A critical period for conversion of ectodermal cells to a neural crest fate

Previously, we found that interactions between neural and nonneural ectoderm can generate neural crest cells, with both the ectodermal and the neuroepithelial cells contributing to induced population (M. A. J. Selleck and M. Bronner-Fraser, 1995, Development 121, 525-538). To further characterize the ability of ectodermal cells to form neural crest, we have challenged their normal fate by transplanting them into the neural tube. To ensure that the ectoderm was from nonneural regions, we utilized extraembryonic ectoderm (the proamnion) and transplanted it into the presumptive midbrain of 1. 5-day-old chick embryos. We observed that the grafted ectoderm has the capacity to adopt a neural crest fate, responding within a few hours of surgery by turning on neural crest markers HNK-1 and Slug. However, the competence of the ectoderm to respond to neural crest-inducing signals is time limited, declining rapidly in donors older than the 10-somite stage. Similarly, the inductive capacity of the host midbrain declines in a time-dependent fashion. Our results show that extraembryonic ectoderm has the capacity to form neural crest cells given proper inducing signals, expressing both morphological and molecular markers characteristic of neural crest cells.     

The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis

Germ cells may be specified through the localization of germ line determinants to specific cells in early embryogenesis, or by inductive signals from neighboring cells to germ cell precursors in later embryogenesis. Such determinants can be produced and localized during or after oogenesis, either autonomously by oocytes or by associated nutritive cells. In Drosophila, each oocyte is connected to nurse cells by cytoplasmic bridges, and determinants synthesized in nurse cells are transported through these bridges to the oocyte. However, the Drosophila model may not be applicable to all arthropods, since in many species of all four extant arthropod classes, gametogenesis functions without nurse cells. In this paper, I use immunodetection of Vasa protein to study germ cell development in the amphipod crustacean Parhyale hawaiensis, a species whose ovaries lack nurse cells and whose eggs lack obvious polarity. Previous cell lineage analyses have shown that all three germ layers and the germ line are exclusively specified by third cleavage. In the present study, I use a molecular marker to follow germ cell development during P. hawaiensis embryogenesis. I determine the capacity of individual blastomeres to form germ cells by isolating blastomeres at early cleavage stages and provide experimental evidence for localized germ cell determinants at the two-cell stage in P. hawaiensis. These experiments indicate that many aspects of early amphipod development, including timing and symmetry of cell division, the transition from holoblastic to superficial cleavage, and possibly some gastrulation movements, are cell autonomous following first cleavage.     

Loss of the beta-catenin homologue aardvark causes ectopic stalk formation in Dictyostelium

Aardvark (Aar) is a Dictyostelium beta-catenin homologue with both cytoskeletal and signal transduction roles during development. Here, we show that loss of aar causes a novel phenotype where multiple stalks appear during late development. Ectopic stalks are preceded by misexpression of the stalk marker ST-lacZ in the surrounding tissue. This process does not involve the kinase GSK-3. Mixing experiments show that ectopic ST-lacZ expression and stalk formation are cell non-autonomous. The protein-cellulose matrix surrounding the stalk of aar mutant fruiting bodies is defective, and damage to the stalk of wild-type fruiting bodies leads to ectopic ST-lacZ expression. We postulate that poor synthesis of the stalk tube matrix allows diffusion of a stalk cell-inducing factor into the surrounding tissue.     

Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formation

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.     

Loss of ectodermal competence for lateral line placode formation in the direct developing frog Eleutherodactylus coqui.

In the direct-developing frog Eleutherodactylus coqui neuromasts and ganglia of the lateral line system never develop. We show here that this absence of the lateral line system, which is evolutionarily derived in anurans, is due to very early changes in development. Ectodermal thickenings, which are typical of lateral line placodes, and from which neuromasts and ganglion cells of the lateral line originate, never form in E. coqui, although other neurogenic placodes are present. Moreover, although NeuroD is expressed in the lateral line placodes of Xenopus laevis, corresponding expression sites are lacking in E. coqui. Heterospecific transplantation experiments show that axolotl ectoderm can be induced to form lateral line placodes after transplantation to E. coqui hosts but that E. coqui ectoderm does not form lateral line placodes on axolotl hosts. This suggests that the loss of the lateral line system in E. coqui is due to the specific loss of ectodermal competence to form lateral line placodes in response to inductive signals. Our results (1) indicate that the competence for lateral line placode formation is distinct and dissociable from the competence to form other neurogenic placodes and (2) support the idea that the lateral line system acts as a module in development and evolution.     

Wnt-YAP interactions in the neural fate of human pluripotent stem cells and the implications for neural organoid formation

Human pluripotent stem cells (hPSCs) have shown the ability to self-organize into different types of neural organoids (e.g., whole brain organoids, cortical spheroids, midbrain organoids etc.) recently. The extrinsic and intrinsic signaling elicited by Wnt pathway, Hippo/Yes-associated protein (YAP) pathway, and extracellular microenvironment plays a critical role in brain tissue morphogenesis. This article highlights recent advances in neural tissue patterning from hPSCs, in particular the role of Wnt pathway and YAP activity in this process. Understanding the Wnt-YAP interactions should provide us the guidance to predict and modulate brain-like tissue structure through the regulation of extracellular microenvironment of hPSCs.     

