Enhancers, development, and evolution.

A single-celled fertilized egg develops into a complex, multicellular animal through a series of selection processes of developmental pathways. During these processes, regulatory genes exhibit spatiotemporally restricted expression under the control of the species-specific genetic program, and dictate developmental processes from germ layer formation to cellular differentiation. Elucidation of molecular mechanisms underlying developmental processes and also of mechanistic bases for morphological diversification during evolution is one of the central issues in contemporary developmental biology. Progress has been made due to recent technological innovations, such as high-throughput nucleotide sequencing, live-cell imaging, efficient genetic manipulation, and establishment of the organoid system, opening new avenues to the above issues.

The absence of SOX2 in the anterior foregut alters the esophagus into trachea and bronchi in both epithelial and mesenchymal components.

In the anterior foregut (AFG) of mouse embryos, the transcription factor SOX2 is expressed in the epithelia of the esophagus and proximal branches of respiratory organs comprising the trachea and bronchi, whereas NKX2.1 is expressed only in the epithelia of respiratory organs. Previous studies using hypomorphic Sox2 alleles have indicated that reduced SOX2 expression causes the esophageal epithelium to display some respiratory organ characteristics. In the present study, we produced mouse embryos with AFG-specific SOX2 deficiency. In the absence of SOX2 expression, a single NKX2.1-expressing epithelial tube connected the pharynx and the stomach, and a pair of bronchi developed in the middle of the tube. Expression patterns of NKX2.1 and SOX9 revealed that the anterior and posterior halves of SOX2-deficient AFG epithelial tubes assumed the characteristics of the trachea and bronchus, respectively. In addition, we found that mesenchymal tissues surrounding the SOX2-deficient NKX2.1-expressing epithelial tube changed to those surrounding the trachea and bronchi in the anterior and posterior halves, as indicated by the arrangement of smooth muscle cells and SOX9-expressing cells and by the expression of Wnt4 (esophagus specific), Tbx4 (respiratory organ specific), and Hoxb6 (distal bronchus specific). The impact of mesenchyme-derived signaling on the early stage of AFG epithelial specification has been indicated. Our study demonstrated an opposite trend where epithelial tissue specification causes concordant changes in mesenchymal tissues, indicating a reciprocity of epithelial-mesenchymal interactions.

Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line.

Transgenic birds are commonly used for time-lapse imaging and fate mapping studies in developmental biology. When researchers use transgenic birds expressing fluorescent protein, they need to understand the integration site of the transgene in the genome and the intensity of fluorescence in the tissues of interest. In this study, we determined the integration site of the transgene and fluorescence property of developing organs in our transgenic chicken line generated by lentivirus infection. The transgene was localized between exons 3 and 4 of MED27. Some homozygotes and heterozygotes appeared to be lethal at early embryonic stages. We performed histological analysis of EGFP expression in transgenic embryos at St. 14, 17, and 24 by immunohistochemistry with anti-GFP antibody on paraffin sections. Next, we cut cryosections and quantified direct EGFP intensity from the transgene in each tissue without performing immunohistochemistry. These results revealed that EGFP intensity in each tissue was unique in developing embryos and changed according to developmental stages. Finally, we demonstrated that EGFP-expressing cells in a micromass culture with co-culturing wild-type cells were clearly distinguishable via live cell imaging. These results provide essential information on the potential of our transgenic line and indicate that these transgenic chicken lines are useful for research associated with developmental biology.

PRDM14 and BLIMP1 control the development of chicken primordial germ cells.

