Xenografts of embryonic nerve tissue from Drosophila neuromutants stimulate development of neural homografts in rat brain and block glial scar formation

The influence of xenografts of Drosophila melanogaster embryonic nerve cells on the development of embryonic neurohomografts in the adult rat brain has been investigated. Embryonic nerve cells, marked with bacterial galactosidase gene (lacZ) from D. melanogaster strain with a mutation in the Delta locus, were transplanted into adult rat brain. Drosophila cells were easily identifiable in brain histological sections by X-gal staining. Xenografts survived for at least 2-3 weeks in the recipient brain after the operation to be subsequently attacked by macrophages. Importantly, no glial scar was formed around the xenograft. The addition of Drosophila embryonic nerve cells to a homograft of rat embryonic neural tissue facilitated the survival and development of this homograft by blocking the glial scar formation, stimulating vascularization of the graft area and differentiation of the implanted embryonic nerve cells.     

Effect of the foreign gene GDNF on development of homo- and xenografts in the rat brain

A transgenic line of Drosophila melanogaster was selected which carried the following genes: Delta, lacZ (for bacterial galactosidase), and human GDNF (for glial cell line-derived neurotrophic factor). Drosophila neuroectodermal embryonic cells were transplanted with the embryonic neurohomografts into the occipital brain region of an adult rat. Xenografts were found to block scar formation at the graft-host tissue boundary, stimulated homograft development (so that it was twice as large as the control homograft transplanted alone with no xenograft added), and noticeably improved vascularization of the homograft area.     

Hsp70 family proteins seem to facilitate allo- and xenotransplantation in the rat brain

Drosophila neuroectodermal embryonic cells were transplanted into the occipital brain region of adult rats. The first series of experiments used a transgenic strain expressing lacZ to detect the presence of Drosophila cells. The second series used a strain carrying a is lethal (ts403) in the X chromosome; this mutation strongly inhibits the synthesis of heat shock proteins (hsps) and their transport into the nuclei. Immunostaining reveals a strong induction of hsp70 in the xenografts in the first series of experiments, in which no glial scar was detectable. By contrast, where the ts mutation was xenotransplanted, the condition of xenografts was worse, and a glial scar was readily evident between the xenograft and host tissue.     

Survival of embryonic amygdala tissue grafted into various parts of the adult rat brain

It was found during the course of histological examination of preparations containing Nissl and Golgi stained neurons that portions of the embryonic amygdala can successfully survive in the intact adult rat brain. A number of parameters were used enabling development of the graft to be assessed objectively: parenchymal integration index, growth potential, cell density, and vascularization index. By comparing qualitative and quantitative findings of our analyses we showed that location within the host brain is one of the principal factors determining success in graft survival. Grafts transplanted into the cortex survived least well, ventricular cavity transplants fared better, and optimum results were observed with tissue grafted onto the subcortical structures. Normal nerve and glial cells were differentiated in grafts which had taken successfully: capillaries grew into the grafted tissue and common neuropil formed between the graft and the host brain. Structural integration between donor tissue and host brain provides a good model for studying both functional interaction and recovery of function impaired by damage to the host amygdala.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 5, pp. 606–612, September–October, 1987.     

Drosophila melanogaster brain invasion: pathogenic Wolbachia in central nervous system of the fly

The pathogenic Wolbachia strain wMelPop rapidly over‐replicates in the brain, muscles, and retina of Drosophila melanogaster, causing severe tissue degeneration and premature death of the host. The unique features of this endosymbiont make it an excellent tool to be used for biological control of insects, pests, and vectors of human diseases. To follow the dynamics of bacterial morphology and titer in the nerve cells we used transmission electron microscopy of 3‐d‐old female brains. The neurons and glial cells from central brain of the fly had different Wolbachia titers ranging from single bacteria to large accumulations, tearing cell apart and invading extracellular space. The neuropile regions of the brain were free of wMelPop. Wolbachia tightly interacted with host cell organelles and underwent several morphological changes in nerve cells. Based on different morphological types of bacteria described we propose for the first time a scheme of wMelPop dynamics within the somatic tissue of the host.     

