capacity of (engineered) cells/tissues to establish cell lineages for regenerative medicine.

Human pancreatic beta-like cells converted from fibroblasts.

Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.

Epigenetic Induction of Definitive and Pancreatic Endoderm Cell Fate in Human Fibroblasts.

Reprogramming can occur by the introduction of key transcription factors (TFs) as well as by epigenetic changes. We demonstrated that histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) combined with a chromatin remodeling medium (CRM) induced expression of a number of definitive endoderm and early and late pancreatic marker genes. When CRM was omitted, endoderm/pancreatic marker genes were not induced. Furthermore, treatment with DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (5AZA) CRM did not affect gene expression changes, and when 5AZA was combined with TSA, no further increase in gene expression of endoderm, pancreatic endoderm, and endocrine markers was seen over levels induced with TSA alone. Interestingly, TSA-CRM did not affect expression of pluripotency and hepatocyte genes but induced some mesoderm transcripts. Upon removal of TSA-CRM, the endoderm/pancreatic gene expression profile returned to baseline. Our findings underscore the role epigenetic modification in transdifferentiation of one somatic cell into another. However, full reprogramming of fibroblasts to β-cells will require combination of this approach with TF overexpression and/or culture of the partially reprogrammed cells under β-cell specific conditions.

SCARECROW-LIKE23 and SCARECROW jointly specify endodermal cell fate but distinctly control SHORT-ROOT movement.

Intercellular signaling through trafficking of regulatory proteins is a widespread phenomenon in plants and can deliver positional information for the determination of cell fate. In the Arabidopsis root meristem, the cell fate determinant SHORT-ROOT (SHR), a GRAS domain transcription factor, acts as a signaling molecule from the stele to the adjacent layer to specify endodermal cell fate. Upon exiting the stele, SHR activates another GRAS domain transcription factor, SCARCROW (SCR), which, together with several BIRD/INDETERMINATE DOMAIN proteins, restricts movement of SHR to define a single cell layer of endodermis. Here we report that endodermal cell fate also requires the joint activity of both SCR and its closest homologue SCARECROW-LIKE23 (SCL23). We show that SCL23 protein moves with zonation-dependent directionality. Within the meristem, SCL23 exhibits short-ranged movement from ground tissue to vasculature. Away from the meristem, SCL23 displays long-range rootward movement into meristematic vasculature and a bidirectional radial spread, respectively. As a known target of SHR and SCR, SCL23 also interacts with SCR and SHR and can restrict intercellular outspread of SHR without relying on nuclear retention as SCR does. Collectively, our data show that SCL23 is a mobile protein that controls movement of SHR and acts redundantly with SCR to specify endodermal fate in the root meristem.

Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst.

The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst-stage embryo. The EPI lineage isdistinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur invivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages invivo. A period of apoptosis occurred concomitantly with ICM cell-fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early mammalian embryonic development prevents fluctuations in cell fate.

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.

GATA6 Levels Modulate Primitive Endoderm Cell Fate Choice and Timing in the Mouse Blastocyst

Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.

Formation of a polarised primitive endoderm layer in embryoid bodies requires fgfr/erk signalling.

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.

Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm

How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.

Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions.

Genome-Wide Direct Target Analysis Reveals a Role for SHORT-ROOT in Root Vascular Patterning through Cytokinin Homeostasis

SHORT-ROOT (SHR) is a key regulator of root growth and development in Arabidopsis (Arabidopsis thaliana). Made in the stele, the SHR protein moves into an adjacent cell layer, where it specifies endodermal cell fate; it is also essential for apical meristem maintenance, ground tissue patterning, vascular differentiation, and lateral root formation. Much has been learned about the mechanism by which SHR controls radial patterning, but how it regulates other aspects of root morphogenesis is still unclear. To dissect the SHR developmental pathway, we have determined the genome-wide locations of SHR direct targets using a chromatin immunoprecipitation followed by microarray analysis method. K-means clustering analysis not only identified additional quiescent center-specific SHR targets but also revealed a direct role for SHR in gene regulation in the pericycle and xylem. Using cell type-specific markers, we showed that in shr, the phloem and the phloem-associated pericycle expanded, whereas the xylem and xylem-associated pericycle diminished. Interestingly, we found that cytokinin level was elevated in shr and that exogenous cytokinin conferred a shr-like vascular patterning phenotype in wild-type root. By chromatin immunoprecipitation-polymerase chain reaction and reverse transcription-polymerase chain reaction assays, we showed that SHR regulates cytokinin homeostasis by directly controlling the transcription of cytokinin oxidase 3, a cytokinin catabolism enzyme preferentially expressed in the stele. Finally, overexpression of a cytokinin oxidase in shr alleviated its vascular patterning defect. On the basis of these results, we suggest that one mechanism by which SHR controls vascular patterning is the regulation of cytokinin homeostasis.

Specific roles for the GATA transcription factors end-1 and end-3 during C. elegans E-lineage development

end-1 and end-3 are GATA transcription factors important for specifying endoderm cell fate in Caenorhabditis elegans. Deletion of both factors together results in larval arrest, 0% survival and a fate change in the endoderm-specifying E lineage. Individual deletions of either factor, however, result in the development of viable, fertile adults, with 100% of worms developing to adults for end-1(−) and 95% for end-3(−). We sought to quantify the variable phenotypes seen in both deletions using automated cell lineaging. We quantified defects in cell lifetime, cell movement and division axis in end-3(−) embryos, while quantifying perturbations in downstream reporter gene expression in strains with homozygous deletions for either gene, showing that each deletion leads to a unique profile of downstream perturbations in gene expression and cellular phenotypes with a high correlation between early and late defects. Combining observations in both cellular and gene expression defects we found that misaligned divisions at the E2 stage resulted in ectopic expression of the Notch target ref-1 in end-3(−) embryos. Using a maximum likelihood phylogenetic approach we found end-1 and end-3 split to form two distinct clades within the Caenorhabditis lineage with distinct DNA-binding structures. These results indicate that end-1 and end-3 have each evolved into genes with unique functions during endoderm development, that end-3(−) embryos have a delay in the onset of E lineage cell fate and that end-1 has only a partially penetrant ability to activate E lineage fate.