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.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary Some aspects of neural and epidermal cell lineages during embryogenesis of Drosophila melanogaster were studied by transplanting horseradish-peroxidase-(HRP-) labelled ectodermal cells from young gastrula donors into host embryos of similar ages. Heterotopic transplantations permitted us to assess the degree of commitment already attained by the transplanted cells. The resulting cell clones showed normal characteristics of cytodifferentiation and cell number. The results indicate that epidermal progenitors perform a maximum of three mitoses during embryonic development, whereas neuroblasts may perform more than ten mitoses. Clone size distribution is in both cases scattered, suggesting either a rather irregular mitotic pattern or cell death. As indicated by heterotopic transplantations, the neurogenic ectoderm for the ventral nervous system exhibits different neurogenic abilities in its different regions, decreasing from medial to lateral; we discuss the hypothesis that some medially located cells of the young gastrulating embryo could be committed towards the neural fate before segregating from the ectoderm. On the other hand, the cells of the dorsal ectodermal regions at the same stage seem to be indifferent with respect to commitment, for they are able to give rise to central neural lineages following their transplantation in the neurogenic region.     

The neuroblast of the grasshopper embryo as a new mutagen test system: I. In vitro radiosensitivity

The neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer) are being studied to determine their suitability for detecting environmental clastogens (chromosome-breaking agents). They are very sensitive to the induction of chromosome breakage by radiation in vivo. Their sensitity, 0.011 break / cell / R, is 4–5 times higher than pollen mother cells of Tradescantia (micronuclei), 10 times higher than either human lymphocytes or Chinese hamster cells (metaphase chromosome aberrations), and 15 times higher than mouse than mouse erythroblasts (micronuclei). Furthermore, they have no spontaneous chromosome breakage, which facilitates the detection of agents that break chromosomes. The present study shows that Chortophaga embryos maintain normal mitotic activity in vitro for 5 cell cycles at 38°C (20 h), and that neuroblasts of embryos grown in vitro have the same radiosensitivity as those of embryos in vivo. Thus in vitro exposure of grasshopper embryos is a promising method for obtaining data on the response of neuroblasts to chemical clastogens.     

The migratory pathway of neural crest cells into the skin of mouse embryos

The migration of neural crest derived melanoblasts into the skin of mice was studied by the ectoderm-mesoderm recombination technique. Dorsolateral skin from albino and black mouse embryos at the time of initial melanoblast invasion was separated into ectoderm and mesoderm components, recombined with each other, and grown in the chick embryo coelom for a sufficient period to allow melanin formation. Recombined skin from embryos 11 days old formed pigment only when the mesodermal component was from a genetically black embryo. The black ectoderm-albino mesoderm recombinations failed to produce pigment in all cases. At this critical age when melanoblasts were first entering the skin, they were present exclusively in the mesodermal component. Skin recombinations made from 12-day mouse embryos showed a spread of melanoblasts into the ectodermal component, and by 13 and 14 days both dermal mesoderm and epidermal ectoderm were populated by melanoblasts.     

Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis

Rohon-Beard mechanosensory neurons (RBs), neural crest cells, and neurogenic placodes arise at the border of the neural- and non-neural ectoderm during anamniote vertebrate development. Neural crest cells require BMP expressing non-neural ectoderm for their induction. To determine if epidermal ectoderm-derived BMP signaling is also involved in the induction of RB sensory neurons, the medial region of the neural plate from donor Xenopus laevis embryos was transplanted into the non-neural ventral ectoderm of host embryos at the same developmental stage. The neural plate border and RBs were induced at the transplant sites, as shown by expression of Xblimp1, and XHox11L2 and XN-tubulin, respectively. Transplantation studies between pigmented donors and albino hosts showed that neurons are induced both in donor neural and host epidermal tissue. Because an intermediate level of BMP4 signaling is required to induce neural plate border fates, we directly tested BMP4′s ability to induce RBs; beads soaked in either 1 or 10 ng/ml were able to induce RBs in cultured neural plate tissue. Conversely, RBs fail to form when neural plate tissue from embryos with decreased BMP activity, either from injection of noggin or a dominant negative BMP receptor, was transplanted into the non-neural ectoderm of un-manipulated hosts. We conclude that contact between neural and non-neural ectoderm is capable of inducing RBs, that BMP4 can induce RB markers, and that BMP activity is required for induction of ectopic RB sensory neurons.     

