Abbreviated Action Potential Kinetics in a Mouse Model of Potassium Channel Overexpression During Hippocampal Development

Loss or "gain" of function mutations in voltage-gated ion channels often results in an adverse neurological phenotype. We have examined the electrical characteristics of hippocampal pyramidal cells in a transgenic mouse model to determine how overexpression of a Shaker-type potassium channel subunit during early postnatal development might alter excitability properties of developing neurons. We found that in CA3 neurons potassium channel overexpression led to a transient shortening in duration of single action potentials during the first two postnatal weeks. There was an increase in maximal repolarization rate, without significant effect on the rate of rise. Transgenic CA3 neurons also showed a decrease of firing frequency in response to sustained depolarizing current injection. In contrast, repolarization of action potentials in CA1 neurons was not significantly altered by trangene expression. Western Blot Analysis of membrane-associated transgene protein indicated that transgene protein levels decreased during development, in agreement with functional measures of spike width. Our data indicate that the functional consequences of potassium channel transgene expression are dependent on cellular environment and developmental stage. A transient period of hypoexcitability during a critical period of development for CA3 neurons may contribute to the hyperexcitable phenotype observed in adult animals.     

Specific neuronal expression of human NGF receptors in the basal forebrain and cerebellum of transgenic mice

Transgenic mice carrying multiple copies of the human NGF receptor gene have been generated. Using a monoclonal antibody specific for the human receptor, we have detected specific expression in cholinergic neurons in the basal forebrain and Purkinje cells in the cerebellum during the postnatal period. Expression in the PNS was exemplified by immunostaining of sympathetic and sensory neurons during an early embryonic age. Transection of the sciatic nerve in transgenic animals resulted in induction of human NGF receptors, indicating that the inserted gene can be appropriately regulated. These transgenic mice will provide an opportunity to study the elements regulating the NGF receptor. Furthermore, the ability to obtain specific expression in transgenic mice will permit directed expression of heterologous genes in discrete cells important in the cholinergic septal-hippocampal pathway and the PNS.     

Emx1-specific expression of foreign genes using "knock-in" approach

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles. Its expression is limited to the neurons in developing and adult cerebral cortex and hippocampus. Because of the highly restricted expression pattern of the Emx1 gene, it would be quite desirable to characterize the promoter of the Emx1 for directing foreign gene expression in the transgenic mouse. We report here that we have achieved the Emx1-specific expression in transgenic mice by inserting the lacZ reporter and cre genes directly into the exon 1 of the Emx1 gene using embryonic stem (ES) cell technology. The distribution of the beta-galactosidase activity in the transgenic mice was consistent with the published results obtained using in situ hybridization and immunohistochemistry. Furthermore, we have demonstrated that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. The creation of this line of cre transgenic mice, and the demonstration that the insertion site located in the exon 1 of the Emx1 gene could render foreign genes a specific expression pattern restricted to the developing and adult cerebral cortex and hippocampus, should be conducive to further studies of the effect of a gene mutation or overexpression upon the development and plasticity of cerebral cortex and hippocampus.     

Constitutive notch signaling in adult transgenic mice inhibits bFGF‐induced angiogenesis and blocks ovarian follicle development

Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock‐out and transgenic mice have precluded studies of the role of Notch post‐natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre‐conditional expression of the constitutively active intracellular domain of Notch1 (IC‐Notch1). Double transgenic IC‐Notch1/Tie2‐Cre embryos with endothelial specific IC‐Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline‐inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC‐Notch1 expression inhibited bFGF‐induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF‐induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies. genesis 52:809–816, 2014. © 2014 Wiley Periodicals, Inc.     

Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo.Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice.The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.     

Increased transgene expression by the mouse tyrosinase enhancer is restricted to neural crest-derived pigment cells

In this study, we have addressed the impact of the mouse tyrosinase enhancer on regulated expression from the mouse tyrosinase promoter during embryonic development. Stable and transient transgenic experiments using the reporter gene lacZ reveal that (1) expression is detected in neural crest-derived melanoblasts from E11.5 onward, (2) the enhancer does not increase transgenic expression in optic cup-derived pigment cells of the retinal pigment epithelium (RPE), and (3) expression in the telencephalon is not any longer detected. The importance of the enhancer for expression in pigment cells of the eye was further investigated in adult mice using an attenuated diphtheria toxin A gene. This demonstrated that in presence of the enhancer the transgene expression is specifically targeted to neural crest-derived melanocytes of the choroid and not, or slightly, to the RPE. This suggests that tyrosinase is differentially regulated in the two pigment cell lineages, and that this promoter can be used to target expression preferentially to the neural crest-derived melanocyte lineage.     