The differentiation of primordial germ cells (PGCs) is a fundamental step in development. PR domain-containing protein 14 (PRDM14) and B lymphocyte-induced maturation protein 1 (BLIMP1) play pivotal roles in mouse PGC specification. In the present study, we assessed the roles of chicken orthologs of PRDM14 and BLIMP1 in PGC development. PRDM14 and BLIMP1 were expressed in blastodermal cells and PGCs. The in vivo knockdown of PRDM14 or BLIMP1 by introducing a replication-competent retroviral vector expressing shRNAs to the blastodermal stage of embryos reduced the number of SSEA-1 or chicken vasa homologue-positive PGCs on day 5.5-6.5. Since the inhibition of Activin receptor-like kinase 4/5/7 in cultured PGCs reduced the expression of PRDM14, BLIMP1, and NANOG, and that of MEK inhibited PRDM14 expression, the expression of these genes seems to be controlled by Activin A and FGF2 signaling. Overall, PRDM14, BLIMP1, and NANOG seem to be involved in the self-renewal of PGCs in cultured PGCs and embryos.

Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage.

To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.

Anatomical integration of the sacral-hindlimb unit coordinated by GDF11 underlies variation in hindlimb positioning in tetrapods.

Elucidating how body parts from different primordia are integrated during development is essential for understanding the nature of morphological evolution. In tetrapod evolution, while the position of the hindlimb has diversified along with the vertebral formula, the mechanism responsible for this coordination has not been well understood. However, this synchronization suggests the presence of an evolutionarily conserved developmental mechanism that coordinates the positioning of the hindlimb skeleton derived from the lateral plate mesoderm with that of the sacral vertebrae derived from the somites. Here we show that GDF11 secreted from the posterior axial mesoderm is a key factor in the integration of sacral vertebrae and hindlimb positioning by inducing Hox gene expression in two different primordia. Manipulating the onset of GDF11 activity altered the position of the hindlimb in chicken embryos, indicating that the onset of Gdf11 expression is responsible for the coordinated positioning of the sacral vertebrae and hindlimbs. Through comparative analysis with other vertebrate embryos, we also show that each tetrapod species has a unique onset timing of Gdf11 expression, which is tightly correlated with the anteroposterior levels of the hindlimb bud. We conclude that the evolutionary diversity of hindlimb positioning resulted from heterochronic shifts in Gdf11 expression, which led to coordinated shifts in the sacral-hindlimb unit along the anteroposterior axis.

A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus.

During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition.

Efficient harvesting methods for early-stage snake and turtle embryos.

Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle.

Inactivation of Sonic Hedgehog Signaling and Polydactyly in Limbs of Hereditary Multiple Malformation, a Novel Type of Talpid Mutant.

Hereditary Multiple Malformation (HMM) is a naturally occurring, autosomal recessive, homozygous lethal mutation found in Japanese quail. Homozygote embryos (hmm-/-) show polydactyly similar to talpid2 and talpid3 mutants. Here we characterize the molecular profile of the hmm-/- limb bud and identify the cellular mechanisms that cause its polydactyly. The hmm-/- limb bud shows a severe lack of sonic hedgehog (SHH) signaling, and the autopod has 4 to 11 unidentifiable digits with syn-, poly-, and brachydactyly. The Zone of Polarizing Activity (ZPA) of the hmm-/- limb bud does not show polarizing activity regardless of the presence of SHH protein, indicating that either the secretion pathway of SHH is defective or the SHH protein is dysfunctional. Furthermore, mesenchymal cells in the hmm-/- limb bud do not respond to ZPA transplanted from the normal limb bud, suggesting that signal transduction downstream of SHH is also defective. Since primary cilia are present in the hmm-/- limb bud, the causal gene must be different from talpid2 and talpid3. In the hmm-/- limb bud, a high amount of GLI3A protein is expressed and GLI3 protein is localized to the nucleus. Our results suggest that the regulatory mechanism of GLI3 is disorganized in the hmm-/- limb bud.

Quantitative analysis of tissue deformation dynamics reveals three characteristic growth modes and globally aligned anisotropic tissue deformation during chick limb development.