Stimulating axonal regeneration of mature retinal ganglion cells and overcoming inhibitory signaling

Like other neurons of the central nervous system (CNS), retinal ganglion cells (RGCs) are normally unable to regenerate injured axons and instead undergo apoptotic cell death. This regenerative failure leads to lifelong visual deficits after optic nerve damage and is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as to an insufficient intrinsic ability for axonal regrowth. In addition to its ophthalmological relevance, the optic nerve has long been used as a favorable paradigm for studying regenerative failure in the CNS as a whole. Findings over the last 15 years have shown that, under certain circumstances, mature RGCs can be transformed into an active regenerative state enabling these neurons to survive axotomy and to regenerate axons in the optic nerve. Moreover, combinatorial treatments overcoming the inhibitory environment of the glial scar and optic nerve myelin, together with approaches activating the intrinsic growth program, can further enhance the amount of regeneration in vivo. These findings are encouraging and open the possibility that clinically meaningful regenerationmay become achievable in the future.     

Commissure formation in the embryonic CNS of Drosophila.

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. In later stages, midline glial cells are required to separate anterior and posterior commissures into distinct axon bundles. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, we conducted a saturating ethylmethane sulfonate mutagenesis, screening for mutations which disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. In addition to commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene, two gene functions are required for the initial formation of commissural tracts. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures.     

The scoop on the fly brain: glial engulfment functions in Drosophila

Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.     

Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery after spinal cord injury in mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.     

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ß-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.     

Segment-specific requirements for dorsoventral patterning genes during early brain development in Drosophila.

An initial step in the development of the Drosophila central nervous system is the delamination of a stereotype population of neural stem cells (neuroblasts, NBs) from the neuroectoderm. Expression of the columnar genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh) subdivides the truncal neuroectoderm (primordium of the ventral nerve cord) into a ventral, intermediate and dorsal longitudinal domain, and has been shown to play a key role in the formation and/or specification of corresponding NBs. In the procephalic neuroectoderm (pNE, primordium of the brain), expression of columnar genes is highly complex and dynamic, and their functions during brain development are still unknown. We have investigated the role of these genes (with special emphasis on the Nkx2-type homeobox gene vnd) in early embryonic development of the brain. We show at the level of individually identified cells that vnd controls the formation of ventral brain NBs and is required, and to some extent sufficient, for the specification of ventral and intermediate pNE and deriving NBs. However, we uncovered significant differences in the expression of and regulatory interactions between vnd, ind and msh among brain segments, and in comparison to the ventral nerve cord. Whereas in the trunk Vnd negatively regulates ind, Vnd does not repress ind (but does repress msh) in the ventral pNE and NBs. Instead, in the deutocerebral region, Vnd is required for the expression of ind. We also show that, in the anterior brain (protocerebrum), normal production of early glial cells is independent from msh and vnd, in contrast to the posterior brain (deuto- and tritocerebrum) and to the ventral nerve cord.     

Neuroblast formation and patterning during early brain development in Drosophila

The Drosophila embryo provides a useful model system to study the mechanisms that lead to pattern and cell diversity in the central nervous system (CNS). The Drosophila CNS, which encompasses the brain and the ventral nerve cord, develops from a bilaterally symmetrical neuroectoderm, which gives rise to neural stem cells, called neuroblasts. The structure of the embryonic ventral nerve cord is relatively simple, consisting of a sequence of repeated segmental units (neuromeres), and the mechanisms controlling the formation and specification of the neuroblasts that form these neuromeres are quite well understood. Owing to the much higher complexity and hidden segmental organization of the brain, our understanding of its development is still rudimentary. Recent investigations on the expression and function of proneural genes, segmentation genes, dorsoventral-patterning genes and a number of other genes have provided new insight into the principles of neuroblast formation and patterning during embryonic development of the fly brain. Comparisons with the same processes in the trunk help us to understand what makes the brain different from the ventral nerve cord. Several parallels in early brain patterning between the fly and the vertebrate systems have become evident.     

Regulation of glial cell number and differentiation by ecdysone and Fos signaling

In the midline glia of the embryonic ventral nerve cord of Drosophila, differentiation as well as the subsequent regulation of cell number is under the control of EGF-receptor signaling. During pupal stages apoptosis of all midline glial cells is initiated by ecdysone signaling. In a genetic screen we have identified mutations in disembodied, rippchen, spook, shade, shadow, shroud and tramtrack that all share a number of phenotypic traits, including defects in cuticle differentiation and nervous system development. Some of these genes were previously placed in the so-called 'Halloween-group' and were shown to affect ecdysone synthesis during embryogenesis. Here we demonstrate that the Halloween mutations not only affect glial differentiation but also lead to an increase in the number of midline glial cells, suggesting that during embryogenesis ecdysone signaling is required to adjust glial cell number similar to pupal stages. Finally we isolated a P-element-induced mutation of shroud, which controls the expression of ecdysone inducible genes. The P-element insertion occurs in one of the promoters of the Drosophila fos gene for which we present a yet undescribed complex genomic organization. The recently described kayak alleles affect only one of the six different Fos isoforms. This work for the first time links ecydsone signaling to Fos function and shows that during embryonic and pupal stages similar developmental mechanisms control midline glia survival.     

