Glypican4 modulates lateral line collective cell migration non cell-autonomously.

Collective cell migration is an essential process during embryonic development and diseases such as cancer, and still much remains to be learned about how cell intrinsic and environmental cues are coordinated to guide cells to their targets. The migration-dependent development of the zebrafish sensory lateral line proves to be an excellent model to study how proteoglycans control collective cell migration in a vertebrate. Proteoglycans are extracellular matrix glycoproteins essential for the control of several signaling pathways including Wnt/ß-catenin, Fgf, BMP and Hh. In the lateral line primordium the modified sugar chains on proteoglycans are important regulators of cell polarity, ligand distribution and Fgf signaling. At least five proteoglycans show distinct expression patterns in the primordium; however, their individual functions have not been studied. Here, we describe the function of glypican4 during zebrafish lateral line development. glypican4 is expressed in neuromasts, interneuromast cells and muscle cells underlying the lateral line. knypekfr6/glypican4 mutants show severe primordium migration defects and the primordium often U-turns and migrates back toward the head. Our analysis shows that Glypican4 regulates the feedback loop between Wnt/ß-catenin/Fgf signaling in the primordium redundantly with other Heparan Sulfate Proteoglycans. In addition, the primordium migration defect is caused non-cell autonomously by the loss of cxcl12a-expressing muscle precursors along the myoseptum via downregulation of Hh. Our results show that glypican4 has distinct functions in primordium cells and cells in the environment and that both of these functions are essential for collective cell migration.

Defining progressive stages in the commitment process leading to embryonic lens formation.

The commitment of regions of the embryo to form particular tissues or organs is a central concept in development, but the mechanisms controlling this process remain elusive. The well-studied model of lens induction is ideal for dissecting key phases of the commitment process. We find in Xenopus tropicalis, at the time of specification of the lens, i.e., when presumptive lens ectoderm (PLE) can be isolated, cultured, and will differentiate into a lens that the PLE is not yet irreversibly committed, or determined, to form a lens. When transplanted into the posterior of a host embryo lens development is prevented at this stage, while ~ 3 h later, using the same assay, determination is complete. Interestingly, we find that specified lens ectoderm, when cultured, acquires the ability to become determined without further tissue interactions. Furthermore, we show that specified PLE has a different gene expression pattern than determined PLE, and that determined PLE can maintain expression of essential regulatory genes (e.g., foxe3, mafB) in an ectopic environment, while specified PLE cannot. These observations set the stage for a detailed mechanistic study of the genes and signals controlling tissue commitment.

Modification of the zone of polarizing activity signal by trypsin.

Patterning of the developing vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) via the expression of Sonic hedgehog (Shh) and along the proximal-distal axis by the apical ectodermal ridge (AER) through the production of fibroblast growth factors (FGFs). ZPA grafting, as well as ectopic application of SHH to the anterior chick limb bud, demonstrate that digit patterning is largely influenced by these secreted factors. Although signal transduction pathways have been well characterized for SHH and for FGFs, little is known of how these signals are regulated extracellularly in the limb. The present study shows that alteration of the extracellular environment through trypsin treatment can have profound effects on digit patterning. These effects appear to be mediated by the induction of Shh in host tissues and by ectopic AER formation, implicating the extracellular matrix in regulating the signaling activities of key patterning genes in the limb.

Unique organization of the frontonasal ectodermal zone in birds and mammals.

The faces of birds and mammals exhibit remarkable morphologic diversity, but how variation arises is not well-understood. We have previously demonstrated that a region of facial ectoderm, which we named the frontonasal ectodermal zone (FEZ), regulates proximo-distal extension and dorso-ventral polarity of the upper jaw in birds. In this work, we examined the equivalent ectoderm in murine embryos and determined that the FEZ is conserved in mice. However, our results revealed that fundamental differences in the organization and constituents of the FEZ in mice and chicks may underlie the distinct growth characteristics that distinguish mammalian and avian embryos during the earliest stages of development. Finally, current models suggest that neural crest cells regulate size and shape of the upper jaw, and that signaling by Bone morphogenetic proteins (Bmps) within avian neural crest helps direct this process. Here we show that Bmp expression patterns in neural crest cells are regulated in part by signals from the FEZ. The results of our work reconcile how a conserved signaling center that patterns growth of developing face may generate morphologic diversity among different animals. Subtle changes in the organization of gene expression patterns in the FEZ could underlie morphologic variation observed among and within species, and at extremes, variation could produce disease phenotypes.