Tissue-level characterization of deformation dynamics is crucial for understanding organ morphogenetic mechanisms, especially the interhierarchical links among molecular activities, cellular behaviors and tissue/organ morphogenetic processes. Limb development is a well-studied topic in vertebrate organogenesis. Nevertheless, there is still little understanding of tissue-level deformation relative to molecular and cellular dynamics. This is mainly because live recording of detailed cell behaviors in whole tissues is technically difficult. To overcome this limitation, by applying a recently developed Bayesian approach, we here constructed tissue deformation maps for chick limb development with high precision, based on snapshot lineage tracing using dye injection. The precision of the constructed maps was validated with a clear statistical criterion. From the geometrical analysis of the map, we identified three characteristic tissue growth modes in the limb and showed that they are consistent with local growth factor activity and cell cycle length. In particular, we report that SHH signaling activity changes dynamically with developmental stage and strongly correlates with the dynamic shift in the tissue growth mode. We also found anisotropic tissue deformation along the proximal-distal axis. Morphogenetic simulation and experimental studies suggested that this directional tissue elongation, and not local growth, has the greatest impact on limb shaping. This result was supported by the novel finding that anisotropic tissue elongation along the proximal-distal axis occurs independently of cell proliferation. Our study marks a pivotal point for multi-scale system understanding in vertebrate development.

Efficient embryonic culture method for the Japanese striped snake, Elaphe quadrivirgata, and its early developmental stages.

The morphogenesis of snake embryos is an elusive yet fascinating research target for developmental biologists. However, few data exist on development of early snake embryo due to limited availability of pregnant snakes, and the need to harvest early stage embryos directly from pregnant snakes before oviposition without knowing the date of fertilization. We established an ex vivo culture method for early snake embryos using the Japanese striped snake, Elaphe quadrivirgata. This method, which we named "sausage-style (SS) culture", allows us to harvest snake embryos at specific stages for each experiment. Using this SS culture system, we calculated somite formation rate at early stages before oviposition. The average somite formation rate between 6/7 and 12/13 somite stages was 145.9 min, between 60/70 and 80/91 somite stages 42.4 min, and between 113-115 and 126/127 somite stages 71 min. Thus, somite formation rate that we observed during early snake embryogenesis was changed over time. We also describe a developmental staging series for E. quadrivirgata. This is the first report of a developmental series of early snake embryogenesis prior to oviposition by full-color images with high-resolution. We propose that the SS culture system is an easy method for treating early snake embryos ex vivo.

Etv1 and Ewsr1 cooperatively regulate limb mesenchymal Fgf10 expression in response to apical ectodermal ridge-derived fibroblast growth factor signal.

Fibroblast growth factors (FGFs) expressed in the apical ectodermal ridge (AER) and FGF10 expressed in the underlying mesoderm are essential for limb bud outgrowth. Their expression is maintained through a positive feedback loop. We identified the cis-regulatory element and trans-acting factors involved in the AER-FGF-dependent transactivation of Fgf10. Etv1 and Ewsr1 stimulated transcription from the Fgf10 promoter in the sub-AER mesenchyme of mouse and chick limb buds in a conserved AGAAAR cluster-dependent manner. We found that both Etv1 and Ewsr1 were necessary for Fgf10 expression and elongation of the limb bud. In addition, Etv1 and AER-FGF synergistically stimulated Fgf10 promoter activity in an Ewsr1-dependent manner. We also found that Etv1 and Ewsr1 bound to the segment of DNA containing the AGAAAR cluster in vivo and in vitro. Moreover, Etv1 directly bound to the AGAAAR sequence in vitro. Our results suggest that Etv1 and Ewsr1 transactivate Fgf10 directly and cooperatively in response to AER-FGFs.

Leucophores are similar to xanthophores in their specification and differentiation processes in medaka.

