Direct development of neurons within foregut endoderm of sea urchin embryos.
ABSTRACT: Although it is well established that neural cells are ectodermal derivatives in bilaterian animals, here we report the surprising discovery that some of the pharyngeal neurons of sea urchin embryos develop de novo from the endoderm. The appearance of these neurons is independent of mouth formation, in which the stomodeal ectoderm joins the foregut. The neurons do not derive from migration of ectoderm cells to the foregut, as shown by lineage tracing with the photoactivatable protein KikGR. Their specification and development depend on expression of Nkx3-2, which in turn depends on Six3, both of which are expressed in the foregut lineage. SoxB1, which is closely related to the vertebrate Sox factors that support a neural precursor state, is also expressed in the foregut throughout gastrulation, suggesting that this region of the fully formed archenteron retains an unexpected pluripotency. Together, these results lead to the unexpected conclusion that, within a cell lineage already specified to be endoderm by a well-established gene regulatory network [Peter IS, Davidson EH (2010) Dev Biol 340:188-199], there also operates a Six3/Nkx3-2-dependent pathway required for the de novo specification of some of the neurons in the pharynx. As a result, neuroendoderm precursors form in the foregut aided by retention of a SoxB1-dependent pluripotent state.
Project description:Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of nkx2.3+ pouch progenitors in zebrafish embryos and demonstrated an essential requirement of ectodermal BMP2b for their specification. At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgf?a rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification.
Project description:BACKGROUND: Metazoan digestive systems develop from derivatives of ectoderm, endoderm and mesoderm, and vary in the relative contribution of each germ layer across taxa and between gut regions. In a small number of well-studied model systems, gene regulatory networks specify endoderm and mesoderm of the gut within a bipotential germ layer precursor, the endomesoderm. Few studies have examined expression of endomesoderm genes outside of those models, and thus, it is unknown whether molecular specification of gut formation is broadly conserved. In this study, we utilize a sequenced genome and comprehensive fate map to correlate the expression patterns of six transcription factors with embryonic germ layers and gut subregions during early development in Capitella teleta. RESULTS: The genome of C. teleta contains the five core genes of the sea urchin endomesoderm specification network. Here, we extend a previous study and characterize expression patterns of three network orthologs and three additional genes by in situ hybridization during cleavage and gastrulation stages and during formation of distinct gut subregions. In cleavage stage embryos, Ct-otx, Ct-blimp1, Ct-bra and Ct-nkx2.1a are expressed in all four macromeres, the endoderm precursors. Ct-otx, Ct-blimp1, and Ct-nkx2.1a are also expressed in presumptive endoderm of gastrulae and later during midgut development. Additional gut-specific expression patterns include Ct-otx, Ct-bra, Ct-foxAB and Ct-gsc in oral ectoderm; Ct-otx, Ct-blimp1, Ct-bra and Ct-nkx2.1a in the foregut; and both Ct-bra and Ct-nkx2.1a in the hindgut. CONCLUSIONS: Identification of core sea urchin endomesoderm genes in C. teleta indicates they are present in all three bilaterian superclades. Expression of Ct-otx, Ct-blimp1 and Ct-bra, combined with previously published Ct-foxA and Ct-gataB1 patterns, provide the most comprehensive comparison of these five orthologs from a single species within Spiralia. Each ortholog is likely involved in endoderm specification and midgut development, and several may be essential for establishment of the oral ectoderm, foregut and hindgut, including specification of ectodermal and mesodermal contributions. When the five core genes are compared across the Metazoa, their conserved expression patterns suggest that 'gut gene' networks evolved to specify distinct digestive system subregions, regardless of species-specific differences in gut architecture or germ layer contributions within each subregion.
Project description:Patterning of the primitive foregut promotes appropriate organ specification along its anterior-posterior axis. However, the molecular pathways specifying foregut endoderm progenitors are poorly understood. We show here that Wnt2/2b signaling is required to specify lung endoderm progenitors within the anterior foregut. Embryos lacking Wnt2/2b expression exhibit complete lung agenesis and do not express Nkx2.1, the earliest marker of the lung endoderm. In contrast, other foregut endoderm-derived organs, including the thyroid, liver, and pancreas, are correctly specified. The phenotype observed is recapitulated by an endoderm-restricted deletion of beta-catenin, demonstrating that Wnt2/2b signaling through the canonical Wnt pathway is required to specify lung endoderm progenitors within the foregut. Moreover, activation of canonical Wnt/beta-catenin signaling results in the reprogramming of esophagus and stomach endoderm to a lung endoderm progenitor fate. Together, these data reveal that canonical Wnt2/2b signaling is required for the specification of lung endoderm progenitors in the developing foregut.
Project description:During gastrulation, distinct lineage specification into three germ layers, the mesoderm, endoderm and ectoderm, occurs through an elaborate harmony between signaling molecules along the embryonic proximo-distal and anterior-posterior axes, and Nodal signaling plays a key role in the early embryonic development governing embryonic axis formation, mesoderm and endoderm specification, and left-right asymmetry determination. However, the mechanism by which Nodal expression is regulated is largely unknown. Here, we show that Meteorin regulates Nodal expression and is required for mesendoderm development. It is highly expressed in the inner cell mass of blastocysts and further in the epiblast and extra-embryonic ectoderm during gastrulation. Genetic ablation of the Meteorin gene resulted in early embryonic lethality, presumably due to impaired lineage allocation and subsequent cell accumulation. Embryoid body culture using Meteorin-null embryonic stem (ES) cells showed reduced Nodal expression and concomitant impairment of mesendoderm specification. Meteorin-null embryos displayed reduced levels of Nodal transcripts before the gastrulation stage, and impaired expression of Goosecoid, a definitive endoderm marker, during gastrulation, while the proximo-distal and anterior-posterior axes and primitive streak formation were preserved. Our results show that Meteorin is a novel regulator of Nodal transcription and is required to maintain sufficient Nodal levels for endoderm formation, thereby providing new insights in the regulation of mesendoderm allocation.