RNA biosynthesis during differentiation of various cell types of chicken embryo in cerebral hemispheres

Summary During the development of the chick embryo from the 6th to the 15th day of incubation, the cell types in cerebral hemispheres undergo differentiation. During this period the indifferent cells of the germinal layer migrate away from the neural cavity to form the mantle layer. These cells differentiate into neuroblasts and spongioblasts.RNA biosynthesis is very active in the cells of the germmal layer of the young embryos. From the 10th day on, it decreased becoming very weak in the 15-days old embryos. The RNA is stored in the nucleus and its passage to cytoplasm is very slow.In 6 and 8-days old embryos the RNA biosynthesis in the mantle layer is not very active but increases during embryonic development as the germinal cells differentiate. The biosynthesis is always more intense in the neuroblasts than in the spongioblasts. The RNA is stored in the nucleus and its passage to cytoplasm is slow in the young neuroblasts and the spongioblasts. The formation of Nissl bodies in neuroblasts and the differentiation of neuroblasts into neurons, which corresponds to the development of axons and dendrites, both are accompanied by an activation of the RNA passage from the nucleus into the cytoplasm.With the technical assistance of A. Brossard.     

Conversion of ectoderm into a neural fate by ATH-3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal.

We have isolated a novel basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed ATH-3, from Xenopus and mouse. ATH-3 is expressed in the developing nervous system, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH-3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non-neural development. This ATH-3-induced neural hyperplasia does not require cell division, indicating that surrounding cells which are normally non-neural types adopt a neural fate. In a Xenopus animal cap assay, ATH-3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker-inducing ability without affecting general neurogenic activities. These results provide evidence that ATH-3 can directly convert non-neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH-3 activity by phosphorylation.     

Complexity of nuclear and polysomal RNAs in growing Dictyostelium discoideum cells

The proliferative activity of undifferentiated brain cells from either 5- or 7-day-old chick embryos has been investigated by labeling the cells with a 24-hr pulse label of [¹⁴C]- or [³H]-thymidine during the early stages (0 to 8 days) of culture. As soon as the neurons and the glial cells could be distinguished (after 4, 7, or 14 days of culture), the cultures were prepared and submitted to the activated autoradiographic method. In some experiments a continuous labeling was applied up to 2 weeks. During the first 48 hr of culture, and for both embryonic ages studied, nearly all neuronal precursors were able to proliferate. After 4 days in culture for the 7-day-old embryo and 7 days in culture for the 5-day-old embryo most of the neuronal cells stopped dividing. These two culture periods correspond to the stage of the embryonic life when the end of the mitotic activity of neuroblasts occurs in vivo. Thus, the proliferation and development in culture of most neuroblasts was found to parallel the in vivo evolution of these cells. Some neuroblasts, however, continued to multiply in vitro for a longer period of time. The astroblasts precursors were found to multiply actively from the 3rd day on, or immediately from time zero, for the 5- and 7-day-old chick embryos, respectively. These observations seem to indicate that the astroblast precursors are in a latent stage until they have reached Day 7. Thereafter, they proliferate actively during the first week of culture and therefore remain in an embryonic stage during this culture period. This fact corresponds also to the in vivo situation, where the glial cell precursors multiply actively around the same time period.     

Male Killing Spiroplasma Preferentially Disrupts Neural Development in the Drosophila melanogaster Embryo

Male killing bacteria such as Spiroplasma are widespread pathogens of numerous arthropods including Drosophila melanogaster. These maternally transmitted bacteria can bias host sex ratios toward the female sex in order to ‘selfishly’ enhance bacterial transmission. However, little is known about the specific means by which these pathogens disrupt host development in order to kill males. Here we show that a male-killing Spiroplasma strain severely disrupts nervous tissue development in male but not female D. melanogaster embryos. The neuroblasts, or neuron progenitors, form properly and their daughter cells differentiate into neurons of the ventral nerve chord. However, the neurons fail to pack together properly and they produce highly abnormal axons. In contrast, non-neural tissue, such as mesoderm, and body segmentation appear normal during this time, although the entire male embryo becomes highly abnormal during later stages. Finally, we found that Spiroplasma is altogether absent from the neural tissue but localizes within the gut and the epithelium immediately surrounding the neural tissue, suggesting that the bacterium secretes a toxin that affects neural tissue development across tissue boundaries. Together these findings demonstrate the unique ability of this insect pathogen to preferentially affect development of a specific embryonic tissue to induce male killing.     