Behavior and Differentiation of the Neural Stem Cells in vivo

We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.     

Nerve cells of Drosophila Notch mutant are differentiated inside amphibian brain: a new approach for the analysis of genetic control of nerve cell differentiation

Fragments of the neural primordium of a new Notch mutant of Drosophila melanogaster produced in our laboratory were transplanted into the neural tube of embryos of 4 amphibian species (caudate and ecaudate) immediately after completion of neurulation. The grafts were identified by using a light microscope, scanning electron miscroscope, and in situ hybridization with mobile genetic elements of Drosophila and fluorescent dyes as markers. As has been shown, Drosophila nerve cells survive and differentiate inside the neural tube of amphibian embryos. The grafts increase in size by twentyfold and the cell proliferation zones are retained during the period of six months. Differentiated cells of the graft formed axon-dendritic contacts with recipient cells and penetrated into the organisms' brain structures. The effect of Drosophila transplants proved to be different for caudate and ecaudate amphibians. The presence of the graft accelerated the development of Xenopus laevis and it also affected their behavior. This approach can be very useful for the study of genetic basis of development and behavior.     

Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: A study using quail-chick transplantation chimeras

Brain capillaries have structural and functional characteristics that constitute a regulatory interface, or “barrier,” between the blood and the brain. We have investigated the role of the neural tissue environment in the differentiation of the endothelial barrier, by transplanting embryonic brain fragments to the coelomic cavity, where they were vascularized by nonneural vessels, and fragments of embryonic mesoderm to the brain, where they were vascularized by neural vessels. A major problem in this approach is that when embryonic tissues are transplanted to an ectopic site, their own blood vessels survive and form a part of the new vascular system. This has made the results of previous experiments difficult to interpret. We overcame this problem by transplanting fragments of tissue that had not yet been vascularized from very young quail embryos to host chick embryos. These grafts did not contain vascular channels that could form part of a new vascular system. Furthermore, the distinctive quail nuclear morphology allowed us to demonstrate that the grafted tissue was, in fact, vascularized by the host vessels. Abdominal vessels vascularizing grafted neural tissue formed structural, functional, and histochemical features of the blood-brain barrier. In contrast, brain vessels vascularizing grafted mesodermal tissue were devoid of barrier characteristics. These results indicate that endothelial blood-brain barrier characteristics develop in response to some aspect of the neural environment.     

The long-term fate of neurons in allografts of ganglia in AG-B-compatible normal and immunologically tolerant rats

Neurons in Ag-B-incompatible allografts of ganglia are acutely rejected while those in Ag-B-compatible grafts are able to survive the immune reaction directed against them. The present study was undertaken to determine the long-term fate of neurons in allografts of ganglia in Ag-B-compatible rats. Isogenic strains of Ag-B-compatible adult Lewis (LE) and Fischer (FR) rats were used. The sensory nodose ganglia were reciprocally exchanged between normal LE and FR and between LE and FR animals rendered immunologically tolerant of each other's histocompatibity antigens. The findings were similar in both rat strains and revealed that although neurons and glial cells (i.e., satellite and Schwann cells) could survive for prolonged periods they were nevertheless eventually rejected by normal (nonimmunosuppressed) recipients. On the other hand, neurons and glial cells survived indefinitely in allografts in tolerant rats. Moreover, these neurons were functional because they regenerated nerve fibers into cotransplanted isografts of tongue and exerted the neurotrophic influence of inducing taste bud regeneration. The results demonstrate that, unlike kidney and heart, neurons in ganglia allografts cannot survive indefinitely without immunosuppression in Ag-B-compatible animals. Nevertheless, the permanent survival and function of neurons in Ag-B-compatible grafts can be achieved, as it is in Ag-B-incompatible ganglia grafts, by rendering the recipient immunologically tolerant.