Animal body color is generated primarily by neural crest-derived pigment cells in the skin. Mammals and birds have only melanocytes on the surface of their bodies; however, fish have a variety of pigment cell types or chromatophores, including melanophores, xanthophores, and iridophores. The medaka has a unique chromatophore type called the leucophore. The genetic basis of chromatophore diversity remains poorly understood. Here, we report that three loci in medaka, namely, leucophore free (lf), lf-2, and white leucophore (wl), which affect leucophore and xanthophore differentiation, encode solute carrier family 2, member 15b (slc2a15b), paired box gene 7a (pax7a), and solute carrier family 2 facilitated glucose transporter, member 11b (slc2a11b), respectively. Because lf-2, a loss-of-function mutant for pax7a, causes defects in the formation of xanthophore and leucophore precursor cells, pax7a is critical for the development of the chromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, as these chromatophores have purine-dependent light reflection. Our identification of slc2a15b and slc2a11b as genes critical for the differentiation of leucophores and xanthophores in medaka led to a further finding that the existence of these two genes in the genome coincides with the presence of xanthophores in nonmammalian vertebrates: birds have yellow-pigmented irises with xanthophore-like intracellular organelles. Our findings provide clues for revealing diverse evolutionary mechanisms of pigment cell formation in animals.

Wnt and BMP signaling cooperate with Hox in the control of Six2 expression in limb tendon precursor.

The number and shape of limb tendons vary along the proximodistal axis, and the autopod contains more tendons than the zeugopod. The transcription factor Six2 is expressed in the developing tendons, and its expression can be traced back to a group of limb mesenchymal cells that are thought to be tendon precursor cells. We tried to elucidate the mechanism controlling position-specific tendon pattern formation using Six2 as a tendon marker. Six2 expression was always found in cells between the limb cartilage and ectoderm. Administration of BMP-2 or BMP antagonist Noggin to the limb bud, respectively repressed or facilitated Six2 expression. Removal of the ectoderm or administration of the Wnt antagonist sFRP-2 abolished Six2 expression and ectopic Wnt expression induced ectopic Six2 expression. Taken together, Six2 expression is induced in the cells located at the point where cartilage-derived Noggin and ectoderm-derived Wnt signals meet. Misexpression of the autopod-specific Hoxa-13 or Hoxd-13 induced ectopic expression of Six2 in the zeugopodal mesenchymal cells of the chick limb bud. Six2 expression in the dorsal autopodal mesenchyme was not detected in Hoxa-13(-/-);HoxD(del/del) mice, indicating that autopod-specific Hox is required for the regulation of Six2 expression. Misexpression of Wnt in the autopod induced ectopic Six2 expression in the autopod. On the other hand, Wnt misexpression alone never induced Six2 expression in the zeugopod, yet co-misexpression of Hoxa-13 and Wnt in the zeugopod enhanced ectopic Six2 expression. Our results indicate that autopodal Hox genes regulate Six2 expression in the autopodal tendon precursor in cooperation with the factors from cartilage and ectoderm.

Fibroblast growth factor 10 gene regulation in the second heart field by Tbx1, Nkx2-5, and Islet1 reveals a genetic switch for down-regulation in the myocardium.

During cardiogenesis, Fibroblast Growth Factor (Fgf10) is expressed in the anterior second heart field. Together with Fibroblast growth factor 8 (Fgf8), Fgf10 promotes the proliferation of these cardiac progenitor cells that form the arterial pole of the heart. We have identified a 1.7-kb region in the first intron of Fgf10 that is necessary and sufficient to direct transgene expression in this cardiac context. The 1.7-kb sequence is directly controlled by T-box transcription factor 1 (Tbx1) in anterior second heart field cells that contribute to the outflow tract. It also responds to both NK2 transcription factor related, locus 5 (Nkx2-5) and ISL1 transcription factor, LIM/homeodomain (Islet1), acting through overlapping sites. Mutation of these sites reduces transgene expression in the anterior second heart field where the Fgf10 regulatory element is activated by Islet1 via direct binding in vivo. Analysis of the response to Nkx2-5 loss- and Isl1 gain-of-function genetic backgrounds indicates that the observed up-regulation of its activity in Nkx2-5 mutant hearts, reflecting that of Fgf10, is due to the absence of Nkx2-5 repression and to up-regulation of Isl1, normally repressed in the myocardium by Nkx2-5. ChIP experiments show strong binding of Nkx2-5 in differentiated myocardium. Molecular and genetic analysis of the Fgf10 cardiac element therefore reveals how key cardiac transcription factors orchestrate gene expression in the anterior second heart field and how genes, such as Fgf10, normally expressed in the progenitor cell population, are repressed when these cells enter the heart and differentiate into myocardium. Our findings provide a paradigm for transcriptional mechanisms that underlie the changes in regulatory networks during the transition from progenitor state to that of the differentiated tissue.

