Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens.

BACKGROUND: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. RESULT: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses. CONCLUSIONS: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.

Activity-dependent neuroprotective protein (ADNP) is an alcohol-responsive gene and negative regulator of alcohol consumption in female mice.

Neuroadaptations in the brain reward system caused by excessive alcohol intake, lead to drinking escalation and alcohol use disorder phenotypes. Activity-dependent neuroprotective protein (ADNP) is crucial for brain development, and is implicated in neural plasticity in adulthood. Here, we discovered that alcohol exposure regulates Adnp expression in the mesolimbic system, and that Adnp keeps alcohol drinking in moderation, in a sex-dependent manner. Specifically, Sub-chronic alcohol treatment (2.5 g/kg/day for 7 days) increased Adnp mRNA levels in the dorsal hippocampus in both sexes, and in the nucleus accumbens of female mice, 24 h after the last alcohol injection. Long-term voluntary consumption of excessive alcohol quantities (~10-15 g/kg/24 h, 5 weeks) increased Adnp mRNA in the hippocampus of male mice immediately after an alcohol-drinking session, but the level returned to baseline after 24 h of withdrawal. In contrast, excessive alcohol consumption in females led to long-lasting reduction in hippocampal Adnp expression. We further tested the regulatory role of Adnp in alcohol consumption, using the Adnp haploinsufficient mouse model. We found that Adnp haploinsufficient female mice showed higher alcohol consumption and preference, compared to Adnp intact females, whereas no genotype difference was observed in males. Importantly, daily intranasal administration of the ADNP-snippet drug candidate NAP normalized alcohol consumption in Adnp haploinsufficient females. Finally, female Adnp haploinsufficient mice showed a sharp increase in alcohol intake after abstinence, suggesting that Adnp protects against relapse in females. The current data suggest that ADNP is a potential novel biomarker and negative regulator of alcohol-drinking behaviors.

Fibroblast Growth Factor 2 in the Dorsomedial Striatum Is a Novel Positive Regulator of Alcohol Consumption.

Repeated alcohol intake leads to mesostriatal neuroadaptations, resulting in drinking escalation and addiction phenotypes. Fibroblast growth factor 2 (FGF2) has been shown to interact with the mesostriatal dopaminergic system, and has been implicated in the actions of psychostimulants in the brain, and in several psychiatric disorders. Here, we report on a positive regulatory feedback loop of alcohol and FGF2 in rodent models. Specifically, we found that acute alcohol exposure (2.5 g/kg, i.p.) increased the mRNA expression of Fgf2 in the dorsal hippocampus, nucleus accumbens, and dorsal striatum. Longer alcohol exposure (7 d × 2.5 g/kg, i.p.) restricted these increases to the dorsal striatum, and the latter effect was blocked by the dopamine D2-like receptor antagonist haloperidol. Voluntary prolonged and excessive alcohol consumption in a 2-bottle choice procedure increased Fgf2 expression selectively in dorsomedial striatum (DMS) of both mice and rats. Importantly, we found that systemic administration of recombinant FGF2 (rFGF2) in mice, or rFGF2 infusion into the dorsal striatum or DMS of rats, increased alcohol consumption and preference, with no similar effects on saccharin or sucrose consumption. Finally, we found that inhibition of the endogenous FGF2 function in the DMS, by an anti-FGF2 neutralizing antibody, suppressed alcohol consumption and preference. Together, our results suggest that alcohol consumption increases the expression of Fgf2 in the DMS, and that striatal FGF2 promotes alcohol consumption, suggesting that FGF2 in the DMS is a positive regulator of alcohol drinking.SIGNIFICANCE STATEMENT Long-term alcohol intake may lead to neuroadaptations in the mesostriatal reward system, resulting in addiction phenotypes. Fibroblast growth factor 2 (FGF2) is crucial for the development and maintenance of the mesostriatal dopaminergic system. Here, we provide evidence for the involvement of FGF2 in alcohol-drinking behaviors. We show that alcohol increases Fgf2 expression in the dorsal striatum, an effect mediated via dopamine D2-like receptors. Importantly, we show that infusion of recombinant FGF2 into the dorsomedial striatum increases alcohol consumption, whereas inhibiting the endogenous FGF2 function suppresses consumption. Thus, FGF2 is an alcohol-responsive gene constituting a positive regulatory feedback loop with alcohol. This loop leads to facilitation of alcohol consumption, marking FGF2 as a potential new therapeutic target for alcohol addiction.

Re-exposure to nicotine-associated context from adolescence enhances alcohol intake in adulthood.

Alcohol and nicotine are the two most commonly-abused substances and are often used together. Nicotine enhances alcohol-drinking behaviors in humans and in animals, and was suggested to enhance the reinforcing properties of other reinforcers. Here, we show that nicotine-associated environment, rather than nicotine itself, enhances alcohol intake in rats. Adolescent rats received repeated intermittent injections of nicotine (0.4 mg/kg, i.p., 5 injections, every 3rd day) or saline. The injection was paired with their home cage, or with the subsequent alcohol self-administration context. Rats were then trained to self-administer 20% alcohol. Nicotine given in the home cage did not alter subsequent alcohol intake. However, pairing nicotine with the operant chamber during adolescence led to a long-lasting increased alcohol self-administration in adulthood, compared to nicotine pre-treatment in other contexts. This effect persisted 3 months after nicotine cessation, in a relapse test after abstinence. Furthermore, re-exposure to the nicotine-associated context in adult rats led to a decrease in glial cell line-derived neurotrophic factor (Gdnf) mRNA expression in the ventral tegmental area, an effect that leads to increased alcohol consumption, as we have previously reported. Our findings suggest that retrieval of nicotine-associated contextual memories from adolescence may gate alcohol intake in adulthood, with a possible involvement of GDNF.

