Region-specific cell clones in the developing spinal cord of the zebrafish

 To analyse the proliferative abilities of cells within particular regions of the zebrafish neural plate, injections of fluorescein-dextran were made into single cells at either medial or intermediary positions in the neural plate region of two-somite stage embryos. The resulting cell clones were analysed in 3.5-day-old embryos. Clones with similar compositions were found among those derived from injections in both regions, and these were grouped into classes. 78 clones 29 obtained following injections in the medial region, and 22 of 59 cell clones derived from injections in the intermediary region, were classifiable into 9 and 10 different classes, respectively, each comprising a variable number of clones. Several identified cell types, as well as each of the clone classes themselves, were specific for the regions of the neural plate from which they derived, i.e. they were not represented among the clones derived from the other region. These results suggest that the composition of the lineages derived from particular cells is constant in different animals. Received: 13 July 1998 / Accepted: 20 October 1998     

Expression of protocadherin 18 in the CNS and pharyngeal arches of zebrafish embryos

Here, we report the results of molecular cloning and expression analyses of a non-clustered protocadherin (pcdh), pcdh18 in zebrafish embryos. The predicted zebrafish pcdh18 protein shows 6566% identity and 7879% homology with its mammalian and Xenopus counterparts. It has a Disabled-1 binding motif in its cytoplasmic domain, which is characteristic of pcdh18. Zebrafish embryos expressed pcdh18 by the early gastrula stage, 6 h post-fertilization (hpf), in their animal cap but not in the germ ring or the shield. pcdh18 was expressed in the neural tube and the central nervous system (CNS) from 12 hpf. Some populations of cells in the lateral neural tube and spinal cord of 1218 hpf embryos expressed pcdh18, but expression in these cells disappeared by 24 hpf. The hindbrain of embryos at 2456 hpf expressed pcdh18 in cells closely adjacent to the rostral and caudal rhombomeric boundaries in a thread-like pattern running in the dorsoventral direction. The pcdh18-positive cells were localized in the ventral part of the hindbrain at 24 hpf and in the dorsal part from 36 hpf. pcdh18 was also expressed in the telencephalon, diencephalon, tectum, upper rhombic lip, retina and otic vesicle. Expression in the CNS decreased markedly before hatching. Pharyngeal arch primordia, arches, jaws and gills expressed pcdh18, and the molecule was also expressed in some endodermal cells in late embryos.     

Segmental lineage restrictions in the chick embryo spinal cord depend on the adjacent somites.

We have investigated whether the developing spinal cord is intrinsically segmented in its rostrocaudal (anteroposterior) axis by mapping the spread of clones derived from single labelled cells within the neural tube of the chick embryo. A single cell in the ventrolateral neural tube of the trunk was marked in situ with the fluorescent tracer lysinated rhodamine dextran (LRD) and its descendants located after two days of further incubation. We find that clones derived from cells labelled before overt segmentation of the adjacent mesoderm do not respect any boundaries within the neural tube. Those derived from cells marked after mesodermal segmentation, however, never cross an invisible boundary aligned with the middle of each somite, and tend to be elongated along the mediolateral axis of the neural tube. When the somite pattern is surgically disturbed, neighbouring clones derived from neuroectodermal cells labelled after somite formation behave like clones derived from younger cells: they no longer respect any boundaries, and are not elongated mediolaterally. These results indicate that periodic lineage restrictions do exist in the developing spinal cord of the chick embryo, but their maintenance requires the presence of the adjacent somite mesoderm.     

