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.

Chicken embryo cultures in the dorsal-upward orientation for the manipulation of epiblasts.

Chicken embryos have many advantages in the study of amniote embryonic development. In particular, culture techniques developed for early-stage embryos have promoted the advancement of modern developmental studies using chicken embryos. However, the standard technique involves placing chicken embryos in the ventral-upward (ventral-up) orientation, limiting manipulation of the epiblast at the dorsal surface, which is the primary source of ectodermal and mesodermal tissues. To circumvent this limitation, we developed chicken embryo cultures in the dorsal-up orientation and exploited this technique to address diverse issues. In this article, we first review the history of chicken embryo culture techniques to evaluate the advantages and limitations of the current standard technique. Then, the dorsal-up technique is discussed. These technological discussions are followed by three different examples of experimental analyses using dorsal-up cultures to illustrate their advantages: (1) EdU labeling of epiblast cells to assess potential variation in the cell proliferation rate; (2) migration behaviors of N1 enhancer-active epiblast cells revealed by tracking cells with focal fluorescent dye labeling in dorsal-up embryo culture; and (3) neural crest development of mouse neural stem cells in chicken embryos.

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.

YAP is essential for tissue tension to ensure vertebrate 3D body shape.

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

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.

Obituary: Tokindo S. Okada (1927-2017).

Hisato Kondoh and Harukazu Nakamura look back at the life and career of their mentor Tokindo S. Okada, a pioneer of Japanese developmental biology.

ChIP-seq analysis of genomic binding regions of five major transcription factors highlights a central role for ZIC2 in the mouse epiblast stem cell gene regulatory network.

To obtain insight into the transcription factor (TF)-dependent regulation of epiblast stem cells (EpiSCs), we performed ChIP-seq analysis of the genomic binding regions of five major TFs. Analysis of in vivo biotinylated ZIC2, OTX2, SOX2, POU5F1 and POU3F1 binding in EpiSCs identified several new features. (1) Megabase-scale genomic domains rich in ZIC2 peaks and genes alternate with those rich in POU3F1 but sparse in genes, reflecting the clustering of regulatory regions that act at short and long-range, which involve binding of ZIC2 and POU3F1, respectively. (2) The enhancers bound by ZIC2 and OTX2 prominently regulate TF genes in EpiSCs. (3) The binding sites for SOX2 and POU5F1 in mouse embryonic stem cells (ESCs) and EpiSCs are divergent, reflecting the shift in the major acting TFs from SOX2/POU5F1 in ESCs to OTX2/ZIC2 in EpiSCs. (4) This shift in the major acting TFs appears to be primed by binding of ZIC2 in ESCs at relevant genomic positions that later function as enhancers following the disengagement of SOX2/POU5F1 from major regulatory functions and subsequent binding by OTX2. These new insights into EpiSC gene regulatory networks gained from this study are highly relevant to early stage embryogenesis.

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.

Nasal and otic placode specific regulation of Sox2 involves both activation by Sox-Sall4 synergism and multiple repression mechanisms.

Transcription factor gene Sox2 is expressed throughout sensory development, but the enhancers that regulate the gene vary depending on the developmental stages and tissues. To gain new insights into the gene regulatory network in sensory placode specification, regulation of the nasal-otic bispecific NOP1 enhancer of Sox2 was investigated in chicken embryos. Deletion and mutational analyses using electroporation showed that transcriptional repression mechanisms in combination with activation mechanisms determine placodal specificity. Activation of the NOP1 enhancer involves synergistic action by Sall4 and SoxB1/SoxE factors that bind to the adjacent sites. Deletion of repressive elements resulted in widening of the tissue area for enhancer activity to a region where the expression of Sall4 and SoxB1/E overlaps, e.g., the CNS and neural crest. Among multiple repressive elements that contribute to the placodal confinement of the NOP1 enhancer activity, CACCT/CACCTG motifs bound by Zeb/Snail family repressors play important roles. Overexpression of δEF1 (Zeb1) or Snail2 (Slug) strongly inhibited NOP1 activity. These data indicate that both activation by Sall4-Sox synergism and multiple repression mechanisms involving Zeb/Snail factors are essential for Sox2 regulation to be confined to the nasal and otic placodes.

Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.

Intrinsic lens potential of neural retina inhibited by Notch signaling as the cause of lens transdifferentiation.

