Dynamic expression of Dab2 in the mouse embryonic central nervous system

Background  

Dab2, one of two mammalian orthologs of Drosophila Disabled, has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis, but its role in neuronal development is not yet fully understood. In this report, we have examined the localization of the Dab2 protein in the mouse embryonic central nervous system (CNS) at different developmental stages.     

Differential expression and function of the Drosophila Pax6 genes eyeless and twin of eyeless in embryonic central nervous system development

We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain.     

Regulatory elements of the bithorax complex that control expression along the anterior-posterior axis

The Drosophila bithorax complex (BX-C) controls segmental development by selectively deploying three protein products, Ubx, abd-A and Abd-B, within specific segments along the body axis. Expression of these products within any one segment (or, more accurately, parasegment) is affected by mutations clustered in a particular region of the BX-C. The regulatory regions defined by this genetic analysis span 20-50 kb and there is one region for each segmental unit. Here we describe regulatory elements from several of these regions, identified by fusion to a Ubx-lacZ gene and analysis in germline transformants. A small DNA fragment from the abx region programs expression with an anterior boundary in the second thoracic segment (parasegment 5). This anterior limit is appropriate, since the abx region normally controls Ubx in parasegment 5. Other regulatory regions of the BX-C that control development of parasegments 6, 7 or 8 contain similar regulatory elements that program expression with anterior limits in parasegments 6, 7 or 8, respectively. These experiments define a class of BX-C regulatory elements that control expression along the anterior-posterior axis. The early appearance of the lacZ patterns in embryos suggests a role for these elements in the initial activation of expression from the BX-C.     

Differential expression of Sonic hedgehog along the anterior-posterior axis regulates patterning of pharyngeal pouch endoderm and pharyngeal endoderm-derived organs

Previous studies have implicated Sonic hedgehog (Shh) as an important regulator of pharyngeal region development. Here we show that Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In Shh-/- mutants, the pharyngeal pouches and arches formed by E9.5 and marker expression showed that initial patterning was normal. However, by E10.5-E11.0, the first arch had atrophied and the first pouch was missing. Although small, the second, third, and fourth arches and pouches were present. The expression patterns of Fgf8, Pax1, and Bmp4 suggested that pouch identity was abnormal at E10.5 and that Shh is a negative regulator of these genes in the pouches. Despite the loss of pouch identity and an increase in mesenchymal cell death, arch identity markers were expressed normally. Our data show that a Shh-dependent patterning mechanism is required to maintain pouch patterning, independent or downstream of arch identity. Changes in the distribution of Bmp4 and Gcm2 in the third pouch endoderm and subsequent organ phenotypes in Shh-/- mutants suggested that exclusion of Shh from the third pouch is required for dorsal-ventral patterning and for parathyroid specification and organogenesis. Furthermore, this function for Shh may be opposed by Bmp4. Our data suggest that, as in the posterior gut endoderm, exclusion of Shh expression from developing primordia is required for the proper development of pharyngeal-derived organs.     

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.     

Heme oxygenase expression in human central nervous system disorders

In the normal mammalian CNS, heme oxygenase-2 (HO-2) is constitutively, abundantly, and fairly ubiquitously expressed, whereas heme oxygenase-1 (HO-1) mRNA and protein are confined to small populations of scattered neurons and neuroglia. Unlike ho-2, the ho-1 gene in neural (and many systemic) tissues is exquisitely sensitive to upregulation by a host of pro-oxidant and other noxious stimuli. In Alzheimer disease, HO-1 immunoreactivity is significantly augmented in neurons and astrocytes of the hippocampus and cerebral cortex relative to age-matched, nondemented controls and colocalizes to senile plaques, neurofibrillary tangles, and corpora amylacea. In Parkinson disease, HO-1 decorates Lewy bodies of affected dopaminergic neurons and is highly overexpressed in astrocytes residing within the substantia nigra. The ho-1 gene is also upregulated in glial cells within multiple sclerosis plaques; in the vicinity of human cerebral infarcts, hemorrhages, and contusions; and in various other degenerative and nondegenerative human CNS disorders. The products of the heme oxygenase reaction, free ferrous iron, carbon monoxide, and biliverdin/bilirubin, are all biologically active molecules that may profoundly influence tissue redox homeostasis under a wide range of pathophysiological conditions. Evidence adduced from whole animal and in vitro studies indicates that enhanced HO-1 activity may either ameliorate or exacerbate neural injury, effects likely contingent upon the specific model employed, the duration and intensity of HO-1 induction, and the chemistry of the local redox microenvironment. HO-1 hyperactivity also promotes mitochondrial sequestration of nontransferrin iron in oxidatively challenged astroglia and may thereby contribute to the pathological iron deposition and bioenergetic failure amply documented in aging and degenerating human neural tissues.     

Differential expression patterns of NDRG family proteins in the central nervous system.