Development of phenobarbital-sensitive control mechanisms for uridine diphosphate glucuronyltransferase activity in chick embryo liver

Uridine diphosphate (UDP) glucuronyltransferase activity in chick liver rises at hatching from near zero to adult levels. This rise will occur prematurely in embryo liver during organ culture. Increase in enzyme activity during organ culture differs with embryo age: in liver from 11-day old embryos it ceases at adult values; in liver from 5-day old embryos it continues to much higher-than-adult levels. Phenobarbital added to culture medium accelerates these rises in enzyme activity and elevates the plateau reached in 11-day embryo liver to that observed in 5-day embryo liver. Kinetic analysis of the changes in enzyme activity induced by phenobarbital during culture suggests that the regulatory mechanisms for enzyme activity are different in 5- and 11-day embryo liver and that these differences reflect developmental changes occurring in ovo.     

Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts

By immunizing mice with cells from embryonic chick motoneuron cultures, an antiserum was produced which recognizes an antigen(s) restricted to cell surfaces of most, or all, neurons. With the use of this antiserum, the appearance of neuron-specific antigenicity in cells of the embryonic spinal cord was examined by indirect immunofluorescence microscopy. The antigen or set of antigens reacting with this antiserum was first detectable in the neural tube of chick embryos at stage 15–16 (V. Hamburger and H. L. Hamilton, 1951, J. Morphol.88, 49–92). In addition to the neuroblasts located in the mantle layer, some mitotic cells as well as some spindle-shaped cells in the germinal layer were antigen positive. Immunofluorescence microscopy combined with autoradiography revealed that none of the antigen-positive cells could be labeled with [³H]thymidine; thus they do not synthesize DNA, and none of the cells in the DNA synthetic phase expressed the antigen(s). As the neuroblasts do not synthesize DNA after they have differentiated from the germinal cells, we believe that the antigen-positive cells are differentiated elements and that the differentiation of membranes specific for neurons begins already before or during the terminal mitosis of cells which will be defined as neuroblasts.     

Histogenesis and dependent glutamine synthetase inducibility in embryonic neural retina. Irreversible inhibition of differentiation by 5-bromodeoxyuridine

Young, mitotically active neural retinas from 7-day chick embryos were cultured with 5-bromodeoxyuridine (BrdU) for 8 hr or more. After this treatment, they failed to differentiate beyond the stage at which they were explanted; there was no histogenesis or increase in glutamine synthetase (GS) inducibility in intact tissues or in aggregates of dissociated cells. Normally GS can be induced in the retina with hydrocortisone as the cells cease to be mitotically active and begin showing histological organization after day 7. This inhibition by BrdU was irreversible even in the presence of excess thymidine. Overall incorporation of ¹⁴C-amino acids into protein was only slightly inhibited, and the ability of cells from treated tissue to aggregate and sort out from nonneural cell types was unaffected. Control cultures without BrdU showed considerable histogenesis and a parallel increase in enzyme inducibility. Postmitotic 10-day retinas appeared to be unaffected by BrdU. The incorporation rates of both tritiated BrdU and thymidine (dT) into DNA were 14× higher in 7- than in 10-day retinas. Simultaneous addition of excess unlabeled dT with either of the labeled nucleosides reduced their incorporation and reduced the inhibitory action of BrdU on differentiation.It is concluded that BrdU irreversibly inhibits the differentiation of retina cell surface properties involved in histogenesis and dependent cytodifferentiation without affecting already differentiated properties of the cell surface. The results support the hypothesis that histogenesis is directed by genes affecting specific cell surface properties.     

Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development

The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin–myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues.     

Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo

$\text{T}\text{T}$ embryos have been first distinguishable at 8 days post coitum by their gross morphological abnormalities. By quantitative morphometry of histological sections, anomalies in the homozygotes were expressed numerically. At 8 days p.c., morphologically identifiable T-homozygotes had an increased number of ectodermal and a reduced number of mesodermal cells compared to the wild type. At 7 days p.c., embryos with a low mesoderm/ectoderm ratio were found only in litters of $\text{T}\text{+}\text{×}\text{T}\text{+}$ matings at the expected frequency. At 6 days p.c., one-fourth of the embryos in $\text{T}\text{+}\text{×}\text{T}\text{+}$ litters showed a delay in the transition from cuboidal to squamous endoderm. No such embryos were found in the +/+ × +/+ matings. In 6-, 7-, and 8-day mutant embryos, cells proliferated at statistically normal rates. Therefore, it may be said that advanced morphological irregularities of 8-day homozygotes cannot be accounted for by anomalies in cell proliferation. When the total cell number was 5 × 10⁴/embryo (8 days), a sudden change was observed in the regional distribution of mesodermal and ectodermal cells along the anteroposterior axis of $\text{T}\text{T}$ embryos. Since no regional difference in the cell cycle time was observed, these abnormalities may best be explained by anomalies in cell migration. These results strongly suggest abnormal morphology of $\text{T}\text{T}$ mutants resulting from defects in morphogenetic movement.     

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.     

Effect of the notochord on proliferation and differentiation in the neural tube of the chick embryo

After implantation of a notochord fragment lateral to the neural tube in a 2-day chick embryo, at 4 days the ipsilateral neural tube half was increased in size and axons left the neural tube in a broad dorsoventral area (van Straaten et al. 1985). This enlargement appears to coincide with an increased area of AChE-positive basal plate neuroblasts, as determined with scan-cytophotometry. The effect was ipsilateral and local: clear effects were seen only when the implant was localized less than 80 microns from the neural tube and over 120 microns from the ventral notochord. In order to investigate the expected enhancement of proliferation, the mitotic density and the number of cells at the site of the implant at 3 days was determined and the mitotic index calculated. All three parameters showed an increase. It was concluded that the cell cycle was shorter in the implant area relative to the control area, at least during the third day. At 4 days the number of cells was still increased, predominantly in the basal plate. It appeared that the numerical increase was for the larger part due to neuroblasts. The synergism of two notochords thus resulted in enhancement of proliferation and differentiation in the neural tube. It is suggested that the notochord merely regulates and arranges the surrounding sclerenchymal cells, which are the effective cells in the regulation of neural tube development.     

Persistent Nucleoli in Early Postimplantation of Rat Embryos

Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely.     

Mitotic sympathetic neuroblasts initiate axonal pathfinding in vivo.

Neuronal precursor proliferation and axodendritic outgrowth have been traditionally regarded as discrete and sequential developmental stages. However, we recently found that sympathetic neuroblasts in vitro often elaborate long neuritic processes before dividing. Furthermore, these "paramitotic" neurites were maintained during cell division and neuritic morphology was consistently preserved by daughter cells after mitosis. This inheritance of neuritic morphology in vitro raised the possibility that proliferating neuroblasts engage in axodendritic outgrowth. To determine whether mitotic superior cervical ganglion (SCG) neuroblasts are engaged in pathfinding in vivo, we have combined retrograde axonal tracing of efferent nerve trunks with bromodeoxyuridine (BrdU) labeling of cells in S-phase. In fact, about 13% of BrdU(+) cells were retrogradely labeled, indicating that mitotic neuroblasts often have extraganglionic axonal projections. Moreover, the presence of axons during S-phase was observed at two developmental ages (E15.5 and E16. 5), implicating an ongoing function of paramitotic axons during neuronal ontogeny. Using a calculation to account for experimental limitations, we estimate that virtually all mitotic SCG neuroblasts have direct access to extraganglionic signals during development. We conclude that mitotic neuronal precursors in vivo engage in pathfinding, raising the possibility that interaction of proliferating populations with distant signals actively coordinates cell division and neural connectivity.