Project description:Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5-11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn-/- mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.
Project description:The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction.
Project description:The current classification of cells in an organism is largely based on their anatomic and developmental origin. Cells types and tissues are traditionally classified into those that arise from the three embryonic germ layers, the ectoderm, mesoderm and endoderm, but this model does not take into account the organization of cell type-specific patterns of gene expression. Here, we present computational models for cell type and tissue specification derived from a collection of 921 RNA-sequencing samples from 272 distinct mouse cell types or tissues. In an unbiased fashion, this analysis accurately predicts the three known germ layers. Unexpectedly, this analysis also suggests that in total there are eight major domains of cell type-specification, corresponding to the neurectoderm, neural crest, surface ectoderm, endoderm, mesoderm, blood mesoderm, germ cells and the embryonic domain. Further, we identify putative genes responsible for specifying the domain and the cell type. This model has implications for understanding trans-lineage differentiation for stem cells, developmental cell biology and regenerative medicine.
Project description:Cnidarians (for example, sea anemones and jellyfish) develop from an outer ectodermal and inner endodermal germ layer, whereas bilaterians (for example, vertebrates and flies) additionally have a mesodermal layer as intermediate germ layer. Currently, cnidarian endoderm (that is, 'mesendoderm') is considered homologous to both bilaterian endoderm and mesoderm. Here we test this hypothesis by studying the fate of germ layers, the localization of gut cell types, and the expression of numerous 'endodermal' and 'mesodermal' transcription factor orthologues in the anthozoan sea anemone Nematostella vectensis. Surprisingly, we find that the developing pharyngeal ectoderm and its derivatives display a transcription-factor expression profile (foxA, hhex, islet, soxB1, hlxB9, tbx2/3, nkx6 and nkx2.2) and cell-type combination (exocrine and insulinergic) reminiscent of the developing bilaterian midgut, and, in particular, vertebrate pancreatic tissue. Endodermal derivatives, instead, display cell functions and transcription-factor profiles similar to bilaterian mesoderm derivatives (for example, somatic gonad and heart). Thus, our data supports an alternative model of germ layer homologies, where cnidarian pharyngeal ectoderm corresponds to bilaterian endoderm, and the cnidarian endoderm is homologous to bilaterian mesoderm.
Project description:A great challenge in development biology is to understand how interacting networks of regulatory genes can direct the often highly complex patterning of cells in a 3D embryo. Here, we detail the gene regulatory network that describes the distribution of ciliary band-associated neurons in the bipinnaria larva of the sea star. This larva, typically for the ancestral deuterostome dipleurula larval type that it represents, forms two loops of ciliary bands that extend across much of the anterior-posterior and dorsal-ventral ectoderm. We show that the sea star first likely uses maternally inherited factors and the Wnt and Delta pathways to distinguish neurogenic ectoderm from endomesoderm. The broad neurogenic potential of the ectoderm persists throughout much of gastrulation. Nodal, bone morphogenetic protein 2/4 (Bmp2/4), and Six3-dependent pathways then sculpt a complex ciliary band territory that is defined by the expression of the forkhead transcription factor, foxg. Foxg is needed to define two molecularly distinct ectodermal domains, and for the formation of differentiated neurons along the edge of these two territories. Thus, significantly, Bmp2/4 signaling in sea stars does not distinguish differentiated neurons from nonneuronal ectoderm as it does in many other animals, but instead contributes to the patterning of an ectodermal territory, which then, in turn, provides cues to permit the final steps of neuronal differentiation. The modularity between specification and patterning likely reflects the evolutionary history of this gene regulatory network, in which an ancient module for specification of a broad neurogenic potential ectoderm was subsequently overlaid with a module for patterning.
Project description:Embryonic development of the respiratory system is regulated by a series of mesenchymal-epithelial interactions that are only partially understood. Mesenchymal FGF and Wnt2/Wnt2b signaling are implicated in specification of mammalian pulmonary progenitors from the ventral foregut endoderm, but their epistatic relationship and downstream targets are largely unknown. In addition, how wnt2 and wnt2b are regulated in the developing foregut mesenchyme is unknown. We show that the Odd-skipped-related (Osr) zinc-finger transcriptional repressors Osr1 and Osr2 are redundantly required for Xenopus lung specification in a molecular pathway linking foregut pattering by FGFs to Wnt-mediated lung specification and RA-regulated lung bud growth. FGF and RA signals are required for robust osr1 and osr2 expression in the foregut endoderm and surrounding lateral plate mesoderm (lpm) prior to respiratory specification. Depletion of both Osr1 and Osr2 (Osr1/Osr2) results in agenesis of the lungs, trachea and esophagus. The foregut lpm of Osr1/Osr2-depleted embryos fails to express wnt2, wnt2b and raldh2, and consequently Nkx2.1(+) progenitors are not specified. Our data suggest that Osr1/Osr2 normally repress bmp4 expression in the lpm, and that BMP signaling negatively regulates the wnt2b domain. These results significantly advance our understanding of early lung development and may impact strategies to differentiate respiratory tissue from stem cells.