Role of interleukin-1beta in the control of neuroepithelial proliferation and differentiation of the spinal cord during development

Interleukin-1beta (IL-1beta) is an important trophic factor in the nervous system (NS). IL-1beta is ubiquitously expressed from very early stages during the development of the amphibian NS and its action has been demonstrated in vitro on survival, proliferation and differentiation in mammalian embryos. In this report, we show that IL-1beta is immunocytochemically expressed in embryonic spinal cord from early stages, both in rat (embryonic day 12) and in chicken (stage 17-HH), in neuroepithelial cells and nerve fibres, dorsal root ganglia, anterior and posterior roots of the spinal nerves, and in the fibres of these nerves. Our in vivo experiments on chick embryos, with microbeads impregnated with IL-1beta implanted laterally to the spinal cord at the level of the wing anlage, demonstrate that this cytokine produces a statistically significant increase in nuclear incorporation of BrdU at the dorsal level and a reduction of this at the ventral level, whereas local immunoblocking with anti-IL-1beta antibodies causes a dorsal reduction of BrdU incorporation and alters ventral differentiation. These data demonstrate that IL-1beta plays a part in controlling proliferation and early differentiation during the development of the spinal cord in chick embryos.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Distinct expression patterns of two zebrafish homologues of the human APP gene during embryonic development

The human amyloid protein precursor (APP) gene correlates with early onset of Alzheimer's disease in humans. We have identified two APP homologues in zebrafish, which we call appa and appb. They show a high degree of identity to human APP particularly in the beta APP42 and the transmembrane domain. Widespread expression of both appa and appb was detected from mid-gastrulation until the bud stage. During segmentation, the two genes diverged in their pattern of expression: at 14 h post-fertilisation (hpf) and 18 hpf both genes were expressed rostrally in the prospective CNS, but only appa was found caudally in the paraxial segmental plate and presomitic mesoderm, excluding the midline. In contrast, appb was found caudally in the neural rod at 14 hpf and the developing spinal cord at 18 hpf. Later, at 24 hpf both genes shared common expression domains, namely the telencephalon, the ventral diencephalon, the trigeminal ganglia, and the posterior lateral line ganglia. Unique expression domains for appa were the lens, the otic vesicles and the somites, while appb was expressed in a serially repeated set of nuclei within the hindbrain, the ventral mesencephalon and the motoneurones of the developing spinal cord.     

Cell lineage analysis of neural induction: origins of cells forming the induced nervous system

Horseradish peroxidase (HRP) was used as an intracellular lineage tracer in two experiments designed to reveal the sites of origin of cells that formed the duplicate embryo which developed in relation to an organizer grafted in the ventral marginal zone (VMZ) of Xenopus laevis embryos. In the first experiment a dorsal blastoporal lip fully labeled with HRP was grafted in the VMZ of an unlabeled embryo at the beginning of gastrulation. This resulted in development of a second embryo in which labeled cells, of graft origin, formed the notochord, and parts of the somites, endoderm, and neural tube. The second experiment was designed to show the sites of origin of the host's cells that formed parts of the induced embryo. HRP was injected into individual blastomeres in a series of Xenopus embryos at the 32-cell stage and each embryo received an unlabeled organizer graft in the VMZ at the beginning of gastrulation. In these embryos the lineages that contributed to the host's primary neural tube did not contribute any cells to the induced neural tube. All the cells in the induced neural tube which originated from the host were descendants of ventral blastomeres that did not contribute to the neural tube normally. This shows that the second neural tube is formed as a result of the action of the organizer on cells in its immediate vicinity which would not normally have entered neural pathways of differentiation.     

Regulation in the neural plate of Xenopus laevis demonstrated by genetic markers

To follow the subsequent history of grafted tissue in experiments designed to study regulation and commitment in the amphibian neural plate, previous workers have relied on graft scars, vital dyes applied externally to cells, or xenoplastic grafts. Each of these methods has been criticized on the grounds that they do not indicate unambiguously the origins of individual cells within the operated host. To overcome these difficulties, homoplastic, genetically marked embryonic grafts were taken from the prospective spinal neuroectoderm of triploid and tetraploid Xenopus laevis frogs and transplanted to presumptive eye and prosencephalic regions of the neural plate of diploid X. laevis embryos. Orthotopic presumptive eye grafts also were done. Marked cells were scored in section either by nucleolar number or computerized nuclear size analysis. Of 28 heterotopically grafted embryos that survived to stage 41, when the retina has differentiated, prospective spinal cord neuroectoderm in eight animals gave rise to cell types unique to the eye. The remaining 20 survivors appeared to be mosaic. These results substantiate claims of regulation in the neural plate and extend these observations to the level of individual cell types, a level of resolution not previously obtained in other studies.     

