Neural expression of alpha-internexin promoter in vitro and in vivo

alpha-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse alpha-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse alpha-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro, and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of EGFP with 1.3 kb mouse alpha-internexin promoter was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb alpha-Internexin promoter. The activity of the Cre recombinase at postnatal day 1 was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that beta-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for alpha-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any beta-galactosidase activity. Therefore, the cre transgene driven by mouse alpha-internexin promoter, described here, provides a useful animal model to specifically manipulate genes in the developing nervous system.     

A Uchl1‐Histone2BmCherry:GFP‐gpi BAC transgene for imaging neuronal progenitors

Summary: Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP‐gpi. The Uchl1‐H2BmCherry:GFP‐gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cells in endocrine tissues where earlier in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1‐H2BmCherry:GFP‐gpi transgene will be a powerful tool for static and live imaging, as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis. genesis 51:852–861. © 2013 Wiley Periodicals, Inc.     

A systematic analysis of <Emphasis Type="Italic">Drosophila</Emphasis> gustatory receptor gene expression in abdominal neurons which project to the central nervous system

In Drosophila, the gustatory receptor (Gr) gene family contains 60 family members that encode 68 proteins through alternative splicing. Some gustatory receptors (Grs) are involved in the sensing of sugars, bitter substrates, CO₂, pheromones, and light. Here, we systematically examined the expression of all 68 Grs in abdominal neurons which project to the abdominal ganglion of the central nervous system using the GAL4/UAS system. Gr gene expression patterns have been successfully analyzed in previous studies by using the GAL4/UAS system to drive reporter gene expression. Interestingly, 21 Gr-GAL4 drivers showed abdominal ganglion projection, and 18 of these 21 Gr-GAL4 drivers labeled multidendritic neurons of the abdominal wall. 4 drivers also labeled neuronal processes innervating the reproductive organs. The peripheral expression of Gr-GAL4 drivers in abdominal multidendritic neurons or neurons innervating the reproductive organs suggests that these Grs have atypical sensory functions in these organs not limited to conventional taste sensing.     

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B‐GFP transgenic mice

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B‐GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (?540 to +31). Using the fresh brain sections of F1B‐GFP transgenic mice, we found that the F1B‐GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B‐GFP⁺ cells existed in the brains of F1B‐GFP transgenic mice. We demonstrated that one population of F1B‐GFP⁺ cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B‐GFP⁺ cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B‐GFP⁺ cells and tyrosine hydroxylase indicated that a subpopulation of F1B‐GFP⁺‐neuronal cells was dopaminergic neurons. Importantly, these F1B‐GFP⁺/TH⁺ cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B‐GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 232–248, 2015     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.     

Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

Background

The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons.

Methods/Findings

Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia.

Conclusions/Significance

We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.     

IKAP/hELP1 downregulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP-downregulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.Key words: Familial Dysautonomia, IKAP/hELP1, neuronal differentiation, laminin, contactin, peripheral nervous system     

Neurotrophin-3 as an essential signal for the developing nervous system

Rapid advances in characterization of the biological actions mediated by the third member of the neurotrophin family, neurotrophin-3 (NT-3), have been made recently in vitro as well asin situ. These have been made possible by the cloning of the genes for NT-3 and for its transducing receptor tyrosine kinase TrkC. This article will focus on the roles of NT-3 in the nervous system.In situ localization of NT-3 consistent with that of its receptor is manifested at all developmental stages studied and into adulthood. Through TrkC, NT-3 signals a number of trophic effects, ranging from mitogenesis, promotion of survival, or differentiation, depending on the developmental stage of the target cells. The sites of action of NT-3 reside primarily in the peripheral nervous system (PNS), various areas of the central nervous system (CNS), and in the enteric nervous system (ENS). Analyses of the phenotypes of transgenic mice lacking NT-3 or injection of embryos with a blocking antibody have so far revealed the essential role of NT-3 in development of specific populations of the PNS, and in particular of proprioceptive, nodose, and auditory sensory neurons and of sympathetic neurons. The actions of NT-3 also extend to modulation of transmitter release at several types of synapses in the periphery as well as in the adult CNS. In addition, NT-3 may play a role in the development of tissues other than the nervous system, such as the cardiovascular system. Future investigations will widen the understanding of the many roles of NT-3 on both neuronal and nonneuronal cells.     