Improvement of peripheral nerve regeneration by a tissue-engineered nerve filled with ectomesenchymal stem cells.

Ectomesenchymal stem cells (EMSCs) originate from the cranial neural crest. They are a potential source of neuronal and Schwann cells (SCs) of the peripheral nervous system (PNS) during embryonic development. The third passage of EMSCs enzymatically isolated from the mandibular processes of Sprague-Dawley rats were cultured in forskolin and bovine pituitary extract for 6 days to generate functional Schwann cell phenotypes. Next, 10-mm defects in the sciatic nerves were bridged with an autograft, tissue-engineered nerve filled with differentiated cells in collagen, or a PLGA conduit alone in 18 rats, and the nerve defects of another four rats were left untreated. The regenerated nerves were evaluated by the sciatic functional index (SFI) monthly and by histological analysis 4 months after grafting. The recovery index of the sciatic nerve improved significantly in the autograft and tissue-engineered nerve groups, both of which were superior to the PLGA group. In animals transplanted with the EMSCs, there was greater regeneration than with conduit alone during the same period of implantation. These results show that when EMSCs are transplanted to a peripheral nerve defect they differentiate into supportive cells that contribute to the promotion of axonal regeneration.

Regionalised signalling within the extraembryonic ectoderm regulates anterior visceral endoderm positioning in the mouse embryo.

The development of the anterior-posterior (AP) axis in the mammalian embryo is controlled by interactions between embryonic and extraembryonic tissues. It is well established that one of these extraembryonic tissues, the anterior visceral endoderm (AVE), can repress posterior cell fate and that signalling from the other, the extraembryonic ectoderm (ExE), is required for posterior patterning. Here, we show that signals from the prospective posterior ExE repress AVE gene expression and affect the distribution of the AVE cells. Surgical ablation of the prospective posterior, but not the anterior, extraembryonic region at 5.5 days of development (E5.5) perturbs the characteristic distal-to-anterior distribution of AVE cells and leads to a dramatic expansion of the AVE domain. Time-lapse imaging studies show that this increase is due to the ectopic expression of an AVE marker, which results in a symmetrical positioning of the AVE. Surgical ablation of this same ExE region after the distal-to-anterior migration has already commenced, at E5.75, does not affect the localisation of the AVE, indicating that this effect takes place within a short time window. Conversely, transplanting the prospective posterior, but not the anterior, extraembryonic region onto isolated E5.5 embryonic explants drastically reduces the AVE domain. Further, transplantation experiments demonstrate that the signalling regulating AVE gene expression originates from the posterior ExE, rather than its surrounding VE. Together, our results show that signals emanating from the future posterior ExE within a temporal window both restrict the AVE domain and promote its specific positioning. This indicates for the first time that the ExE is already regionalised a day before the onset of gastrulation in order to correctly set the orientation of the AP axis of the mouse embryo. We propose a reciprocal function of the posterior ExE and the AVE in establishing a balance between the antagonistic activities of these two tissues, essential for AP patterning.

Neuronal leucine-rich repeat 6 (XlNLRR-6) is required for late lens and retina development in Xenopus laevis.

Leucine-rich repeat proteins expressed in the developing vertebrate nervous system comprise a complex, multifamily group, and little is known of their developmental function in vivo. We have identified a novel member of this group in Xenopus laevis, XlNLRR-6, and through sequence and phylogenetic analysis, have placed it within a defined family of vertebrate neuronal leucine-rich repeat proteins (NLRR). XlNLRR-6 is expressed in the developing nervous system and tissues of the eye beginning at the neural plate stage, and expression continues throughout embryonic and larval development. Using antisense morpholino oligonucleotide (MO) -mediated knockdown of XlNLRR-6, we demonstrate that this protein is critical for development of the lens, retina, and cornea. Reciprocal transplantation of presumptive lens ectoderm between MO-treated and untreated embryos demonstrate that XlNLRR-6 plays autonomous roles in the development of both the lens and retina. These findings represent the first in vivo functional analysis of an NLRR family protein and establish a role for this protein during late differentiation of tissues in the developing eye.

The dissociation of the Fgf-feedback loop controls the limbless state of the neck.

