Fgf10 mutant newts regenerate normal hindlimbs despite severe developmental defects.

In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

Live imaging of avian epiblast and anterior mesendoderm grafting reveals the complexity of cell dynamics during early brain development.

Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations.

Sox2 gene regulation via the D1 enhancer in embryonic neural tube and neural crest by the combined action of SOX2 and ZIC2.

The transcription factor (TF) SOX2 regulates various stem cells and tissue progenitors via functional interactions with cell type-specific partner TFs that co-bind to enhancer sequences. Neural progenitors are the major embryonic tissues where SOX2 assumes central regulatory roles. In order to characterize the partner TFs of SOX2 in neural progenitors, we investigated the regulation of the D1 enhancer of the Sox2 gene, which is activated in the embryonic neural tube (NT) and neural crest (NC), using chicken embryo electroporation. We identified essential TF binding sites for a SOX, and two ZIC TFs in the activation of the D1 enhancer. By comparison of dorso-ventral and antero-posterior patterns of D1 enhancer activation, and the effect of mutations on the enhancer activation patterns with TF expression patterns, we determined SOX2 and ZIC2 as the major D1 enhancer-activating TFs. Binding of these TFs to the D1 enhancer sequence was confirmed by chromatin immunoprecipitation analysis. The combination of SOX2 and ZIC2 TFs activated the enhancer in both the NT and NC. These results indicate that SOX2 and ZIC2, which have been known to play major regulatory roles in neural progenitors, do functionally cooperate. In addition, the recently demonstrated SOX2 expression during the NC development is accounted for at least partly by the D1 enhancer activity. Deletion of the D1 enhancer sequence from the mouse genome, however, did not affect the mouse development, indicating functional redundancies of other enhancers.

The formation of multiple pituitary pouches from the oral ectoderm causes ectopic lens development in hedgehog signaling-defective avian embryos.

BACKGROUND: Hedgehog signaling has various regulatory functions in tissue morphogenesis and differentiation. To investigate its involvement in anterior pituitary precursor development and the lens precursor potential for anterior pituitary precursors, we investigated Talpid mutant Japanese quail embryos, in which hedgehog signaling is defective. RESULTS: Talpid mutants develop multiple pituitary precursor-like pouches of variable sizes from the oral ectoderm (OE). The ectopic pituitary pouches initially express the pituitary-associated transcription factor (TF) LHX3 similarly to Rathke's pouch, the genuine pituitary precursor. The pouches coexpress the TFs SOX2 and PAX6, a signature of lens developmental potential. Most Talpid mutant pituitary pouches downregulate LHX3 expression and activate the lens-essential TF PROX1, leading to the development of small lens tissue expressing α-, ß-, and δ-crystallins. In contrast, mutant Rathke's pouches express a lower level of LHX3, which is primarily localized in the cytoplasm, and activate the lens developmental pathway. CONCLUSIONS: Hedgehog signaling in normal embryos regulates the development of Rathke's pouch in two steps. First, by confining Rathke's pouch development in a low hedgehog signaling region of the OE. Second, by sustaining LHX3 activity to promote anterior pituitary development, while inhibiting ectopic lens development.

Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.

Endoderm precursors expressing FoxA2 and Sox17 develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm-generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with Foxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate Foxa2 expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g., Eomes, Gsc, and Sox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E-cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin-rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E-cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post-gastrulation stage embryos: (1) definitive endoderm co-expressing high SOX17, GATA4, and E-cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E-cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E-cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin-rich matrix models the gastrulation-dependent endoderm precursor development.

Molecular markers for X-ray-insensitive differentiated cells in the Inner and outer regions of the mesenchymal space in planarian Dugesia japonica.

Planarian's strong regenerative ability is dependent on stem cells (called neoblasts) that are X-ray-sensitive and proliferative stem cells. In addition to neoblasts, another type of X-ray-sensitive cells was newly identified by recent research. Thus, planarian's X-ray-sensitive cells can be divided into at least two populations, Type 1 and Type 2, the latter corresponding to planarian's classically defined "neoblasts". Here, we show that Type 1 cells were distributed in the outer region (OR) immediately underneath the muscle layer at all axial levels from head to tail, while the Type 2 cells were distributed in a more internal region (IR) of the mesenchymal space at the axial levels from neck to tail. To elucidate the biological significance of these two regions, we searched for genes expressed in differentiated cells that were locate close to these X-ray-sensitive cell populations in the mesenchymal space, and identified six genes mainly expressed in the OR or IR, named OR1, OR2, OR3, IR1, IR2 and IR3. The predicted amino acid sequences of these genes suggested that differentiated cells expressing OR1, OR3, IR1, or IR2 provide Type 1 and Type 2 cells with specific extracellular matrix (ECM) environments.

Epiblast cells gather onto the anterior mesendoderm and initiate brain development without the direct involvement of the node in avian embryos: Insights from broad-field live imaging.

Live imaging of migrating and interacting cells in developing embryos has opened a new means for deciphering fundamental principles in morphogenesis and patterning, which was not possible with classic approaches of experimental embryology. In our recent study, we devised a new genetic tool to sparsely label cells with a green-fluorescent protein in the broad field of chicken embryos, enabling the analysis of cell migration during the early stages of brain development. Trajectory analysis indicated that anterior epiblast cells from a broad area gather to the head axis to form the brain primordia or brain-abutting head ectoderm. Grafting the mCherry-labeled stage (st.) 4 node in an anterior embryonic region resulted in the anterior extension of the anterior mesendoderm (AME), the precursor for the prechordal plate and anterior notochord, from the node graft at st. 5. Grafting the st. 4 node or st. 5 AME at various epiblast positions that otherwise develop into the head ectoderm caused local cell gathering to the graft-derived AME. The node was not directly associated with this local epiblast-gathering activity. The gathered anterior epiblast cells developed into secondary brain tissue consisting of consecutive brain portions, e.g., forebrain and midbrain or midbrain and hindbrain, reflecting the brain portion specificities inherent to the epiblast cells. The observations indicated the bipotentiality of all anterior epiblast cells to develop into the brain or head ectoderm. Thus, a new epiblast brain field map is proposed, allowing the reinterpretation of classical node graft data, and the role of the AME is highlighted. The new model leads to the conclusion that the node does not directly participate in brain development.

DrosoPhyla: Resources for Drosophilid Phylogeny and Systematics.

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

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.