Animal Cap Research Articles

Sizzled (Frzb3) physically interacts with noncanonical Wnt ligands to inhibit gastrulation cell movement

The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The non-canonical Wnt signaling cascade serves as a pivotal regulator of convergent and extension cellular movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled, to assess its potential role in inhibiting non-canonical Wnt signaling. In our current study, we demonstrated that ectopic expression of sizzled led to gastrulation defects in a dose-dependent manner, without altering cell fate specification. Overexpression of sizzled resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Sizzled with non-canonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon sizzled overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing sizzled expression during early Xenopus gastrulation.

Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation

The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) interacts with the Ldb1–Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1–Lhx1-Ssbp transcriptional complexes in embryonic development.

NANOG is required to establish the competence for germ-layer differentiation in the basal tetrapod axolotl.

Pluripotency defines the unlimited potential of individual cells of vertebrate embryos, from which all adult somatic cells and germ cells are derived. Understanding how the programming of pluripotency evolved has been obscured in part by a lack of data from lower vertebrates; in model systems such as frogs and zebrafish, the function of the pluripotency genes NANOG and POU5F1 have diverged. Here, we investigated how the axolotl ortholog of NANOG programs pluripotency during development. Axolotl NANOG is absolutely required for gastrulation and germ-layer commitment. We show that in axolotl primitive ectoderm (animal caps; ACs) NANOG and NODAL activity, as well as the epigenetic modifying enzyme DPY30, are required for the mass deposition of H3K4me3 in pluripotent chromatin. We also demonstrate that all 3 protein activities are required for ACs to establish the competency to differentiate toward mesoderm. Our results suggest the ancient function of NANOG may be establishing the competence for lineage differentiation in early cells. These observations provide insights into embryonic development in the tetrapod ancestor from which terrestrial vertebrates evolved.

Microsurgical Methods to Make the Keller Sandwich Explant and the Dorsal Isolate.

This protocol summarizes preparation of the dorsal marginal zone sandwich explant (a.k.a. the "Keller sandwich") and the dorsal isolate from Xenopus embryos. The Keller sandwich is assembled from two early gastrula stage dorsal marginal zone (DMZ) explants. DMZ explants isolated before involution maintain planar patterning processes and block radial signals that might be exchanged between pre- and postinvolution tissues. DMZ explants isolated later in gastrulation, but subsequently opened and flattened may have both planar and radial patterning. The epithelial margins of DMZ explants in Keller sandwiches heal and basal contacts form between the deep layers of the two DMZ explants. The dorsal isolate is dissected from mid- to late-gastrula stage embryos after involution and archenteron formation. Germ-layer contacts between dorsal endoderm, mesoderm, and ectoderm generated by gastrulation movements are maintained in the dorsal isolate. These two explants can be used to study tissue, cell, and subcellular processes relevant to convergent extension, from patterning to cell behaviors, and their collective biomechanics. Skills needed to dissect the Keller sandwich are greater than those needed to dissect animal cap ectoderm and can be mastered in a few weeks; skills needed to dissect the dorsal isolate are similar to those needed to dissect animal caps and can be learned in a week.

Microsurgical Manipulations to Isolate Collectively Migrating Mesendoderm.

Mesendoderm mantle closure completes the gastrulation movements of the <i>Xenopus laevis</i> embryo and provides an unparalleled opportunity to study collective cell behaviors within a mesenchymal tissue. Free-edge sheet-like collective movements of these tissues contrast with movements of epithelial tissues in that mesendodermal cells are not constrained by tight junctions or adherens junctions, yet migrate in a coherent and persistent mode over several hours. Mesendoderm cells are the largest motile cells in the <i>Xenopus</i> embryo and complete a 500-µm migratory path. When mesendoderm is cultured on rigid glass substrates, these cells can exceed 100 µm in length and show a highly persistent leading lamellipodia that can exceed 20 µm from tip to base. These large collectively migrating cells provide a unique imaging opportunity to visualize polarized adhesive and cytoskeletal structures with high-numerical-aperture objectives. Mesendodermal cells in the early embryo originate from around the entirety of the marginal zone and may also be distinguished by their source along the animal-vegetal axis. Here we use the term mesendoderm but note alternative terms for these cells can include head mesoderm, endomesoderm, and prechordal mesoderm. This protocol summarizes microsurgical preparation of mesendoderm tissue explants and "windowed" embryos. Skills needed to dissect fragments of the mesendoderm mantle are marginally greater than those needed to isolate animal cap ectoderm and can be mastered within 2 weeks; skills needed to isolate the mesendoderm "donut" or "ring" or to prepare windowed embryos are significantly greater and may require several additional weeks of training.