The N-myc downstream-regulated gene (NDRG) family consists of four proteins: NDRG1, NDRG2, NDRG3, and NDRG4 in mammals. NDRG1 has been thoroughly studied as an intracellular protein associated with stress response, cell growth, and differentiation. A nonsense mutation in the NDRG1 gene causes hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D. We previously generated Ndrg1-deficient mice and found that they exhibited peripheral nerve degeneration caused by severe demyelination, but that the complicated motor abilities were retained. These results implied that other NDRG family proteins may compensate for the NDRG1 deficiency in the central nervous system. In this study we raised specific antibodies against each member of the NDRG protein family and examined their cellular expression patterns in the mouse brain. In the cerebrum, NDRG1 and NDRG2 were localized in oligodendrocytes and astrocytes, respectively, whereas NDRG3 and NDRG4 were ubiquitous. In the cerebellum, NDRG1 and NDRG4 were localized in Purkinje cells and NDRG2 in Bergmann glial cells. NDRG3 was detected in the nuclei in most cells. These expression patterns demonstrated the cell type-specific and ubiquitous localization of the NDRG family proteins. Each NDRG may play a partially redundant role in specific cells in the brain.     

DNA synthesis in the embryonic chick central nervous system

Summary DNA synthesis has been studied in chick embryos age between 2 and 10 days, using labelling with tritiated thymidine and stripping film autoradiography. The observations made earlier in the literature on a premitotic migration of the nuclei in the neural epithelium have been verified. In young stages (before day 7) peripherally migrated cells do not synthesize DNA, but after day 7 such a synthesis occurs. In spite of this, few mitoses are seen. The interpretation of these facts is discussed.The costs of this investigation were defrayed by grants from the Swedish Medical Research Council, the Medical Faculty of Lund, and the Royal Physiographic Society.     

Dissecting planarian central nervous system regeneration by the expression of neural-specific genes

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.     

Differential distribution of aldolase A and C in the human central nervous system

We have analyzed the distribution of aldolase A and C mRNAs and proteins in various areas of the human brain using Northern blot analyses and immunohistochemistry. Aldolase A mRNA expression was higher than aldolase C mRNA expression in all areas of the brain examined. Aldolase C mRNA expression was highest in the cerebellum. Aldolase C protein was present in well-delimited regions of the CNS, and was distributed in stripes in the Purkinje cell layer of the cerebellum, in the inferior olives and in the sensory neurons of the posterior horn of the spinal cord. The novel finding of aldolase C in well-delimited cell compartments of the human cerebellum and in several other areas of the CNS lends weight to the hypothesis that this protein exerts other functions (e.g. sensory transmission) besides those characteristic of a glycolytic enzyme.     

Differential expression of the Tmem132 family genes in the developing mouse nervous system

The family of novel transmembrane proteins (TMEM) 132 have been associated with multiple neurological disorders and cancers in humans, but have hardly been studied in vivo. Here we report the expression patterns of the five Tmem132 genes (a, b, c, d and e) in developing mouse nervous system with RNA in situ hybridization in wholemount embryos and tissue sections. Our results reveal differential and partially overlapping expression of multiple Tmem132 family members in both the central and peripheral nervous system, suggesting potential partial redundancy among them.     

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.     

Lipofection of cDNAs in the embryonic vertebrate central nervous system.

Neurons from the embryonic brain of Xenopus were transfected in vivo with a vector expressing luciferase cDNA using a simple lipofection procedure. Luciferase activity was monitored quantitatively, and the protein was immunolocalized in whole-mount embryonic brains. Luciferase-expressing neurons were often intensely labeled, displaying a Golgi-like filling of their dendrites, axons, and growth cones. Luciferase expression could be targeted to the retina by simply removing the skin epidermis covering the area and exposing the whole embryo to the DNA-lipofectin mixture. Luciferase activity in transfected embryos rose to peak values during the first 48 hr posttransfection and was still detectable 28 days later. Cotransfection experiments in which embryonic nervous tissue was exposed simultaneously to two different genes, luciferase and chloramphenicol acetyl-transferase, showed that transfected cells coexpressed the two genes at an extremely high frequency (85%-100%). This offers the possibility of targeting functionally significant genes along with benign reporter genes in the developing CNS.     

Retinoic acid,HOX genes and the anterior-posterior axis in chordates

In vertebrate development, the HOX genes act to specify cell identity along much of the anterior-posterior axis of the embryonic central nervous system. In all vertebrates examined to date, the vitamin A metabolite retinoic acid is implicated in the patterning of the anterior posterior axis and the induction of HOX gene expression. Two recent papers have extended the study of retinoic acid induction of HOX genes to the closest relatives of the vertebrates, amphioxus and tunicates^(1,2). In both these species, exogenous retinoic acid is able to induce ectopic expression of HOX 1 genes in the anterior central nervous system. This suggests that retinoic acid control of anterior-posterior axis formation and HOX induction is not specific to vertebrates. However, in the more distantly related echinoderms and arthropods, retinoic acid does not seem to act in the same way. Thus the role of retinoic acid in anterior-posterior axis specification may be a chordate innovation, perhaps linked to the evolution of another chordate character, the dorsal neural tube.