DEAD-box protein Ddx46 is required for the development of the digestive organs and brain in zebrafish.

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.

Zebrafish Dmrta2 regulates neurogenesis in the telencephalon.

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis.

FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion.

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9(Eks)) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.

Sdf1/Cxcr4 signaling controls the dorsal migration of endodermal cells during zebrafish gastrulation.

During vertebrate gastrulation, both mesodermal and endodermal cells internalize through the blastopore beneath the ectoderm. In zebrafish, the internalized mesodermal cells move towards the dorsal side of the gastrula and, at the same time, they extend anteriorly by convergence and extension (C&E) movements. Endodermal cells showing characteristic filopodia then migrate into the inner layer within the hypoblast next to the yolk syncytial layer (YSL). However, little is known about how the movement of endodermal cells is regulated during gastrulation. Here we show that sdf1a- and sdf1b-expressing mesodermal cells control the movements of the cxcr4a-expressing endodermal cells. The directional migration of endodermal cells during gastrulation is inhibited by knockdown of either cxcr4a or sdf1a/sdf1b (sdf1). We also show that misexpressed Sdf1 acts as a chemoattractant for cxcr4a-expressing endodermal cells. We further found, using the endoderm-specific transgenic line Tg(sox17:EGFP), that Sdf1/Cxcr4 signaling regulates both the formation and orientation of filopodial processes in endodermal cells. Moreover, the accumulation of phosphoinositide 3,4,5-trisphosphate (PIP(3)), which is known to occur at the leading edge of migrating cells, is not observed at the filopodia of endodermal cells. Based on our results, we propose that sdf1-expressing mesodermal cells, which overlie the endodermal layer, guide the cxcr4a-expressing endodermal cells to the dorsal side of the embryo during gastrulation, possibly through a PIP(3)-independent pathway.

Foregut endoderm is specified early in avian development through signal(s) emanating from Hensen's node or its derivatives.

In this study, the initial specification of foregut endoderm in the chick embryo was analyzed. A fate map constructed for the area pellucida endoderm at definitive streak-stage showed centrally-located presumptive cells of foregut-derived organs around Hensen's node. Intracoelomic cultivation of the area pellucida endoderm at this stage combined with somatic mesoderm resulted in the differentiation predominantly into intestinal epithelium, suggesting that this endoderm may not yet be regionally specified. In vitro cultivation of this endoderm for 1-1.5 day combined with Hensen's node or its derivatives but not with other embryonic structures/tissues elicited endodermal expression of cSox2 but not of cHoxb9, which is characteristic of specified foregut endoderm. When the anteriormost or posteriormost part of the area pellucida endoderm at this stage, whose fate is extraembryonic, was combined with Hensen's node or its derivatives for 1 day, then enwrapped with somatic mesoderm and cultivated for a long period intracoelomically, differentiation of various foregut organ epithelia was observed. Such epithelia never appeared in the endoderm associated with other embryonic structures/tissues and cultured similarly. Thus, Hensen's node and its derivatives that lie centrally in the presumptive endodermal area of the foregut are likely to play an important role in the initial specification of the foregut. Chordin-expressing COS cells or noggin-producing CHO cells transplanted into the anteriormost area pellucida of the definitve streak-stage embryo could induce endodermal expression of cSox2 but not of cHoxb9, suggesting that chordin and noggin that emanate from Hensen's node and its derivatives, may be involved in this process.