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 ​kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 ​kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Information Theory as an Experimental Tool for Integrating Disparate Biophysical Signaling Modules.

There is a growing appreciation in the fields of cell biology and developmental biology that cells collectively process information in time and space. While many powerful molecular tools exist to observe biophysical dynamics, biologists must find ways to quantitatively understand these phenomena at the systems level. Here, we present a guide for the application of well-established information theory metrics to biological datasets and explain these metrics using examples from cell, developmental and regenerative biology. We introduce a novel computational tool named after its intended purpose, calcium imaging, (CAIM) for simple, rigorous application of these metrics to time series datasets. Finally, we use CAIM to study calcium and cytoskeletal actin information flow patterns between Xenopus laevis embryonic animal cap stem cells. The tools that we present here should enable biologists to apply information theory to develop a systems-level understanding of information processing across a diverse array of experimental systems.

Ccr7 affects both morphogenesis and differentiation during early Xenopus embryogenesis.

Chemokines play important roles in early embryogenesis, including morphogenesis and cell differentiation, before the immune system is established. We characterized Xenopus laevis CC-type chemokine receptor 7S (ccr7.S) to clarify its role during early development. ccr7 transcripts were detected ubiquitously in early embryos. Dorsal overexpression of ccr7.S inhibited gastrulation, and ccr7.S mRNA-injected embryos had short axes and widely opened neural folds. Because the Keller sandwich explants of the injected embryos elongated well, ccr7.S might affect cell migration, but not convergent extension movements. Ventral ccr7.S overexpression induced secondary axes and chrd.1 upregulation in gastrula-stage embryos. Animal cap assays showed increased expression of neural and cement gland marker genes at later stages. Ccr7.S knockdown reduced chrd.1 expression and inhibited gastrulation at the dorsal side. Our findings suggest that ccr7.S plays important roles in morphogenetic movement and cell differentiation.

The Biological Robots Are Coming! But Note They Have Been Here for ∼3.5 Billion Years

The Biological Robots Are Coming! But Note They Have Been Here for ∼3.5 Billion Years

Goosecoid Controls Neuroectoderm Specification via Dual Circuits of Direct Repression and Indirect Stimulation in Xenopus Embryos.

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.

Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment

Activin, a member of the transforming growth factor-β (TGF-β) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann’s organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Molecular mechanisms of hearing loss in Nager syndrome

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.

Combinatorial transcription factor activities on open chromatin induce embryonic heterogeneity in vertebrates

During vertebrate gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal‐ventral patterning and establishing of the dorsal axis. We applied single‐cell chromatin accessibility and transcriptome analyses to explore the emergence of cellular heterogeneity during gastrulation in Xenopus tropicalis. Transcriptionally inactive lineage‐restricted genes exhibit relatively open chromatin in animal caps, whereas chromatin accessibility in dorsal marginal zone cells more closely reflects transcriptional activity. We characterized single‐cell trajectories and identified head and trunk organizer cell clusters in early gastrulae. By integrating chromatin accessibility and transcriptome data, we inferred the activity of transcription factors in single‐cell clusters and tested the activity of organizer‐expressed transcription factors in animal caps, alone or in combination. The expression profile induced by a combination of Foxb1 and Eomes most closely resembles that observed in the head organizer. Genes induced by Eomes, Otx2, or the Irx3‐Otx2 combination are enriched for maternally regulated H3K4me3 modifications, whereas Lhx8‐induced genes are marked more frequently by zygotically controlled H3K4me3. Taken together, our results show that transcription factors cooperate in a combinatorial fashion in generally open chromatin to orchestrate zygotic gene expression.