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.     

Generation of BAF53b‐Cre transgenic mice with pan‐neuronal Cre activities

Molecular and functional studies of genes in neurons in mouse models require neuron‐specific Cre lines. The current available neuronal Cre transgenic or knock‐in lines either result in expression in a subset of neurons or expression in both neuronal and non‐neuronal tissues. Previously we identified BAF53b as a neuron‐specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b‐Cre is largely neuron‐specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b‐Cre functioned in primary cultures in a pan‐neuron‐specific manner. Thus, BAF53b‐Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies. genesis, 53:440–448, 2015. © 2015 Wiley Periodicals, Inc.     

IKAP/hELP1 down-regulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor, and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP – down - regulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.     

A zebrafish SKIV2L2-enhancer trap line provides a useful tool for the study of peripheral sensory circuit development

The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)^j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.     

Evaluation of an FRDA–EGFP genomic reporter assay in transgenic mice

Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide expansion in the first intron of the Friedreich ataxia gene (FRDA) that causes reduced synthesis of frataxin, a mitochondrial protein likely to be involved in biosynthesis of iron–sulfur clusters. This leads to increased oxidative stress, progressive loss of large sensory neurons, and hypertrophic cardiomyopathy. To elucidate the mechanisms regulating FRDA expression and to develop an in vivo assay for agents that might upregulate FRDA expression in a therapeutically relevant manner, we have generated transgenic mice with a BAC genomic reporter construct consisting of an in-frame fusion between FRDA and the gene coding for enhanced green fluorescent protein (EGFP). Production of full-length frataxin–EGFP fusion protein was demonstrated by immunoblotting. EGFP expression was observed as early as day E3.5 of development. Most tissues of adult transgenic mice were fluorescent. The level of FRDA–EGFP expression in peripheral blood, bone marrow, and cells obtained from enzymatically disaggregated tissues was quantitated by flow cytometry. There was a twofold increase in EGFP expression in mice homozygous for the transgene when compared to hemizygous mice. These transgenic mice are a valuable tool for the examination of spatial and temporal aspects of FRDA gene expression and for the preclinical evaluation of pharmacological inducers of FRDA expression in a whole-animal model. In addition, tissues from these mice should also be valuable for stem cell transplantation studies.     

Generation of the tamoxifen-inducible DBH-Cre transgenic mouse line DBH-CT

We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the dopamine-β-hydroxylase promoter. By crossing to the ROSA26 reporter mice we show that tamoxifen-induced Cre recombinase in adult mice specifically activates β-galactosidase expression in differentiated noradrenergic neurons of the central and peripheral nervous system. Tamoxifen application in adult mice did not induce β-galactosidase activity in parasympathetic neurons that transiently express DBH during development. Thus, this transgenic mouse line represents a valuable tool to study gene function in mature noradrenergic neurons by conditional inactivation.     

A transgenic mouse class-III beta tubulin reporter using yellow fluorescent protein

A yellow fluorescence protein (YFP) reporter construct was cloned downstream of the beta-tubulin III promoter and injected to produce two founder lines of transgenic mice. YFP expression was observed in many regions of the developing peripheral and central nervous system. YFP expression was first observed in the peripheral and central nervous system as early as embryonic day 9.0. There was a dramatic increase in the number of neuronal systems expressing YFP through P0. Then as the animals reached adult age, the expression levels decreased, but many neurons still show YFP expression, notably in regions of the brain undergoing adult neurogenesis, i.e., the rostral migratory stream and subgranular layer of the dentate gyrus. This reporter-based staining was compared with anti-class-III beta-tubulin immunocytochemistry and shown to closely parallel the expression of the endogenous protein. These transgenic lines should provide unique models to study in vivo and in vitro neurodevelopment.