Pax6 regulates gene expression in the vertebrate lens through miR-204.

During development, tissue-specific transcription factors regulate both protein-coding and non-coding genes to control differentiation. Recent studies have established a dual role for the transcription factor Pax6 as both an activator and repressor of gene expression in the eye, central nervous system, and pancreas. However, the molecular mechanism underlying the inhibitory activity of Pax6 is not fully understood. Here, we reveal that Trpm3 and the intronic microRNA gene miR-204 are co-regulated by Pax6 during eye development. miR-204 is probably the best known microRNA to function as a negative modulator of gene expression during eye development in vertebrates. Analysis of genes altered in mouse Pax6 mutants during lens development revealed significant over-representation of miR-204 targets among the genes up-regulated in the Pax6 mutant lens. A number of new targets of miR-204 were revealed, among them Sox11, a member of the SoxC family of pro-neuronal transcription factors, and an important regulator of eye development. Expression of Trpm/miR-204 and a few of its targets are also Pax6-dependent in medaka fish eyes. Collectively, this study identifies a novel evolutionarily conserved mechanism by which Pax6 controls the down-regulation of multiple genes through direct up-regulation of miR-204.

Pax6: a multi-level regulator of ocular development.

Eye development has been a paradigm for the study of organogenesis, from the demonstration of lens induction through epithelial tissue morphogenesis, to neuronal specification and differentiation. The transcription factor Pax6 has been shown to play a key role in each of these processes. Pax6 is required for initiation of developmental pathways, patterning of epithelial tissues, activation of tissue-specific genes and interaction with other regulatory pathways. Herein we examine the data accumulated over the last few decades from extensive analyses of biochemical modules and genetic manipulation of the Pax6 gene. Specifically, we describe the regulation of Pax6's expression pattern, the protein's DNA-binding properties, and its specific roles and mechanisms of action at all stages of lens and retinal development. Pax6 functions at multiple levels to integrate extracellular information and execute cell-intrinsic differentiation programs that culminate in the specification and differentiation of a distinct ocular lineage.

The mechanism of lens placode formation: a case of matrix-mediated morphogenesis.

Although placodes are ubiquitous precursors of tissue invagination, the mechanism of placode formation has not been established and the requirement of placode formation for subsequent invagination has not been tested. Earlier measurements in chicken embryos supported the view that lens placode formation occurs because the extracellular matrix (ECM) between the optic vesicle and the surface ectoderm prevents the prospective lens cells from spreading. Continued cell proliferation within this restricted area was proposed to cause cell crowding, leading to cell elongation (placode formation). This view suggested that continued cell proliferation and adhesion to the ECM between the optic vesicle and the surface ectoderm was sufficient to explain lens placode formation. To test the predictions of this "restricted expansion hypothesis," we first confirmed that the cellular events that accompany lens placode formation in chicken embryos also occur in mouse embryos. We then showed that the failure of lens placode formation when the transcription factor, Pax6 was conditionally deleted in the surface ectoderm was associated with greatly diminished accumulation of ECM between the optic vesicle and ectoderm and reduced levels of transcripts encoding components of the ECM. In accord with the "restricted expansion hypothesis," the Pax6-deleted ectoderm expanded, rather than being constrained to a constant area. As a further test, we disrupted the ECM by deleting Fn1, which is required for matrix assembly and cell-matrix adhesion. As in Pax6(CKO) embryos, the Fn1(CKO) lens ectoderm expanded, rather than being constrained to a fixed area and the lens placode did not form. Ectoderm cells in Fn1(CKO) embryos expressed markers of lens induction and reorganized their cytoskeleton as in wild type ectoderm, but did not invaginate, suggesting that placode formation establishes the minimal mechanical requirements for invagination.

Pax6 is essential for lens fiber cell differentiation.

The developing ocular lens provides an excellent model system with which to study the intrinsic and extrinsic cues governing cell differentiation. Although the transcription factors Pax6 and Sox2 have been shown to be essential for lens induction, their later roles during lens fiber differentiation remain largely unknown. Using Cre/loxP mutagenesis, we somatically inactivated Pax6 and Sox2 in the developing mouse lens during differentiation of the secondary lens fibers and explored the regulatory interactions of these two intrinsic factors with the canonical Wnt pathway. Analysis of the Pax6-deficient lenses revealed a requirement for Pax6 in cell cycle exit and differentiation into lens fiber cells. In addition, Pax6 disruption led to apoptosis of lens epithelial cells. We show that Pax6 regulates the Wnt antagonist Sfrp2 in the lens, and that Sox2 expression is upregulated in the Pax6-deficient lenses. However, our study demonstrates that the failure of differentiation following loss of Pax6 is independent of beta-catenin signaling or Sox2 activity. This study reveals that Pax6 is pivotal for initiation of the lens fiber differentiation program in the mammalian eye.

Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing.

Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3' splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events.