Differentiation of lens- and Iris-like tissue in explants of the anterior part of the frog neural plate

Summary The differentiation was studied of presumptive eye material developing in the absence of ectoderm. Explants were made of the anterior (forebrain- and eye-forming) part of the neural plate, without the lateral neural folds, of early to mid-neurulae ofRana temporaria andR. esculenta. The underlying endomesoderm as well as the outer layer of the neural plate were removed prior to explantation. Consequently the explants did not become surrounded by epidermis. The explants segregated into a mass of forebrain tissue and a single retina, which did not assume the typical cup shape. In between these two components an interzone developed, consisting of incompletely differentiated layers of iris tissue. In the interzone typical lentoids, as well as lentoids continuous with other tissue components, differentiated. The formation of lentoids in the absence of ectoderm is discussed in terms of the availability of a lens-inducing agent. It is assumed that in the interzone the lens-inducing agent acts on tissue components which are competent for lens formation. The formation of lens-like tissue may be regarded as analogous to lens regeneration in newts.The author wishes to express her sincere appreciation to Prof. G. V. Lopashov for his advice and encouragement throughout the course of this study, to Mrs. Nina A. Ivanova for expert technical assistance, and to Dr. J. Faber (Hubrecht Laboratory, Utrecht) for the correction of the English.     

Suillus variegatus causes significant changes in the content of individual polyamines and flavonoids in Scots pine seedlings during mycorrhiza formation in vitro

Changes in the concentrations of individual flavonoids and polyamines (PAs) in Scots pine (Pinus sylvestris L.) cotyledonary seedlings were studied during the establishment of an ectomycorrhizal (ECM) symbiosis with two Suillus variegatus strains in vitro. Both flavonoids and PAs were analysed after 3, 7, and 14 d in dual culture, and changes in concentrations were compared with growth of the seedlings. Both S. variegatus strains caused similar responses in Scots pine seedlings. Free putrescine accumulated immediately but only transiently after inoculation. This was followed by continuous accumulation of PA conjugates in needles and stems, and free spermidine and spermine in roots, which was accompanied by mycorrhiza formation and improved growth. The fungi induced lateral root formation and main root and primary needle elongation. Inoculation caused no qualitative changes in flavonoid composition, while quantitative changes in flavonols, catechins, and condensed tannins were observed in shoots during mycorrhiza formation. These results indicate that in this in vitro system conjugated PAs and specific flavonoids, generally related to the plant's defence reactions, did not play a major role in the regulation of the establishment of the ectomycorrhizal (ECM) symbiosis in Scots pine roots. The results also clearly show that positive growth responses in shoots and roots due to S. variegatus were supported by different and highly specific changes in the synthesis of both primary and secondary metabolites in these parts of the seedling.     

Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate

We correlated available fate maps for the avian neural plate at stages HH4 and HH8 with the progress of local molecular specification, aiming to determine when the molecular specification maps of the primary longitudinal and transversal domains of the anterior forebrain agree with the fate mapped data. To this end, we examined selected gene expression patterns as they normally evolved in whole mounts and sections between HH4 and HH8 (or HH10/11 in some cases), performed novel fate-mapping experiments within the anterior forebrain at HH4 and examined the results at HH8, and correlated grafts with expression of selected gene markers. The data provided new details to the HH4 fate map, and disclosed some genes (e.g., Six3 and Ganf) whose expression domains initially are very extensive and subsequently retract rostralwards. Apart from anteroposterior dynamics, some genes soon became downregulated at the prospective forebrain floor plate, or allowed to identify an early roof plate domain (dorsoventral pattern). Peculiarities of the telencephalon (initial specification and differentiation of pallium versus subpallium) are contemplated. The basic anterior forebrain subdivisions seem to acquire correlated specification and fate mapping patterns around stage HH8.     

Notochordal induction of cell wedging in the chick neural plate and its role in neural tube formation

Cells in the median hinge point (MHP) of the bending chick neural plate are tightly apposed to the underlying notochord. These cells differ from those in adjacent lateral neuroepithelial areas (L) in that MHP cells are short and mainly wedge-shaped and line a furrow, whereas L cells are tall and mainly spindle-shaped and do not line a furrow. Cell generation time also differs in these regions. These consistent differences are detectable only after the notochord has formed and established contact with the neural plate; it is unclear whether they result from self-differentiation or induction. Two experiments were performed to evaluate the hypothesis that MHP characteristics develop owing to inductive interactions between the notochord and overlying neuroepithelial cells. First, notochordless chick embryos were generated to determine whether midline neuroepithelial cells still developed typical MHP characteristics. In the absence of the notochord, such characteristics did not develop. Second, isolated segments of quail notochord were transplanted subjacent to L of chick hosts to ascertain whether the notochord is capable of inducing MHP characteristics in L cells. When transplanted notochordal segments established apposition with host L cells, the apposing L cells usually developed typical MHP characteristics. Collectively, these results provide strong evidence that the notochord plays an inductive role in the formation of MHP characteristics. This investigation further revealed that bending can occur in the absence of MHP characteristics, forming a neural tube with an abnormal morphology. Thus, the formation of such characteristics, particularly cell wedging, is not required for bending but plays a major role in generating the normal cross-sectional morphology of the neural tube.     

