Claudio D. Stern

Initiation of neural induction by FGF signalling before gastrulation

During neural induction, the ‘organizer’ of the vertebrate embryo instructs neighbouring ectodermal cells to become nervous system rather than epidermis. This process is generally thought to occur around the mid-gastrula stage of embryogenesis. Here we report the isolation of ERNI, an early response gene to signals from the organizer (Hensens node). Using ERNI as a marker, we present evidence that neural induction begins before gastrulation—much earlier in development than previously thought. We show that the organizer and some of its precursor cells produce a fibroblast growth factor signal, which can initiate, and is required for, neural induction.

Contact inhibition of locomotion in vivo controls neural crest directional migration

Contact inhibition of locomotion was discovered by Abercrombie more than 50 years ago and describes the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction on contact. Its failure was suggested to contribute to malignant invasion. However, the molecular basis of contact inhibition of locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion, demonstrate contact inhibition of locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, in the same manner as metastatic cancer cells. We show that inhibition of non-canonical Wnt signalling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt-signalling members localize at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of contact inhibition of locomotion in vivo, provide an explanation for coherent directional migration of groups of cells and establish a previously unknown role for non-canonical Wnt signalling.

Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.

The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm

The chick homeobox gene goosecoid (gsc) is first expressed in a barely noticeable cell population near the posterior margin (Kollers sickle) of the unincubated egg. Then it is detected in Hensens node, traditionally considered the chick organizer. Later, gsc-expressing cells leave the node with the prechordal plate. Fate mapping indicates that these three regions are related by cell lineage, and transplantation experiments suggest that they all have inducing activity. Quail posterior margin and anterior primitive streak grafts (gsc expressing) induce gsc transcription in neighboring chick host cells. We propose that development of the chick organizer starts earlier than previously thought and that gsc marks this changing cell population.

Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity

We have investigated the cell interactions and signalling molecules involved in setting up and maintaining the border between the neural plate and the adjacent non-neural ectoderm in the chick embryo at primitive streak stages. msx-1, a target of BMP signalling, is expressed in this border at a very early stage. It is induced by FGF and by signals from the organizer, Hensens node. The node also induces a ring of BMP-4, some distance away. By the early neurula stage, the edge of the neural plate is the only major site of BMP-4 and msx-1 expression, and is also the only site that responds to BMP inhibition or overexpression. At this time, the neural plate appears to have a low level of BMP antagonist activity. Using in vivo grafts and in vitro assays, we show that the position of the border is further maintained by interactions between non-neural and neural ectoderm. We conclude that the border develops by integration of signals from the organizer, the developing neural plate, the paraxial mesoderm and the non-neural epiblast, involving FGFs, BMPs and their inhibitors. We suggest that BMPs act in an autocrine way to maintain the border state.

Churchill, a Zinc Finger Transcriptional Activator, Regulates the Transition between Gastrulation and Neurulation

Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the neural plate is established within the epiblast-both events require FGF signaling. We describe a zinc finger transcriptional activator, Churchill (ChCh), which acts as a switch between different roles of FGF. FGF induces ChCh slowly; this activates Smad-interacting-protein-1 (Sip1), which blocks further induction of the mesoderm markers brachyury and Tbx6L by FGF. ChCh is first expressed as cells stop migrating through the primitive streak, and we show that it regulates cell ingression. We propose a simple mechanism by which FGF sensitizes cells to BMP signals. These results reveal that neural induction requires cessation of mesoderm formation at the midline in addition to the decision between epidermis and neural plate.

The amniote primitive streak is defined by epithelial cell intercalation before gastrulation

During gastrulation, a single epithelial cell layer, the ectoderm, generates two others: the mesoderm and the endoderm. In amniotes (birds and mammals), mesendoderm formation occurs through an axial midline structure, the primitive streak, the formation of which is preceded by massive ‘polonaise’ movements of ectoderm cells. The mechanisms controlling these processes are unknown. Here, using multi-photon time-lapse microscopy of chick (Gallus gallus) embryos, we reveal a medio-lateral cell intercalation confined to the ectodermal subdomain where the streak will later form. This intercalation event differs from the convergent extension movements of the mesoderm described in fish and amphibians (anamniotes): it occurs before gastrulation and within a tight columnar epithelium. Fibroblast growth factor from the extraembryonic endoderm (hypoblast, a cell layer unique to amniotes) directs the expression of Wnt planar-cell-polarity pathway components to the intercalation domain. Disruption of this Wnt pathway causes the mesendoderm to form peripherally, as in anamniotes. We propose that the amniote primitive streak evolved from the ancestral blastopore by acquisition of an additional medio-lateral intercalation event, preceding gastrulation and acting independently of mesendoderm formation to position the primitive streak at the midline.

The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells

Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.

A mechanism regulating the onset of Sox2 expression in the embryonic neural plate.

In vertebrate embryos, the earliest definitive marker for the neural plate, which will give rise to the entire central nervous system, is the transcription factor Sox2. Although some of the extracellular signals that regulate neural plate fate have been identified, we know very little about the mechanisms controlling Sox2 expression and thus neural plate identity. Here, we use electroporation for gain- and loss-of-function in the chick embryo, in combination with bimolecular fluorescence complementation, two-hybrid screens, chromatin immunoprecipitation, and reporter assays to study protein interactions that regulate expression of N2, the earliest enhancer of Sox2 to be activated and which directs expression to the largest part of the neural plate. We show that interactions between three coiled-coil domain proteins (ERNI, Geminin, and BERT), the heterochromatin proteins HP1α and HP1γ acting as repressors, and the chromatin-remodeling enzyme Brm acting as activator control the N2 enhancer. We propose that this mechanism regulates the timing of Sox2 expression as part of the process of establishing neural plate identity.

Cerberus regulates left–right asymmetry of the embryonic head and heart

BACKGROUND Most of the molecules known to regulate left-right asymmetry in vertebrate embryos are expressed on the left side of the future trunk region of the embryo. Members of the protein family comprising Cerberus and the putative tumour suppressor Dan have not before been implicated in left-right asymmetry. In Xenopus, these proteins have been shown to antagonise members of the transforming growth factor beta (TGF-beta) and Wnt families of signalling proteins. RESULTS Chick Cerberus (cCer) was found to be expressed in the left head mesenchyme and in the left flank of the embryo. Expression on the left side of the head was controlled by Sonic hedgehog (Shh) acting through the TGF-beta family member Nodal; in the flank, cCer was also regulated by Shh, but independently of Nodal. Surprisingly, although no known targets of Cerberus are expressed asymmetrically on the right side of the embryo at these stages, misexpression of cCer on this side of the embryo led to upregulation of the transcription factor Pitx2 and reversal of the direction of heart and head turning, apparently as independent events. Consistent with the possibility that cCer may be acting on bilaterally expressed TGF-beta family members such as the bone morphogenetic proteins (BMPs), this result was mimicked by right-sided misexpression of the BMP antagonist, Noggin. CONCLUSIONS Our findings suggest that cCer maintains a delicate balance of different TGF-beta family members involved in laterality decisions, and reveal the existence of partially overlapping molecular pathways regulating left-right asymmetry in the head and trunk of the embryo.