Embryonic neural retinas of avians produce lenses under spreading culture conditions. This phenomenon has been regarded as a paradigm of transdifferentiation due to the overt change in cell type. Here we elucidated the underlying mechanisms. Retina-to-lens transdifferentiation occurs in spreading cultures, suggesting that it is triggered by altered cell-cell interactions. Thus, we tested the involvement of Notch signaling based on its role in retinal neurogenesis. Starting from E8 retina, a small number of crystallin-expressing lens cells began to develop after 20 days in control spreading cultures. By contrast, addition of Notch signal inhibitors to cultures after day 2 strongly promoted lens development beginning at day 11, and a 10-fold increase in δ-crystallin expression level. After Notch signal inhibition, transcription factor genes that regulate the early stage of eye development, Prox1 and Pitx3, were sequentially activated. These observations indicate that the lens differentiation potential is intrinsic to the neural retina, and this potential is repressed by Notch signaling during normal embryogenesis. Therefore, Notch suppression leads to lens transdifferentiation by disinhibiting the neural retina-intrinsic program of lens development.

Cooperation of Sall4 and Sox8 transcription factors in the regulation of the chicken Sox3 gene during otic placode development.

To elucidate the transcriptional regulation that underlies specification of the otic placode, we investigated the Sox3 downstream enhancer Otic1 of the chicken, the activity of which is restricted to and distributed across the entire otic placode. The 181-bp Otic1 enhancer sequence was dissected into a 68-bp minimal activating sequence, which exhibited dimer enhancer activity in the otic placode and cephalic neural crest, and this was further reduced to a 25-bp Otic1 core sequence, which also showed octamer enhancer activity in the same regions. The Otic1 core octamer was activated by the combined action of Sall4 and the SoxE transcription factors (TFs) Sox8 or Sox9. Binding of Sall4, Sox8 and Sox9 to the Otic1 sequence in embryonic tissues was confirmed by ChIP-qPCR analysis. The core-adjoining 3' side sequences of Otic1 augmented its enhancer activity, while inclusion of the CAGGTG sequence in the immediate 3' end of the 68-bp sequence repressed its enhancer activity outside the otic placode. The CAGGTG sequence likely serves as the binding sites of the repressor TFs δEF1 (Zeb1), Sip1 (Zeb2), and Snail2, all of which are expressed in the cephalic neural crest but not in the otic placode. Therefore, the combination of Sall4-Sox8-dependent activation and CAGGTG sequence-dependent repression determines otic placode development. Although the Otic1 sequence is not conserved in mammals or fishes, the activation mechanism is, as Otic1 was also activated in otic placode tissues developed from mouse embryonic stem cells and transient transgenic zebrafish embryos.

Roles of ZIC2 in Regulation of Pluripotent Stem Cells.

Pioneered by the classical mouse embryonic stem cells (ESCs), various stem cell lines representing the peri- and postimplantation stages of embryogenesis have been established. To gain insight into the gene regulatory network operating in these cells, we first investigated epiblast stem cells (EpiSCs), performing ChIP-seq analysis for five major transcription factors (TFs) involved in epiblast regulation. The analysis indicated that SOX2-POU5F1 TF pairs highlighted in mouse ESCs are not the major players in other stem cells. The major acting transcription factors shift from SOX2/POU5F1 in mouse ESCs to ZIC2/OTX2 in EpiSCs, and this shift is primed in ESCs by binding of ZIC2 at relevant genomic positions that later function as enhancers.

R26-WntVis reporter mice showing graded response to Wnt signal levels.

The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a(-/-) and Wnt3a(vt/-) mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes.

Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells.

The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.

Sox proteins: regulators of cell fate specification and differentiation.

Sox transcription factors play widespread roles during development; however, their versatile funtions have a relatively simple basis: the binding of a Sox protein alone to DNA does not elicit transcriptional activation or repression, but requires binding of a partner transcription factor to an adjacent site on the DNA. Thus, the activity of a Sox protein is dependent upon the identity of its partner factor and the context of the DNA sequence to which it binds. In this Primer, we provide an mechanistic overview of how Sox family proteins function, as a paradigm for transcriptional regulation of development involving multi-transcription factor complexes, and we discuss how Sox factors can thus regulate diverse processes during development.

Axial level-dependent molecular and cellular mechanisms underlying the genesis of the embryonic neural plate.

The transcription factor gene Sox2, centrally involved in neural primordial regulation, is activated by many enhancers. During the early stages of embryonic development, Sox2 is regulated by the enhancers N2 and N1 in the anterior neural plate (ANP) and posterior neural plate (PNP), respectively. This differential use of the enhancers reflects distinct regulatory mechanisms underlying the genesis of ANP and PNP. The ANP develops directly from the epiblast, triggered by nodal signal inhibition, and via the combined action of TFs SOX2, OTX2, POU3F1, and ZIC2, which promotes the the ANP development and inhibits other cell lineages. In contrast, the PNP is derived from neuromesodermal bipotential axial stem cells that develop into the neural plate when Sox2 is activated by the N1 enhancer, whereas they develop into the paraxial mesoderm when the N1 enhancer is repressed by the action of TBX6. The axial stem cells are maintained by the activity of WNT3a and T (Brachyury). However, at axial levels more anterior to the 8th somites (cervical levels), the development of both the neural plate and somite proceeds in the absence of WNT3a, T, or TBX6. These observations indicate that distinct molecular and cellular mechanisms determine neural plate genesis based on the axial level, and contradict the classical concept of the term "neural induction," which assumes a pan-neural plate mechanism.