In tetrapods, limbs develop at two specific positions along the anteroposterior axis of the embryo, whereas other regions of the embryo, most prominently the neck and the flank, are limbless. However, the flank can generate an ectopic limb when the Fgf-feedback loop crucial for the initiation of limb budding is activated. Thus, despite its limblessness, the flank is a limb-competent area. Using the chick embryo as model, we investigated whether the neck, as the flank, has the competence to form a limb, and what mechanism may regulate its limblessness. We show that forelimb lateral mesoderm plus ectoderm grafted into the neck can continue limb development, suggesting that the neck does not actively inhibit this process. However, neck tissues themselves do not support or take part in limb formation. Hence, the neck is limb-incompetent. This is due to the dismantling of Fgf signalling at distinct points of the MAPK signalling cascade in the neck lateral mesoderm and ectoderm.

Studies on the use of NE-4C embryonic neuroectodermal stem cells for targeting brain tumour.

Neural stem cells were suggested to migrate to and invade intracranial gliomas. In the presented studies, interactions of NE-4C embryonic neural stem cells were investigated with C6 and Gl261, LL and U87, glioblastoma cells or with primary astrocytes. Glioma-derived humoral factors did not influence the proliferation of stem cells. NE-4C-derived humoral factors did not alter the proliferation of Gl261 and U87 cells, but increased the mitotic activity of C6 cells and that of astrocytes. In chimera-aggregates, all types of glioma cells co-aggregated with astrocytes, but most of them segregated from stem cells. Complete intercalation of stem and tumour cells was detected only in chimera-aggregates of Gl261 glioma and NE-4C cells. If mixed suspensions of NE-4C and Gl261 cells were injected into the brain, stem cells survived and grew inside the tumour mass. NE-4C stem cells, however, did not migrate towards the tumour, if implanted near to Gl261 tumours established in the adult mouse forebrain. The observations indicate that not all types of stem cells could be used for targeting all sorts of brain tumours.

Premature regression of the leg apical ectodermal ridge in the Japanese chick wingless mutant.

The autosomal dominant Japanese wingless mutant has varying degrees of wing and leg truncations. The wing defects range from complete loss to negligible defects, whereas leg abnormalities are usually restricted to loss of the phalanges. Further analyses of the mutant focusing on the leg, which has been relatively uncharacterized, were performed. The expression pattern of Fgf8, a marker gene for the apical ectodermal ridge (AER) that controls outgrowth of the limbs, revealed premature regression at stage 28. Electron microscopy study showed abnormalities in the basement membrane all through the AER in the same stage. In the mutant, cell death was observed in the mesenchyme underlying AER between stages 31 and 32, although in the wild-type leg, AER regression and cell death occurred almost simultaneously at stages 33-34. To know if the cell death and cessation of the outgrowth are common mechanisms of wild-type and the mutant, we removed the AER in wild-type embryos at stage 28 and followed the fate of the limb. This also resulted in premature cell death 48 h after AER removal (equivalent to stage 32) and limb truncations similar to those observed in mutant limbs. To confirm whether either AER or underlying mesenchyme is responsible for the truncation, transplantation of the AER between the wild-type and the mutant was performed. This revealed that AER is the defective tissue in this mutant.

The acquisition of neural fate in the chick.

Neural development in the chick embryo is now understood in great detail on a cellular and a molecular level. It begins already before gastrulation, when a separation of neural and epidermal cell fates occurs under the control of FGF and BMP/Wnt signalling, respectively. This early specification becomes further refined around the tip of the primitive streak, until finally the anterior-posterior level of the neuroectoderm becomes established through progressive caudalization. In this review we focus on processes in the chick embryo and put classical and more recent molecular data into a coherent scenario.

Fate of cloned embryonic neuroectodermal cells implanted into the adult, newborn and embryonic forebrain.

NE-4C, one-cell derived neuroectodermal stem cells expressing a reporter gene--green fluorescent protein (GFP) or heat-resistant alkaline phosphatase (PLAP)--or prelabeled with bromodeoxyuridine (BrdU) were implanted into the forebrain of adult, new-born and fetal mice and into the mid- and forebrain vesicles of early chick embryos. The fate of implanted cells in the mouse and chick hosts was followed up to 6 and 2 weeks, respectively. Neural differentiation was monitored by detecting the expression of neuron-specific markers and GFAP. NE-4C cells integrated into the early embryonic brain tissue and developed into morphologically differentiated neurons. The same cells produced expanding tumor-like aggregates in the newborn forebrain and were expelled from the adult forebrain parenchyma. In the adult brain, long-term survival and integration of stem cells were revealed only in neurogenic zones. The data suggest that noncommitted, proliferating neuroectodermal progenitors can integrate into the brain tissue at time and site of tissue genesis.

