Germ Layer Origin Research Articles

Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis

The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air–liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.

Chemical genomics reveals targetable programs of human cancers rooted in pluripotency

Overlapping principles of embryonic and tumor biology have been described, with recent multi-omics campaigns uncovering shared molecular profiles between human pluripotent stem cells (hPSCs) and adult tumors. Here, using a chemical genomic approach, we provide biological evidence that early germ layer fate decisions of hPSCs reveal targets of human cancers. Single-cell deconstruction of hPSCs-defined subsets that share transcriptional patterns with transformed adult tissues. Chemical screening using a unique germ layer specification assay for hPSCs identified drugs that enriched forcompounds that selectively suppressed the growth of patient-derived tumors corresponding exclusively to their germ layer origin. Transcriptional response of hPSCs to germ layer inducing drugs couldbe used to identify targets capable of regulating hPSC specification as well as inhibiting adult tumors. Our study demonstrates properties of adult tumors converge with hPSCs drug induced differentiation in a germ layer specific manner, thereby expanding our understanding of cancer stemness and pluripotency.

Alliance of Heart and Endoderm: Multilineage Organoids to Model Co-development.

Studies in animal models tracing organogenesis of the mesoderm-derived heart have emphasized the importance of signals coming from adjacent endodermal tissues in coordinating proper cardiac morphogenesis. Although in vitro models such as cardiac organoids have shown great potential to recapitulate the physiology of the human heart, they are unable to capture the complex crosstalk that takes place between the co-developing heart and endodermal organs, partly due to their distinct germ layer origins. In an effort to address this long-sought challenge, recent reports of multilineage organoids comprising both cardiac and endodermal derivatives have energized the efforts to understand how inter-organ, cross-lineage communications influence their respective morphogenesis. These co-differentiation systems have produced intriguing findings of shared signaling requirements for inducing cardiac specification together with primitive foregut, pulmonary, or intestinal lineages. Overall, these multilineage cardiac organoids offer an unprecedented window into human development that can reveal how the endoderm and heart cooperate to direct morphogenesis, patterning, and maturation. Further, through spatiotemporal reorganization, the co-emerged multilineage cells self-assemble into distinct compartments as seen in the cardiac-foregut, cardiac-intestine, and cardiopulmonary organoids and undergo cell migration and tissue reorganization to establish tissue boundaries. Looking into the future, these cardiac incorporated, multilineage organoids will inspire future strategies for improved cell sourcing for regenerative interventions and provide more effective models for disease investigation and drug testing. In this review, we will introduce the developmental context of coordinated heart and endoderm morphogenesis, discuss strategies for in vitro co-induction of cardiac and endodermal derivatives, and finally comment on the challenges and exciting new research directions enabled by this breakthrough.

Association between cancer genes and germ layer specificity.

Cancer signaling pathways defining cell fates are related to differentiation. During the developmental process, three germ layers (endoderm, mesoderm, and ectoderm) are formed during embryonic development that differentiate into organs via the epigenetic regulation of specific genes. To examine the relationship, the specificities of cancer gene mutations that depend on the germ layers are studied. The major organs affected by cancer were determined based on statistics from the National Cancer Information Center of Korea, and were grouped according to their germ layer origins. Then, the gene mutation frequencies were evaluated to identify any bias based on the differentiation group using the Catalogue of Somatic Mutations in Cancer (COSMIC) database. The chi-square test showed that the p-value of 152 of 166 genes was less than 0.05, and 151 genes showed p-values of less than 0.05 even after adjusting for the false discovery rate (FDR). The germ layer-specific genes were evaluated using visualization based on basic statistics, and the results matched the top ranking genes depending on organs in the COSMIC database.The current study confirmed the germ layer specificity of major cancer genes. The germ layer specificity of mutated driver genes is possibly important in cancer treatments because each mutated gene may react differently depending on the germ layer of origin. By understanding the mechanism of gene mutation in the development and progression of cancer in the context of cell-fate pathways, a more effective therapeutic strategy for cancer can be established.

Animal evolution: Of flame and collar cells

Nephridia are small excretory organs that function in waste disposal and osmoregulation in a wide range of animals but differ in type and germ-layer origin. A new comparison of gene expression in disparate species supports homology of nephridia across bilaterian animals, and their origin from ciliated epidermal cells.