F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension.

The floor plate is a cell group implicated in the control of neural cell pattern and axonal growth in the developing vertebrate nervous system. To identify molecules that might mediate the functions of the floor plate, we have used subtractive hybridization techniques to isolate floor plate-enriched cDNA clones. One such clone encodes a novel secreted protein, F-spondin, which is expressed at high levels in the floor plate. The C-terminal half of the protein contains six repeats identified previously in thrombospondin and other proteins implicated in cell adhesion. F-spondin is expressed in the floor plate at the time that axons first extend and at lower levels in the peripheral nerve. Recombinant F-spondin promotes the attachment of spinal cord and sensory neuron cells and the outgrowth of neurites in vitro. F-spondin may contribute to the growth and guidance of axons in both the spinal cord and the PNS.     

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells

Background aimsThe widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2⁺ populations in vitro and in vivo.MethodsTwenty-four clones from embryonic cerebral cortex-derived NG2⁺ cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2⁺ clones into the spinal cord was used to examine their lineage potential in vivo.ResultsIn vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal–glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environmentConclusionsThese results suggest that NG2⁺ cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2⁺ cell lines might provide a cell source for treating spinal cord disorders.     

Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish

In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 h post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.     

Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

Time course of the development of motor behaviors in the zebrafish embryo

The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5°C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 622–632, 1998     

Lineage of neurons and glia in chick dorsal root ganglia: analysis in vivo with a recombinant retrovirus

We used retrovirus-mediated gene transfer to study the lineage of neural crest cells in chick embryos. Individual crest cells were infected before they migrated from the neural tube, and their clonal progeny were subsequently revealed in sensory ganglia and associated structures by a histochemical stain for the viral gene product (lacZ). We found that crest cells were multipotential in several respects. (1) Many clones contained both ventrolateral (VL) and dorsomedial (DM) neurons, which had been suggested to be lineally distinct. (2) Many clones contained both large and small neurons, which are known to innervate distinct targets. (3) Many clones contained multiple glial subtypes, e.g. both Schwann cells, which ensheath axons, and satellite cells, which ensheath neuronal somata. (4) Many clones contained both neurons and glial cells. On the other hand, a sizeable minority of clones was homogenous, e.g. they contained only neurons or only glial cells--suggesting that some progenitors may be, or become, restricted in potential. Finally, this study provides the first opportunity to compare directly the two methods currently available for tracing cell lineage in vertebrate embryos, retroviral infection and tracer injection: our results and those of Bronner-Fraser and Fraser (1989), who used the latter method, provide complementary but consistent views of crest lineage.     

Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.     

Development and characterization of commissural interneurones in the spinal cord of Xenopus laevis embryos revealed by antibodies to glycine

By using an antibody to glutaraldehyde fixation products of glycine we have been able to observe the development of a defined population of spinal interneurones in the CNS of Xenopus laevis embryos. The first glycine immunoreactive (GLY) somata appeared at stage 22 in the caudal hindbrain within a few hours of neural tube closure. The population then increased by extending caudally into the spinal cord and by infill. It was followed up to the time of hatching, stage 37/38. By observing GLY cells at early stages in their differentiation, the normal sequence of cell process formation was deduced. A ventral axon is formed, extends dendrites laterally into the marginal zone and forms a commissure by growing through the ventral ependymal cell floor of the neural tube. On the opposite side, growth cones turn longitudinally and TEM observations show that they make en-passant synaptic contacts. All GLY cells have decussating axons and some grow secondary axons on the same side as the soma. To establish the identity of GLY cells, a detailed comparison was made with commissural and dorsolateral commissural interneurones defined by retrograde and intracellular HRP staining. The GLY cells are identified with the commissural interneurones which are known to serve a glycinergic reciprocal inhibitory function. By showing that these interneurones have a clearly defined group identity and programme of development, this study opens the way to further experiments on factors controlling spinal cord pathway determination.     

Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.