Anterior-posterior patterning of the avian neuraxis.

Nieuwkoop and Nigtevecht's (1954) model of anterior-posterior patterning of the neuraxis states that the naive ectoderm first receives an 'activation' signal giving it an anterior, neural character and subsequently a 'transformation' signal that progressively caudalises the axis resulting in the full rostro-caudal pattern. Stern (2001) proposed a modification to this model that divides the first step into two: a transient 'activation' step and a subsequent 'stabilisation' step. The hypoblast has been implicated as the transient inducer of a pre-neural, pre-forebrain state. In this thesis, the molecular nature of this induction is investigated by grafting the hypoblast into the area opaca to look at the induction of the genes Sox3, Otx2, ERNI and Cyp26Al. It was found that FGFs recapitulate the induction of the first three markers and retinoic acid (RA) can induce Cyp26Al whilst loss of function experiments show that both FGF and RA are required for hypoblast- mediated induction. In the epiblast, these induced genes are maintained as the future forebrain develops. Potential stabilising signals were tested by combining hypoblast grafts with cells secreting various proteins. By antagonising Wnts and/or BMPs and/or Nodal, Sox3 and ERNI can be maintained, whilst Otx2 maintenance requires combined Wnt- and BMP-inhibition, but the definitive neural marker, Sox2, is never induced. This suggests that a further 'neuralising' step might be required. Unlike regions of the epiblast fated to form head structures, the cells that will contribute to the remainder of the neuraxis reside within a small population of progenitors near the node. This indicates that a different mechanism might be responsible for patterning more caudal regions mediated by a qualitative or quantitative mechanism. To test this, secondary axes were generated by grafting progressively older donor nodes but the patterning of these ectopic axes suggests that the node might caudalise in conjunction with the pre-somitic mesoderm (PSM). Indeed, homotopic PSM grafts between different staged embryos do affect the neural tube boundary of Hoxb9. PSM can caudalise the neurectoderm, an effect that increases with age of the donor and decreases with the age of the PSM cells. An interesting conclusion is that some of the same signals are responsible both for the initial activation stages and for the subsequent transformation steps. This highlights the importance of timing as to the response of a particular cell to a particular signal

A new method to transfect the hypoblast of the chick embryo reveals conservation of the regulation of an Otx2 enhancer between mouse and chick extraembryonic endoderm

BACKGROUND: The mouse anterior visceral endoderm (AVE) and the chick hypoblast are thought to have homologous roles in the early stages of neural induction and primitive streak formation. In mouse, many regulatory elements directing gene expression to the AVE have been identified. However, there is no technique to introduce DNA into the chick hypoblast that would enable a comparison of their activity and this has hampered a direct comparison of the regulation of gene expression in the mouse and chick extraembryonic endoderm. RESULTS: Here we describe a new method to introduce DNA into the chick hypoblast, using lipofectamine-mediated transfection. We show that the hypoblast can be easily transfected and that it starts to express a luciferase reporter within 2 hours of transfection. The validity of technique is tested by following the movement and fate of hypoblast cells, which reveals their translocation to the anterior germinal crescent. We then introduce a vector containing GFP driven by the mouse VEcis-Otx2 enhancer (which directs gene expression to the mouse AVE) and we detect activity in the hypoblast. CONCLUSION: The new technique for delivering expression constructs to the chick hypoblast will enable studies on gene activity and regulation to be performed in this tissue, which has proved difficult to transfect by electroporation. Our findings also reveal that regulatory elements that direct gene expression to the mouse AVE are active in chick hypoblast, supporting the idea that these two tissues have homologous functions

The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue

<p>Abstract</p> <p>Background</p> <p>Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized <it>in vitro </it>and <it>in vivo </it>the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells.</p> <p>Results</p> <p>We show that <it>Ens-1 </it>LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the <it>Ens-1 </it>gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of <it>Ens-1</it>.</p> <p>Conclusion</p> <p>Our results show that <it>Ens-1 </it>LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, <it>Ens-1 </it>LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.</p

A role for the hypoblast (AVE) in the initiation of neural induction, independent of its ability to position the primitive streak

AbstractThe mouse anterior visceral endoderm (AVE) has been implicated in embryonic polarity: it helps to position the primitive streak and some have suggested that it might act as a “head organizer”, inducing forebrain directly. Here we explore the role of the hypoblast (the chick equivalent of the AVE) in the early steps of neural induction and patterning. We report that the hypoblast can induce a set of very early markers that are later expressed in the nervous system and in the forebrain, but only transiently. Different combinations of signals are responsible for different aspects of this early transient induction: FGF initiates expression of Sox3 and ERNI, retinoic acid can induce Cyp26A1 and only a combination of low levels of FGF8 together with Wnt- and BMP-antagonists can induce Otx2. BMP- and Wnt-antagonists and retinoic acid, in different combinations, can maintain the otherwise transient induction of these markers. However, neither the hypoblast nor any of these factors or combinations thereof can induce the definitive neural marker Sox2 or the formation of a mature neural plate or a forebrain, suggesting that the hypoblast is not a head organizer and that other signals remain to be identified. Interestingly, FGF and retinoids, generally considered as caudalizing factors, are shown here to play a role in the induction of a transient “pre-neural/pre-forebrain” state

Early patterning in a chondrichthyan model, the small spotted dogfish: towards the gnathostome ancestral state

International audienceIn the past few years, the small spotted dogfish has become the primary model for analyses of early development in chondrichthyans. Its phylogenetic position makes it an ideal outgroup to reconstruct the ancestral gnathostome state by comparisons with established vertebrate model organisms. It is also a suitable model to address the molecular bases of lineage-specific diversifications such as the rise of extraembryonic tissues, as it is endowed with a distinct extraembryonic yolk sac and yolk duct ensuring exchanges between the embryo and a large undivided vitelline mass. Experimental or functional approaches such as cell marking or in ovo pharmacological treatments are emerging in this species, but recent analyses of early development in this species have primarily concentrated on molecular descriptions. These data show the dogfish embryo exhibits early polarities reflecting the dorso-ventral axis of amphibians and teleosts at early blastula stages and an atypical anamniote molecular pattern during gastrulation, independently of the presence of extraembryonic tissues. They also highlight unexpected relationships with amniotes, with a strikingly similar Nodal-dependent regional pattern in the extraembryonic endoderm. In this species, extraembryonic cell fates seem to be determined by differential cell behaviors, which lead to cell allocation in extraembryonic and embryonic tissues, rather than by cell regional identity. We suggest that this may exemplify an early evolutionary step in the rise of extraembryonic tissues, possibly related to quantitative differences in the signaling activities, which shape the early embryo. These results highlight the conservation across gnathostomes of a highly constrained core genetic program controlling early patterning. This conservation may be obscured in some lineages by taxa-specific diversifications such as specializations of extraembryonic nutritive tissues