Resolving homology in the face of shifting germ layer origins: Lessons from a major skull vault boundary.

The vertebrate skull varies widely in shape, accommodating diverse strategies of feeding and predation. The braincase is composed of several flat bones that meet at flexible joints called sutures. Nearly all vertebrates have a prominent ‘coronal’ suture that separates the front and back of the skull. This suture can develop entirely within mesoderm-derived tissue, neural crest-derived tissue, or at the boundary of the two. Recent paleontological findings and genetic insights in non-mammalian model organisms serve to revise fundamental knowledge on the development and evolution of this suture. Growing evidence supports a decoupling of the germ layer origins of the mesenchyme that forms the calvarial bones from inductive signaling that establishes discrete bone centers. Changes in these relationships facilitate skull evolution and may create susceptibility to disease. These concepts provide a general framework for approaching issues of homology in cases where germ layer origins have shifted during evolution.

Sox2 and Klf4 as the Functional Core in Pluripotency Induction without Exogenous Oct4

Ectopic expression of Oct4, Sox2, Klf4, and c-Myc can reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs). For years, Oct4 has been considered the key reprogramming factor core of the four factors. Here, we challenge this view by reporting a core function of Sox2 and Klf4 in reprogramming. We found that polycistronic expression of Sox2 and Klf4 was sufficient to induce pluripotency in the absence of exogenous Oct4, and the stoichiometry of Sox2 and Klf4 was essential. Sox2 and Klf4 cooperatively bound across the genome, leading to epigenetic remodeling of their targets, including pluripotency genes and gradual activation of the pluripotency network. Interestingly, cells of different germ layer origins, fibroblasts (mesoderm) and neural progenitor cells (ectoderm), showed convergent reprogramming trajectories and similar efficiency. This work demonstrates a core function of Sox2 and Klf4 in pluripotency induction and shows that this mechanism is independent of germ layer origin.

Conventional and Emerging Markers in Stem Cell Isolation and Characterization.

Stem cells have emerged as a promising source of cell-based therapy in regenerative medicine with several stem cell-based products currently in clinical trials. Despite the immense therapeutic potential, their isolation from some of the emerging sources and their characterization has been naïve owing to the lack of standard markers for the same. Some biomarkers have now been well established for the isolation and characterization of stem cells. However, there are emerging markers that can be used in addition to these conventional markers or independent of them to establish the identity of the stem cells. In this review, an attempt has been made to describe a few conventionally used markers and emerging markers for the identification, isolation and characterization of stem cells from various niches across the three germ layer origins.

Olfr603, an orphan olfactory receptor, is expressed in multiple specific embryonic tissues

BackgroundOlfactory receptors were initially believed to be expressed specifically within the olfactory neurons. However, accumulating genome-scale data has recently demonstrated more extensive expression. There are hundreds of olfactory receptor family members and the realisation of their widespread expression provides an opportunity to reveal new biology. However, existing data is predominantly based on RT-PCR, microarray and RNA-seq approaches and the details of tissue and cell-type specific expression are lacking. ResultsAs a proof of principle, we selected Olfr603 for expression analysis. We generated an antibody against a non-conserved epitope of Olfr603 and characterised its expression in E8.5-E12.5 mouse embryos using immunohistochemistry. This analysis demonstrated a dynamic pattern of expression in diverse cell types within the developing embryo unrelated to the olfactory system. Expression was detected in migrating neural crest, endothelial precursors and vascular endothelium, endocardial cells, smooth muscle, neuroepithelium and within the ocular tissues. This complex distribution does not conform to any apparent germ layer or tissue origin. ConclusionsThis initial characterisation of Olfr603 expression highlights the potential for a broad role for this receptor in the development of many tissues.