Specification of polarity of teloblastogenesis in the oligochaete annelid Tubifex: cellular basis for bilateral symmetry in the ectoderm.

Ectodermal teloblastogenesis in the oligochaete annelid Tubifex is a spatiotemporally regulated process that gives rise to four bilateral pairs of ectoteloblasts (N, O, P, and Q) that assume distinct fates. Ectoteloblasts on either side of the embryo arise from an invariable sequence of asymmetric cell divisions of a proteloblast, NOPQ, which occur with a defined orientation with respect to the embryonic axes: the N teloblast is generated first and located ventralmost, and the Q teloblast, which is generated next, is located dorsalmost; finally, the O and P teloblasts are generated by almost equal division of their precursor cell, OP. Polarity of teloblastogenesis on one side of the embryo is a mirror image of the other; this mirror symmetry of ectoteloblasts about the embryo's midline gives rise to the bilaterally symmetric organization of the ectoderm. In this study, we examined whether cellular interactions are involved in specification of polarity of asymmetric cell divisions in NOPQ cells. A set of cell transplantation experiments demonstrated that NOPQ cells are initially uncommitted in terms of division pattern and cell fates: If a left NOPQ cell is transplanted to the right side of a host embryo, it exhibits a polarity comparable to that of right NOPQ cells. The results of another set of cell transplantation experiments suggest that contact between NOPQ cells serves as an external cue for their polarization, irrespective of their position in the embryo, and that in the absence of host NOPQ cells, transplanted NOPQ cells can be polarized according to positional information residing in the host embryo. The competence of NOPQ cells to respond to external cues tapers down before their division into N and OPQ. A set of cell ablation experiments demonstrated that neighboring cells such as posteriorly located M teloblasts and anterolaterally located micromeres play a role in controlling spatial aspects of NOPQ's behavior that gives rise to their division along the dorsoventral axis. These results suggest that NOPQ cells, which do not initially have a rigidly fixed polarity, become polarized through external cues. Possible sources of signals for this polarizing induction are discussed in the light of the present results.

Identification of neural crest competence territory: role of Wnt signaling.

In recent years, research on neural crest induction has allowed the identification of several molecules as candidates for neural crest inducers. Although many of these molecules have the ability to induce neural crest in different assays, a general mechanism of neural crest induction that includes a description of the tissues that produce the inductive signals and the time and steps in which this process takes place remains elusive. To better understand the mechanism of neural crest induction, we developed an assay that has been used previously by Nieuwkoop to study anterior-posterior pattern of the neural plate. Folds of competent ectoderm were implanted in different positions of a young neurula embryo, and the induction of neural crest was analyzed using the expression of the neural crest marker Xslug. We identified a very localized region of the early neurula where it is possible to get neural crest induction, whereas all of the regions tested showed a clear induction of the neural plate marker Xsox2. These results indicate that there is a region in the embryo with the appropriate combination of signals needed to induce neural crest cells; we called this region the neural crest competence territory. In addition, our results show that neural crest induction is always accompanied by neural plate induction, but there are many cases where neural plate was induced without neural crest. These results support the model in which the neural crest is induced by an interaction between neural plate and epidermis, but they also suggest that additional signals are required. By making grafts of different sizes and implanting them in the epidermis or the neural plate, we concluded that one of the inductive signals is produced in the dorsal region of the embryo and travels into the ectoderm. Finally, by performing gain- and loss-of-function of Wnt signaling experiments, we show that this pathway plays an important role not only in neural crest induction but also in the specification of the neural crest competence territory. Developmental Dynamics 229:109-117, 2004.

The use of real-time PCR with fluorogenic probes for the rapid selection of mutant neuroectodermal grafts.