Regulation of trunk neural crest delamination by δEF1 and Sip1 in the chicken embryo.

The vertebrate Zfhx1 transcription factor family comprises δEF1 and Sip1, which bind to CACCT-containing sequences and act as transcriptional repressors. It has been a longstanding question whether these transcription factors share the same regulatory functions in vivo. It has been shown that neural crest (NC) delamination depends on the Sip1 activity at the cranial level in mouse and chicken embryos, and it remained unclear how NC delamination is regulated at the trunk level. We observed that the expression of δEF1 and Sip1 overlaps in many tissues in chicken embryos, including NC cells at the trunk level. To clarify the above questions, we separately knocked down δEF1 and Sip1 or in combination in NC cells by electroporation of vectors expressing short hairpin RNAs (shRNAs) against respective mRNAs on the dorsal side of neural tubes that generate NC cells. In all cases, the migrating NC cell population was significantly reduced, paralleled by the decreased expression of δEF1 or Sip1 targeted by shRNAs. Expression of Sox10, the major transcription factor that regulates NC development, was also decreased by the shRNAs against δEF1 or Sip1. We conclude that the trunk NC delamination is regulated by both δEF1 and Sip1 in an analogous manner, and that these transcription factors can share equivalent regulatory functions in embryonic tissues.

Transcriptional regulatory networks in epiblast cells and during anterior neural plate development as modeled in epiblast stem cells.

Somatic development initiates from the epiblast in post-implantation mammalian embryos. Recent establishment of epiblast stem cell (EpiSC) lines has opened up new avenues of investigation of the mechanisms that regulate the epiblast state and initiate lineage-specific somatic development. Here, we investigated the role of cell-intrinsic core transcriptional regulation in the epiblast and during derivation of the anterior neural plate (ANP) using a mouse EpiSC model. Cells that developed from EpiSCs in one day in the absence of extrinsic signals were found to represent the ANP of ~E7.5 embryos. We focused on transcription factors that are uniformly expressed in the E6.5 epiblast but in a localized fashion within or external to the ANP at E7.5, as these are likely to regulate the epiblast state and ANP development depending on their balance. Analyses of the effects of knockdown and overexpression of these factors in EpiSCs on the levels of downstream transcription factors identified the following regulatory functions: cross-regulation among Zic, Otx2, Sox2 and Pou factors stabilizes the epiblastic state; Zic, Otx2 and Pou factors in combination repress mesodermal development; Zic and Sox2 factors repress endodermal development; and Otx2 represses posterior neural plate development. All of these factors variably activate genes responsible for neural plate development. The direct interaction of these factors with enhancers of Otx2, Hesx1 and Sox2 genes was demonstrated. Thus, a combination of regulatory processes that suppresses non-ANP lineages and promotes neural plate development determines the ANP.

Dkk1-dependent inhibition of Wnt signaling activates Hesx1 expression through its 5' enhancer and directs forebrain precursor development.

Development of the anterior forebrain precursor (AFBP) in the anterior neural plate (ANP) depends on the activation of the Hesx1 transcription factor gene. The Hesx1-expression domain of the ANP is underlain by Dkk1-expressing tissues, initially proximal-most anterior visceral endoderm (AVE), and later anterior mesendoderm (AME). As Dkk1-null embryos fail to develop the Hesx1-expressing domain, it is likely that Wnt signal inhibition in the ANP is required for the Hesx1 activation. To investigate the regulation of the AFBP development, we took advantage of epiblast stem cells (EpiSCs), which develop into the ANP in the absence of activin signaling. Expression of Hesx1 and Six3, both involved in the AFBP development, was strongly activated 2 days after activin removal and concomitant addition of Wnt signal inhibitors, Dkk1 or XAV939. Furthermore, we showed that activation of the 720-bp Hesx1 5' enhancer is responsible for Hesx1 expression in the AFBP and depends on Wnt signal inhibition. In addition, we showed that Wnt inhibition during the first day has larger impact on the activation of Hesx1 and Six3 than the second day, suggesting that in embryos Wnt inhibition caused by the AVE-derived Dkk1, rather than the AME-derived Dkk1, contributes greatly in the establishment of the AFBP.