A Model of the Anterior Esophagus in Snakes, with Functional and Developmental Implications

The gross anatomy of the mouth of snakes has always been interpreted as an evolutionary response to feeding demands. In most alethinophidian species, their anatomy allows limited functional independence of right and left sides and the roof and floor of the mouth as well as wide separation of the tips of the mandibles. However, locations of the tongue and glottis in snakes suggest extraordinary rearrangement of pharyngeal structures characteristic of all vertebrates. Serial histological sections through the heads of a number of colubroid species show muscularis mucosal smooth muscle fibers appearing in the paratracheal gutter of the lower jaw at varying levels between the eye and ear regions. Incomplete muscularis externa elements appear beneath the paratracheal gutter more caudally but typically at otic levels. Both muscle layers encompass more of the gut wall at more posterior levels, encircling the gut at the level of the atlas or axis. The pattern in snakes suggests developmental dissociation of dorsal and ventral splanchnic derivatives and extensive topological rearrangements of some ventral pharyngeal arch derivatives typical of most tetrapods. When snakes swallow large prey, the effective oral cavity becomes extremely short ventrally. The palatomaxillary arches function as ratchets packing the prey almost directly into the esophagus. Our findings raise questions about germ layer origins and regulation of differentiation of gut regions and derivatives in snakes and suggest that significant aspects of the evolution of lepidosaurs may be difficult to recover from bones or molecular sequence data alone.

Exposure to Simulated Microgravity Affects Development of the Skull in Danio rerio

To determine how exposure to simulated microgravity (SMG) affects development of the skull, zebrafish embryos were exposed to SMG starting at one of three developmental stages: 10, 12 or 14 hours post‐fertilization. Juvenile and adult fish were analyzed after exposure to SMG using whole‐mount staining and geometric morphometrics. Fish were examined for changes in melanophore surface area and number, in addition to changes in skull morphology. Analyses reveal an overall decrease in the surface area of melanophores and an increase in melanophore number over the period of a week in experimental and control groups; with significant differences in the number of melanophores on the first day after exposure. These effects are short‐term and do not persist in adults. Gross morphological analyses of whole‐mount stained adults, however, identified buckling in several (but not all) craniofacial bones. In conclusion, exposure to SMG affects both skeletal tissues and pigmentation in the head. These results will be discussed in context of germ layer origin. This is the first study examining the long‐term effects of exposure to SMG on zebrafish and provides valuable insight into phenotypic variation.Grant Funding Source : NSERC, Canada; and CSA, Canada

Comparative Study of Hematopoietic Lineage Potential of Ips Cells Derived From Two Mesodermal Cell Lineages

Abstract 1285Identifying the key regulatory genes that direct human ES/iPS cell differentiation to certain lineages is required before considering these cells for their potential clinical application. It has been proposed that the cellular starting material used to generate novel iPS cell lines imparts an epigenetic memory which in turn influences the iPS lines’ potential to differentiate towards certain developmental lineages. To determine novel key regulators that direct ES/iPS cell differentiation to hematopoietic lineages, we decided to compare differentiation capacity of multiple iPS cell lines generated from two neonatal mesodermal tissue derived cell types. For this, we generated multiple iPS cell lines from umbilical cord blood derived endothelial cells and amniotic fluid derived mesenchymal stem cells, using an identical dedifferentiation system/protocol. Despite the similar germ layer origin of these iPS derived cell lines, these lines showed significant differences in blood generation efficiency and lineage specification capacity. From the 8 cord blood derived iPS cell lines we tested, the average efficiency of hematopoietic (CD45+) cell generation was 34.6+/−21.2% (range 9.8 to 65.8%), of which 5.8+/− 4% were CD45+CD34+ hematopoietic progenitors. From the 9 mesenchymal stem cell derived iPS lines, the average efficiency of CD45+ hematopoietic cells was 14.2+/− 9% (range 1.6 to 26.3%), of which 2.9+/− 1.9% were CD45+CD34+ hematopoietic progenitors (Figure 1A). Note, using our standardized iPS-2-Blood differentiation protocol the efficiency of blood differentiation for individual iPS cell lines was highly reproducible. These results indicate a significant increase in the efficiency of blood generation from the endothelial derived iPS cell lines compared to the mesenchymal stem cell derived iPS cell lines, and suggest that the close ontological relationship between hematopoietic cells and endothelial cells results in the increased efficiency of blood generation seen here. We further compared the iPS cell lines for their potential to differentiate into hematopoietic cells of the myeloid, erythroid, and lymphoid lineages. Both mesoderm lineage derived iPS cell lines showed myeloid and lymphoid lineage differentiation potential as evidenced by CFU assay for presence of macrophages and granulocytes, and by 4-week OP9, and OP9-DL1 culture assays showing CD19, CD10, CD34 expression for B cells, CD1a, CD7, CD5, CD4, TCRb, CD3 expression for T cells, and CD56, CD16 for NK cells. However, further comparative analysis revealed a limited erythroid (BFU-E) potential of cord blood derived iPS cell lines compared to amniotic fluid derived iPS cell lines (Figure 1B). Most amniotic fluid derived iPS lines showed significantly erythroid cell potential, where as the cord blood derived iPS lines showed significantly reduced or no erythroid cell potential. These results suggest that the endothelial origin of cord blood derived iPS lines imparts an epigenetic memory that results in the reduced efficiency of erythroid cell generation compared with mesenchymal stem cell derived iPS lines. The difference in the efficiency of hematopoietic cell generation and lineage specification between the cord blood and amniotic fluid derived iPS lines suggests that iPS reprogramming to a pluripotent state is not complete (despite iPS cell line validation of pluripotency via teratocarcinoma assay), and significant gene expression differences exist between the lines. We are currently performing gene expression and epigenetic profile analyses of these closely related mesodermal lines, and expect that the close ontological relationship of the two mesoderm derived cell types will facilitate the deconvolution of the molecular basis of the differences. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Retracted:The Potential of Stem Cells in Adult Tissues Representative of the Three Germ Layers