Dusp1 modulates activin/smad2 mediated germ layer specification via FGF signal inhibition in Xenopus embryos

ABSTRACT Activin, a member of the transforming growth factor (TGF-β) superfamily, induces mesoderm, endoderm and neuro-ectoderm formation in Xenopus embryos. Despite several previous studies, the complicated gene regulatory network and genes involved in this induction await more elaboration. We identified expression of various fibroblast growth factor (FGF) genes in activin/smad2 treated animal cap explants (AC) of Xenopus embryos. Activin/smad2 increased fgf3/8 expression, which was reduced by co-injection of dominant negative activin receptor (DNAR) and dominant negative Fgf receptor (DNFR). Interestingly, activin/smad2 also increased expression of dual specificity phosphatase 1 (dusp1) which has been known to inhibit Fgf signaling. Dusp1 overexpression in dorsal marginal zone caused gastrulation defect and decreased Jnk/Erk phosphorylation as well as Smad1 linker region phosphorylation. Dusp1 decreased neural and organizer gene expression with increasing of endodermal and ventral gene expression in smad2 treated AC, indicating that dusp1 modulates germ layer specification. Dusp1 decreased neural gene expression in fgf8 treated AC, suggesting that Erk and/or Jnk phosphorylation may be involved in fgf8 induced neural induction. In addition, dusp1 decreased the reporter gene activities of activin response element (ARE) and increased it for bmp response element (BRE), indicating that dusp1 modulates two opposite morphogen signaling of dorsal (activin/Smad2) and ventral (bmp/Smad1) tracks, acting to fine tune the Fgf/Erk pathway.

Foxd4l1.1 negatively regulates transcription of neural repressor ventx1.1 during neuroectoderm formation in Xenopus embryos

Neuroectoderm formation is the first step in development of a proper nervous system for vertebrates. The developmental decision to form a non-neural ectoderm versus a neural one involves the regulation of BMP signaling, first reported many decades ago. However, the precise regulatory mechanism by which this is accomplished has not been fully elucidated, particularly for transcriptional regulation of certain key transcription factors. BMP4 inhibition is a required step in eliciting neuroectoderm from ectoderm and Foxd4l1.1 is one of the earliest neural genes highly expressed in the neuroectoderm and conserved across vertebrates, including humans. In this work, we focused on how Foxd4l1.1 downregulates the neural repressive pathway. Foxd4l1.1 inhibited BMP4/Smad1 signaling and triggered neuroectoderm formation in animal cap explants of Xenopus embryos. Foxd4l1.1 directly bound within the promoter of endogenous neural repressor ventx1.1 and inhibited ventx1.1 transcription. Foxd4l1.1 also physically interacted with Xbra in the nucleus and inhibited Xbra-induced ventx1.1 transcription. In addition, Foxd4l1.1 also reduced nuclear localization of Smad1 to inhibit Smad1-mediated ventx1.1 transcription. Foxd4l1.1 reduced the direct binding of Xbra and Smad1 on ventx1.1 promoter regions to block Xbra/Smad1-induced synergistic activation of ventx1.1 transcription. Collectively, Foxd4l1.1 negatively regulates transcription of a neural repressor ventx1.1 by multiple mechanisms in its exclusively occupied territory of neuroectoderm, and thus leading to primary neurogenesis. In conjunction with the results of our previous findings that ventx1.1 directly represses foxd4l1.1, the reciprocal repression of ventx1.1 and foxd4l1.1 is significant in at least in part specifying the mechanism for the non-neural versus neural ectoderm fate determination in Xenopus embryos.

Dach1 regulates neural crest migration during embryonic development

Dachshund 1(Dach1) is a key component of the retinal determination gene network that plays significant roles in cell fate regulation. The vertebrate homolog of Drosophila dachshund has gained considerable importance as an essential regulator of development, but its functions during embryonic development remain elusive. We investigated the functional significance of dach1 during Xenopus embryogenesis using loss-of-function studies. Reverse transcription-polymerase chain reaction demonstrated the maternal nature of dach1, showing enhanced expression at the neurula stage of development, and morpholino oligonucleotide injection of dach1 induced phenotypic anomalies of microcephaly and reduced body length. Animal cap assays followed by whole-mount in-situ hybridization indicated the perturbed expression of neural and neural crest (NC) markers. Our data suggest the prerequisite functions of dach1 in NC migration during Xenopus embryogenesis. However, the developmental pathways regulated by dach1 during embryogenesis require further elucidation.

Applying Tensile and Compressive Force to Xenopus Animal Cap Tissue.