Adenosine is an efficient inhibitor of neuronal activity with the ability to suppress seizure activity in various animal models of epilepsy. In the present study adenosine-releasing neuronal cells were generated as a potential source for therapeutically active grafts. Mice with a genetic disruption of the gene encoding adenosine kinase (Adk(-/-))-the major adenosine metabolizing enzyme-were used as a source for the derivation of adenosine releasing neuronal cells. Since homozygous Adk(-/-) mice constitute a lethal phenotype, embryonic neuroectoderm was derived from intercrosses of Adk(+/-)-mice. Therefore, a rapid genotyping procedure had to be developed using a fluorescent 5'-exonuclease (TaqMan) assay, which permitted the genotyping of embryonic cell material within 3 h. During this time period the cells to be grafted displayed an unaltered viability. Cultured neuroectodermal Adk(-/-) cells released up to 2 micro g adenosine per mg protein per hour. Adk(-/-) neuroectoderm grafted into the lateral brain ventricle of adult mice was found to survive for at least 6 weeks. The method described here suggests the feasibility to graft adenosine releasing neuroectodermal cells as a potential therapeutic approach for the treatment of pharmacoresistant epilepsy.

Comm sorts robo to control axon guidance at the Drosophila midline.

Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. We show here that comm is required in neurons, not in midline cells as previously thought, and that it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor.

Neuroectodermal origin of brain pericytes and vascular smooth muscle cells.

The origin of vascular pericytes (PCs) and smooth muscle cells (vSMCs) in the brain has hitherto remained an open question. In the present study, we used the quail-chick chimerization technique to elucidate the lineage of cranial PCs/vSMCs. We transplanted complete halves of brain anlagen, or dorsal (presumptive neural crest [NC]) or ventral cranial neural tube. Additional experiments included transplantations of neuroectoderm into limb mesenchyme, and of head mesoderm or limb mesenchyme into paraxial head mesoderm. After interspecific transplantation of quail brain rudiment, graft-derived vSMCs were found in the vessel walls of the grafted brain. Notably, transplanted ventral neural tube also gave rise to vSMCs. After grafting of quail head mesoderm, quail endothelial cells were found in the host brain, but no vSMCs of donor origin. Grafting of quail whole or ventral neural tube into the limb bud led to endowment of graft and host vessels with graft-derived vSMCs. Quail limb bud mesenchyme contributed to vSMCs in the ectopic neural graft, but, when transplanted into paraxial head mesenchyme, it did not form intraneural vSMCs. After orthotopic transplantation of cranial NC, graft-derived vSMCs were not only found in meninges and brain of the operated side, but also on the contralateral side. Our results show that 1) avian cranial neuroectoderm is able to differentiate into vSMCs of the brain; 2) this potential is not restricted to the prospective NC; and 3) neither cranial mesoderm nor cranially transplanted limb bud mesoderm can give rise to brain vSMC.

Cornea-lens transdifferentiation in the anuran, Xenopus tropicalis.

Previously, the only anuran amphibian known to regenerate the lens of the eye was Xenopus laevis. This occurs during larval stages through transdifferentiation of the outer cornea epithelium under control of factors presumably secreted by the neural retina. This study demonstrates that a distantly related species, X. tropicalis, is also able to regenerate lenses through this process. A transgenic line of X. tropicalis was used to examine the process of cornea-lens transdifferentiation in which green fluorescent protein (GFP) is expressed in differentiated lens cells under the control of the Xenopus gamma1-crystallin promoter element. Unlike X. laevis, the process of cornea-lens transdifferentiation typically occurs at a very low frequency in X. tropicalis due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening. The inner cornea endothelium serves as a key physical barrier that normally prevents retinal signals from reaching the outer cornea epithelium. If this barrier is circumvented by implanting outer cornea epithelium of transgenic tadpoles directly into the vitreous chamber of non-transgenic X. tropicalis larval eyes, a higher percentage of cases formed lenses expressing GFP. Lenses were also formed if these tissues were implanted into X. laevis larval eyes, suggesting the same or similar inducing factors are present in both species. When pericorneal ectoderm and posteriolateral flank ectoderm were implanted into the vitreous chamber, only in rare cases did pericorneal ectoderm form lens cells. Thus, unlike the case in X. laevis, competence to respond to the inducing factors is tightly restricted to the cornea epithelium in X. tropicalis. As controls, all these tissues were implanted into the space located between the inner and outer corneas. None of these implants, including outer cornea epithelium, exhibited GFP expression. Thus, the essential inductive factors are normally contained within the vitreous chamber. One explanation why this type of lens regeneration is not seen in some other anurans could be due to the rapid rate at which the inner cornea endothelium heals to recover the pupillary opening once the original lens is removed. These findings are discussed in terms of the evolution of this developmental process within the anurans.