Mature adult tissues contain stem cells that express many genes normally associated with the early stage of embryonic development, when maintained in appropriate environments. Cells procured from adult tissues representative of the three germ layers (spinal cord, muscle, and lung), each exhibiting the potential to mature into cells representative of all three germ layers. Cells isolated from adult tissues of different germ layer origin were propagated as nonadherent clusters or spheres that were composed of heterogeneous populations of cells. When the clusters or spheres were dissociated, the cells had the ability to reform new, nonadherent spheres for several generations. When implanted in vivo, in association with biodegradable scaffolds, into immunodeficient mice, tissue containing cells characteristic of the three germ layers was generated. These findings suggest the existence of a population of stem cells in adult tissues that is quite different and distinct from embryonic stem cells that demonstrate a greater potency for differentiation across germ lines than previously believed. Such cells could potentially be as useful as embryonic stem cells in tissue engineering and regenerative medicine.

O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation

Thrombospondin type 1 repeat (TSR) superfamily members regulate diverse biological activities ranging from cell motility to inhibition of angiogenesis. In this study, we verified that mouse protein O-fucosyltransferase-2 (POFUT2) specifically adds O-fucose to TSRs. Using two Pofut2 gene-trap lines, we demonstrated that O-fucosylation of TSRs was essential for restricting epithelial to mesenchymal transition in the primitive streak, correct patterning of mesoderm, and localization of the definitive endoderm. Although Pofut2 mutant embryos established anterior/posterior polarity, they underwent extensive mesoderm differentiation at the expense of maintaining epiblast pluripotency. Moreover, mesoderm differentiation was biased towards the vascular endothelial cell lineage. Localization of Foxa2 and Cer1 expressing cells within the interior of Pofut2 mutant embryos suggested that POFUT2 activity was also required for the displacement of the primitive endoderm by definitive endoderm. Notably, Nodal, BMP4, Fgf8, and Wnt3 expression were markedly elevated and expanded in Pofut2 mutants, providing evidence that O-fucose modification of TSRs was essential for modulation of growth factor signaling during gastrulation. The ability of Pofut2 mutant embryos to form teratomas comprised of tissues from all three germ layer origins suggested that defects in Pofut2 mutant embryos resulted from abnormalities in the extracellular environment. This prediction is consistent with the observation that POFUT2 targets are constitutive components of the extracellular matrix (ECM) or associate with the ECM. For this reason, the Pofut2 mutants represent a valuable tool for studying the role of O-fucosylation in ECM synthesis and remodeling, and will be a valuable model to study how post-translational modification of ECM components regulates the formation of tissue boundaries, cell movements, and signaling.