Over many years, the Xenopus laevis embryo has provided a powerful model system to investigate how mechanical forces regulate cellular function. Here, we describe a system to apply reproducible tensile and compressive force to X. laevis animal cap tissue explants and to simultaneously assess cellular behavior using live confocal imaging.

Super-resolution imaging of densely packed DNA in nuclei of zebrafish embryos using stimulated emission double depletion microscopy

Knowledge about the spatial distribution of DNA in the cell nucleus is an essential aspect of understanding how cells control gene expression. Here, we describe a protocol for the preparation of samples for super-resolution stimulated emission depletion (STED) fluorescence imaging of bulk DNA in nuclei of blastomeres within animal caps of zebrafish embryos. We evaluated different mounting media and DNA stains. With samples stained with the silicon-rhodamine dye JF646 conjugated to Hoechst 33342 as a DNA tag and mounted in glycerol, we compared confocal, STED and stimulated emission double depletion (STEDD), in particular with respect to background fluorescence and image resolution. We show that super-resolved images of lateral planes through the cell nuclei can be collected up to imaging depths of several tens of micrometers with greatly reduced background using STEDD. Acquisition of axial image stacks using both lateral and axial STED confinement was also feasible, though at reduced resolution due to photobleaching. Further optimization steps are discussed to obtain a robust experimental platform for imaging the three-dimensional distribution of DNA inside zebrafish embryo tissue.

Liver Specification in the Absence of Cardiac Differentiation Revealed by Differential Sensitivity to Wnt/β Catenin Pathway Activation.

Embryonic precursors of liver and heart, whilst not sharing cellular origin, develop in close proximity through a dynamic series of inductive signaling events. During gastrulation anterior endoderm (AE) provides cardiogenic signals that act on adjacent mesoderm, resulting in induction of cardiac precursors. Subsequently cardiogenic mesoderm generates a FGF signal that acts on adjacent AE to induce foregut organ specification. Additional signals such as BMP and Wnt provide further information required for liver specification. Most findings on liver specification were derived from mouse explant studies as well as experiments with Xenopus and zebrafish embryos. To address some of the limitations of these models, here we used two complementary ex vivo models based on Xenopus embryos: pluripotent animal cap explants expressing Gata4 transcription factor and conjugates of gastrula-stage AE with animal caps (AC). We show that in these models liver specification is not sensitive to Wnt signaling manipulation, in contrast to the requirement for Wnt antagonism shown in vivo. FGF pathway is not necessary for Gata4-induced liver specification in animal cap explants but is required for prolonged period in sandwiches of AE and AC. In contrast, BMP signaling is shown to be essential for Gata4-induced liver specification. Our findings may have implications for research on liver differentiation from embryonic stem cells.

XNgn2 induces expression of predominantly sensory neuron markers in Xenopus whole embryo ectoderm but induces mixed subtype expression in isolated ectoderm explants

Proneural basic-helix-loop-helix (bHLH) proteins, such as Neurogenin2 (Ngn2) and Ascl1, are critical regulators at the onset of neuronal differentiation. Endogenously they have largely complementary expression patterns, and have conserved roles in the specification of distinct neuronal subtypes. InXenopusembryos, xNgn2 is the master regulator of primary neurogenesis forming sensory, inter- and motor neurons within the neural plate, while xAscl1 is the master regulator of autonomic neurogenesis, forming noradrenergic neurons in the antero-ventral region of the embryo. Here we characterise neuronal subtype identity of neurons induced by xNgn2 in the ectoderm of wholeXenopusembryos in comparison with xAscl1, and in ectodermal “animal cap” explants. We find that the transcriptional cascades mediating primary and autonomic neuron formation are distinct, and while xNgn2 and xAscl1 can upregulate genes associated with a non-endogenous cascade, this expression is spatially restricted within the embryo. xNgn2 is more potent than xAscl1 at inducing primary neurogenesis as assayed by neural-β-tubulin. In ectoderm of the intact embryo, these induced primary neurons have sensory characteristics with no upregulation of motor neuron markers. In contrast, xNgn2 is able to up-regulate both sensory and motor neuron markers in naïve ectoderm of animal cap explants, suggesting a non-permissive environment for motor identity in the patterned ectoderm of the whole embryo.