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.     

Nestin expression in pancreatic exocrine cell lineages

Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule beta-galactosidase only upon Cre-mediated recombination, thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, beta-galactosidase was observed in mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in addition to fibroblasts and vascular cells, but no endocrine cells were tagged by beta-galactosidase. This correlated with the transient expression, observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore beta-galactosidase. Together, these findings show that nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet endocrine progenitor cells.     

Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice

New neurons are continually generated in the adult hippocampus, but the important question, whether adult neurogenesis is transient or leads to the lasting presence of new neurons, has not yet been answered. Dividing cells were labeled with bromodeoxyuridine (BrdU) and were investigated by means of immunofluorescence and confocal microscopy at several time-points 1 day to 11 months thereafter. BrdU-labeled neurons remained stable in number and in their relative position in the granule cell layer over at least 11 months. This finding implies that the addition of new neurons is not transient and that their final number and localization are determined early. By contrast, expression of immature markers beta-III-tubulin and doublecortin in BrdU-labeled cells, peaked early after division and was not detectable after 4 weeks. In transgenic mice expressing enhanced green fluorescent protein under the nestin promoter none of the BrdU/nestin-positive cells early after division expressed the mature marker NeuN, confirming that no dividing neurons were detected. These new data suggest that new neurons are recruited early from the pool of proliferating progenitor cells and lead to a lasting effect of adult neurogenesis.     

Transient expression of somatostatin peptide is a widespread feature of developing sensory and sympathetic neurons in the embryonic rat.

Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined (somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)), only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double-labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types.     

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.     

Temporal expression of mutant LRRK2 in adult rats impairs dopamine reuptake

Parkinson's disease (PD) results from progressive degeneration of dopaminergic neurons. Most PD cases are sporadic, but some have pathogenic mutation in the individual genes. Mutation of the leucine-rich repeat kinase-2 (LRRK2) gene is associated with familial and sporadic PD, as exemplified by G2019S substitution. While constitutive expression of mutant LRRK2 in transgenic mice fails to induce neuron death, transient expression of the disease gene by viral delivery causes a substantial loss of dopaminergic neurons in mice. To further assess LRRK2 pathogenesis, we created inducible transgenic rats expressing human LRRK2 with G2019S substitution. Temporal overexpression of LRRK2(G2019S) in adult rats impaired dopamine reuptake by dopamine transporter (DAT) and thus enhanced locomotor activity, the phenotypes that were not observed in transgenic rats constitutively expressing the gene throughout life time. Reduced DAT binding activity is an early sign of dopaminergic dysfunction in asymptomatic subjects carrying pathogenic mutation in LRRK2. Our transgenic rats recapitulated the initiation process of dopaminergic dysfunction caused by pathogenic mutation in LRRK2. Inducible transgenic approach uncovered phenotypes that may be obscured by developmental compensation in constitutive transgenic rats. Finding in inducible LRRK2 transgenic rats would guide developing effective strategy in transgenic studies: Inducible expression of transgene may induce greater phenotypes than constitutive gene expression, particularly in rodents with short life time.     

Cell-type-specific expression of a TESK1 promoter-linked lacZ gene in transgenic mice

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase highly expressed in testicular germ cells and has the potential to phosphorylate cofilin and induce actin cytoskeletal reorganization. We examined the expression of a lacZ reporter gene linked to a 9.0-kb 5'-flanking region of TESK1 gene in transgenic mice. A high level of lacZ expression was observed in testicular germ cells only at stages after pachytene spermatocytes, the expression patterns being similar to those of TESK1 mRNA in rat testis, determined by in situ hybridization. Expression of lacZ was also detected in renal proximal tubules, cardiac myocytes, and specific neurons in the central nervous system in adult transgenic mice. Whole-mount staining revealed the expression of lacZ in neural tissues in embryonic mice. These results suggest the cell-type- and stage-specific expression of TESK1 gene and the diverse and specific physiological functions of TESK1, including those in spermatogenesis and neural development.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

F-spondin/spon1b expression patterns in developing and adult zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.