Lineage Enforcement by Inductive Mesenchyme on Adult Epithelial Stem Cells across Developmental Germ Layers

During development, cell differentiation is accompanied by the progressive loss of pluripotent gene expression and developmental potential, although de-differentiation in specialized cells can be induced by reprogramming strategies, indicating that transdifferentiation potential is retained in adult cells. The stromal niche provides differentiating cues to epithelial stem cells (SCs), but current evidence is restricted to tissue types within the same developmental germ layer lineage. Anticipating the use of adult SCs for tissue regeneration, we examined if stroma can enforce lineage commitment across germ layer boundaries and promote transdifferentiation of adult epithelial SCs. Here, we report tissue-specific mesenchyme instructing epithelial cells from a different germ layer origin to express dual phenotypes. Prostatic stroma induced mammary epithelia (or enriched Lin(-)CD29(HI)CD24(+/MOD) mammary SCs) to generate glandular epithelia expressing both prostatic and mammary markers such as steroid hormone receptors and transcription factors including Foxa1, Nkx3.1, and GATA-3. Array data implicated Hh and Wnt pathways in mediating stromal-epithelial interactions (validated by increased Cyclin D1 expression). Other recombinants of prostatic mesenchyme and skin epithelia, or preputial gland mesenchyme and bladder or esophageal epithelia, showed foci expressing new markers adjacent to the original epithelial differentiation (e.g., sebaceous cells within bladder urothelium), confirming altered lineage specification induced by stroma and evidence of cross-germ layer transdifferentiation. Thus, stromal cell niche is critical in maintaining (or redirecting) differentiation in adult epithelia. In order to use adult epithelial SCs in regenerative medicine, we must additionally regulate their intrinsic properties to prevent (or enable) transdifferentiation in specified SC niches.

Thymus Organogenesis

The epithelial architecture of the thymus fosters growth, differentiation, and T cell receptor repertoire selection of large numbers of immature T cells that continuously feed the mature peripheral T cell pool. Failure to build or to maintain a proper thymus structure can lead to defects ranging from immunodeficiency to autoimmunity. There has been long-standing interest in unraveling the cellular and molecular basis of thymus organogenesis. Earlier studies gave important morphological clues on thymus development. More recent cell biological and genetic approaches yielded new and conclusive insights regarding the germ layer origin of the epithelium and the composition of the medulla as a mosaic of clonally derived islets. The existence of epithelial progenitors common for cortex and medulla with the capacity for forming functional thymus after birth has been uncovered. In addition to the thymus in the chest, mice can have a cervical thymus that is small, but functional, and produces T cells only after birth. It will be important to elucidate the pathways from putative thymus stem cells to mature thymus epithelial cells, and the properties and regulation of these pathways from ontogeny to thymus involution.

A conserved role of the VEGF pathway in angiogenesis of an ectodermally-derived vasculature

Angiogenesis, the growth and remodeling of a vascular network, is an essential process during development, growth and disease. Here we studied the role of the vascular endothelial growth factor receptor (VEGFR) in experimentally-induced angiogenesis in the colonial ascidian Botryllus schlosseri (Tunicata, Ascidiacea). The circulatory system of B. schlosseri is composed of two distinct, but interconnected regions: a plot of sinuses and lacunae which line the body, and a transparent, macroscopic extracorporeal vascular network. The vessels of the extracorporeal vasculature are morphologically inverted in comparison to the vasculature in vertebrates: they consist of a single layer of ectodermally-derived cells with the basal lamina lining the lumen of the vessel. We found that when the peripheral circulatory system of a colony is surgically removed, it can completely regenerate within 24 to 48 h and this regeneration is dependent on proper function of the VEGF pathway: siRNA-mediated knockdown of the VEGFR blocked vascular regeneration, and interfered with vascular homeostasis. In addition, a small molecule, the VEGFR kinase inhibitor PTK787/ZK222584, phenocopied the siRNA knockdown in a reversible manner. Despite the disparate germ layer origins and morphology of the vasculature, the developmental program of branching morphogenesis during angiogenesis is controlled by similar molecular mechanisms, suggesting that the function of the VEGF pathway may be co-opted during the regeneration of an ectoderm-derived tubular structure.

