Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

The pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

Establishing embryonic territories in the context of Wnt signaling

This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish Xenopus laevis experimental model in Brazil we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, Wnt pathway appears as a major player and has been much explored by us and other research groups. Here we chose to review three published works that we consider landmarks within the history of our research on the developmental biology field and the neural induction and patterning modern findings. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue. Being those: 1. Establishing Head organizer in contrast to trunk organizer at early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties at late gastrula/early neurula.

The BMP ligand Pinhead together with Admp supports the robustness of embryonic patterning

Vertebrate embryonic dorsoventral axis is robustly stable in the face of variations in bone morphogenetic protein (BMP) signaling. However, the molecular mechanism behind this robustness remains uncharacterized. In this study, we show that zebrafish Pinhead, together with Admp, plays an important compensatory role in ensuring the robustness of axial patterning through fine-tuning of BMP signaling. pinhead encodes a BMP-like ligand expressed in the ventrolateral margin of the early gastrula. Transcription of pinhead and admp is under opposing regulation, where pinhead depletion results in a compensatory increase in admp transcription and vice versa, leading to normal axis formation in pinhead or admp mutants. Expression of pinhead and admp is directly repressed by the BMP/Smad pathway. When BMP signals were inhibited or excessively activated, pinhead/admp expression changed accordingly, allowing for self-regulation. Thus, this study reveals a negative feedback loop between BMP signaling and pinhead/admp that effectively stabilizes embryonic patterning by buffering against fluctuations in BMP signaling.

Mapping cell migrations and fates from a gastruloid model to the human primitive streak

Although fate maps of early gastrula embryos exist for nearly all model organisms, a fate map of the gastrulating human embryo remains elusive. Here we use human gastruloids to piece together part of a rudimentary fate map of the human primitive streak (PS). This is possible because stimulation with differing levels of BMP, WNT, and NODAL leads to self-organization of gastruloids into large and homogenous different subpopulations of endoderm and mesoderm, and comparative parallel analysis of these gastruloids, together with the fate map of the mouse embryo, allows the organization of these subpopulations along an anterior-posterior axis. We also developed a novel cell tracking technique that allowed the detection of robust fate-dependent cell migrations in our gastruloids comparable to those found in the mouse embryo. Taken together, our gastruloid derived fate map and recording of cell migrations provides a first coarse view of the embryonic human PS.

Transcriptome analysis of regeneration during Xenopus laevis experimental twinning

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.

A dual function of FGF signaling in Xenopus left-right axis formation

Organ left-right (LR) asymmetry is a conserved vertebrate feature, which is regulated by left-sided activation of Nodal signaling. Nodal asymmetry is established by a leftward fluid-flow generated at the ciliated LR organizer (LRO). While the role of fibroblast growth factor (FGF) signaling pathways during mesoderm development are conserved, diverging results from different model organisms suggested a non-conserved function in LR asymmetry. Here, we demonstrate that FGF is required during gastrulation in a dual function at consecutive stages of Xenopus embryonic development. In the early gastrula, FGF is necessary for LRO precursor induction, acting in parallel to FGF-mediated mesoderm induction. During late gastrulation, the FGF/Ca2+-branch is required for specification of the flow sensing lateral LRO cells, a function related to FGF-mediated mesoderm morphogenesis. This second function in addition requires input from the calcium channel Polycystin-2. Thus, analogous to mesoderm development, FGF activity is required in a dual role for laterality specification, namely for generating and sensing of leftward flow. Moreover, our data show that FGF functions in LR asymmetric development are conserved across vertebrate species, from fish to mammals.

INDUCCIÓN DE LA MADUREZ GONADAL DEL PARGO PALMERO LUTJANUS ANALIS (PISCES: LUTJANIDAE) MEDIANTE LA APLICACIÓN DE UN FOTOTERMOPERIODO ARTIFICIAL DE ACONDICIONAMIENTO

An experimental group (N=7) of three females and four males of mutton snapper Lutjanus analis captured in the wild, whose gonadal development entered in a state of latency because of its confinement in the lab, was maintained under an artificial photothermal cycle of 10 months between October 2001 and July 2002 in order to unblock and estimulate again their sexual maturation. Another group of equal number of fish was used as a control, being maintained during the same time without manipulation of temperature and photoperiod parameters. After eight months the three females of the experimental group showed positive gonadal development signs, their oocyte mean diameters (Ø) reaching 122 ± 76; 86 ± 40; and 122 ± 87 μm respectively. During the tenth month one female reached the state III of gonadal development (Ø = 327 ± 67 μm) and was induced to final maturation and spawning by a single injection of 1,000 IU/kg of HCG. The spawning occurred 64 h after the injection with approximately 25,000 eggs (Ø = 713 ± 56 μm) which reached the early gastrula stage 4:35 h after fertilization. The artificial photothermal cycle applied showed to be effective to unblock and stimulate gonadal development of the captive fish, preliminary result that must be confirmed by further research and which could constitute an important step in the development of a technology for the artificial reproduction of the mutton snapper.