Suramin changes the fate of Spemann's organizer and prevents neural induction in Xenopus laevis.

Suramin, a polyanionic compound, which has previously shown to dissociate platelet derived growth factor (PDGF) from its receptor, prevents the differentiation of neural (brain) structures of recombinants of dorsal blastopore lip (Spemann's organizer) and competent neuroectoderm. Furthermore, the suramin treatment changes the prospective differentiation pattern of isolated blastopore lip. While untreated dorsal blastopore lip will differentiate into dorsal mesodermal structures (notochord and somites), suramin treated dorsal blastopore lip will form ventral mesoderm structures, especially heart structures. The results are discussed in the context of the current opinion about the mode of action of different growth factor superfamilies.     

The roles of phenotype and position in guiding the fate of 16-cell mouse blastomeres

Newly formed 16-cell blastomeres were typed as larger or smaller, labelled with the short-term lineage marker FITC, and aggregated in various spatial arrays with 15 other age-matched unlabelled 16-cell blastomeres. The aggregates were cultured for 8 or 24 hr and the fluorescently labelled progeny identified. In all but 6 of 185 cases, the progeny developed as a physically coherent patch. Labelled larger cells placed on the outside of the aggregate generated mainly trophectoderm; when placed on the inside or randomly they always generated at least one trophectodermal offspring and in some cases also contributed cells to the inner cell mass (ICM). Labelled smaller cells placed on the inside of the aggregate generated mainly ICM; when placed on the outside or randomly they generated cells in the ICM alone, in trophectoderm alone, or in both tissues. From these results we conclude that phenotype is of major importance in determining the fate of larger cells whereas position strongly influences the fate of smaller cells.     

Ventricular pump function under ectopic excitation of the frog heart

The ventricular pump function under ectopic excitation of the heart was studied in decapitated and pithed adult frogs Rana temporaria (n = 21) at 18-19 degrees C. The intraventricular pressure was recorded with a catheter via ventricular wall. During pacing of the ventricular base and apex, the systolic pressure decreased (6.1 +/- 4.5 mm Hg and 8.9 +/- 5.0 mm Hg, respectively) as compared to the supraventricular rhythm (8.9 +/- 5.0 mm Hg, p < 0.05). The end-diastolic pressure decreased insignificantly both under basal and apical pacing. The systolic rate of pressure rise during dP/dtmax decreased under ventricular pacing, especially during pacing of the ventricular apex, as compared to the supraventricular rhythm (14.4 +/- 6/9 mm Hg/s and 22.1 +/- 11.2 mm Hg/s, respectively, p < 0.003). The isovolumetric relaxation (dP/dtmin) slowed during apical pacing as compared to the supraventricular rhythm (-25.1 +/- 13.6 and -35.6 +/- 18.3 mm Hg/s, respectively, p < 0.03). Ectopic excitation of the ventricular base and apex resulted in increase of the QRS duration (93 +/- 33 ms and 81 +/- 30 ms, respectively) as compared to the supraventricular rhythm (63 +/- 13 ms, p < 0.05). Thus, pacing of different ventricular areas ventricular myocardium with the ventricular pump function being reduced more obviously during the apical pacing compared to the pacing of ventricular base.     

Quantitative analyses of changes in cell shapes during bending of the avian neural plate

It is widely believed that changes in cell shapes play important roles in the bending or folding of epithelial sheets, but few studies have actually examined cell shapes in such systems. We have determined the percentages of four types of neuroepithelial cells (i.e., spindle, flask, inverted flask, and globular) present during bending of the avian neural plate. Serial transverse plastic sections through seven craniocaudal levels of the neuroepithelium were examined. Four distinct periods of bending were chosen based on the morphology of the neuroepithelium: period I, flat neural plate; period II, midline furrow without elevation of the neural folds; period III, midline furrow with elevation; and period IV, bilateral furrows with convergence of the neural folds. We compared statistically the percentages of different cell types in bending (furrowed) and nonbending regions of the neuroepithelium, as well as changes in cell shapes with time. Our results demonstrate that dramatic changes in cell shapes occur in the midline and bilateral furrows during bending of the neural plate, such that as many as 70% of the neuroepithelial cells in the midline and 55% in the bilateral furrows are wedge shaped by the end of bending. In contrast, less than 35% of the neuroepithelial cells are wedge shaped outside of the three morphological loci of bending. These results support the hypothesis that localized changes in cell morphologies have roles in bending and shaping of the neural plate, but exactly how cells change shapes and what precise roles such changes play in bending remain to be determined.