Pluripotentiality and Conditional Transgene Regulation in Human Embryonic Stem Cells Expressing Insulated Tetracycline-ON Transactivator

Conditional manipulation of gene expression by using tetracycline (TET)-ON based approaches has proven invaluable to study fundamental aspects of biology; however, the functionality of these systems in human embryonic stem cells (hESC) has not been established. Given the sensitivity of these cells to both genetic manipulation and variations of culture conditions, constitutive expression of TET transactivators might not only be toxic for hESC but might also impair their ability to self-renew or differentiate into multiple tissues. Therefore, the effect of these transactivators on the biology and pluripotentiality of hESC must first be evaluated before broad use of TET-ON methodologies is applied in these cells. Improved insulated lentivectors that display stable transgene expression and minimal insertional transactivation have been described for hESC. By using insulated lentivectors that allow simultaneous expression of TET components and fluorescent reporters, here we demonstrate that hESC constitutively expressing the TET-ON transactivator rtTA2SM2 can be derived and expanded in culture while retaining inducible transgene expression and pluripotentiality, including marker expression, a normal karyotype, and the ability to generate multiple tissues of different germ layer origin in teratomas. We also show that these cells retain the ability to control the expression of a stable integrated transgene in a doxycycline-dependent manner, which demonstrates that an insulated TET-ON lentiviral system is functional in hESC. Together, our results indicate that improved TET regulators like rtTA2SM2 in combination with insulated lentiviral-based systems offer alternative strategies for conditional gene expression in hESC. Disclosure of potential conflicts of interest is found at the end of this article.

Limited Contribution of Cells of Intact Extrahepatic Tissue Origin to Hepatocyte Regeneration in Transplanted Rat Liver

It is now well established that various adult somatic tissues harbor multipotent stem cells that can differentiate into a broad variety of cell types of all three germ layer origins. It remains controversial, however, whether they are a reservoir of cells utilized for emergent tissue repair or simply a vestige of evolution and, if the former is the case, to what extent they can potentially contribute to reconstitution of a specific organ. To get an insight in such a direction, we examined the extent of contribution of naive intact cells of extrahepatic origin to hepatocyte reconstitution in the transplanted liver with or without injury in the rat. Liver from wild-type donor rats was transplanted to green fluorescent protein (GFP)-transgenic rats, and GFP-positive hepatocytes were examined with or without liver injury. The proportion of GFP-positive hepatocytes in the transplanted noninjured liver linearly increased by 0.0048% per week, that is, approximately 5 x 10(3) hepatocytes of extrahepatic origin were generated per day. Liver injury induced by treatment with 2-acetylaminofluorene and CCl4 or the additional application of hepatocyte growth factor did not further increase the percentage of GFP-positive hepatocytes. The present results indicate that cells derived from nonmanipulated extrahepatic tissues appreciably contribute, though limitedly, to hepatocyte reconstitution in the liver of the rat.

Specification of germ layer identity in the chick gastrula

BackgroundChick definitive endoderm is an important source of signals that pattern the early embryo forming a central structure around which the body plan is constructed. Although the origin of definitive endoderm has been mapped in the chick, arising principally from rostral streak at elongating streak stages, it is not known when this layer first becomes fully committed to its germ layer fate, an important issue to resolve in light of its critical role in subsequent patterning of the early embryo.ResultsThrough gene expression screening of chick gastrula, we identified molecular markers of definitive endoderm restricted to rostral (Sox17) and caudal (Gata5/6) regions, suggesting that at least two subpopulations of definitive endodermal cells exist during ingression. We show (1) that presumptive mesoderm cells migrate to the middle layer and remain mesenchymal when transplanted to rostral primitive streak, and prospective endoderm cells enter the lower layer and become epithelial when transplanted to caudal primitive streak; and (2) that presumptive endoderm cells and mesoderm cells lose normal gene expression (Sox17 and Wnt8c, respectively) when transplanted outside of their normal position of origin. Moreover, when rostral or caudal primitive streak segments are transplanted into rostral blastoderm isolates (RBIs), both types of transplants express Sox17 4–6 hours later–consistent with their new position, regardless of their presumptive germ layer origin–and prospective mesoderm transplants, which normally express Wnt8c, turn off expression, suggesting that signals within the rostral blastoderm induce endoderm gene expression, and repress mesoderm gene expression, during gastrulation.ConclusionOur results demonstrate that germ layer identity is fixed at the time populations of endoderm and mesoderm cells ingress through the primitive streak, whereas their gene expression patterns remain labile. In addition, our results show that inductive and repressive signals are present, and that these signals regulate gene expression of both ingressed endoderm and mesoderm cells. Thus, gastrula cells display elements of both pre-patterning and plasticity, with endoderm the first germ layer becoming committed to its fate during early gastrulation stages.