Cell-specific three-photon-fluorescence brain imaging: neurons, astrocytes, and gliovascular interfaces.

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.

Stain-free subcellular-resolution astrocyte imaging using third-harmonic generation.

We demonstrate stain-free, high-contrast, subcellular-resolution imaging of astroglial cells using epi-detected third-harmonic generation (THG). The astrocyte-imaging capability of THG is verified by colocalizing THG images with fluorescence images of astrocytes expressing a genetically encodable fluorescent reporter. We show that THG imaging with an optimized point-spread function can reliably detect significant subcellular features of astrocytes, including cell nuclei, as well as the soma shape and boundaries.

NOMA-GAP/ARHGAP33 regulates synapse development and autistic-like behavior in the mouse.

Neuropsychiatric developmental disorders, such as autism spectrum disorders (ASDs) and schizophrenia, are typically characterized by alterations in social behavior and have been linked to aberrant dendritic spine and synapse development. Here we show, using genetically engineered mice, that the Cdc42 GTPase-activating multiadaptor protein, NOMA-GAP, regulates autism-like social behavior in the mouse, as well as dendritic spine and synapse development. Surprisingly, we were unable to restore spine morphology or autism-associated social behavior in NOMA-GAP-deficient animals by Cre-mediated deletion of Cdc42 alone. Spine morphology can be restored in vivo by re-expression of wild-type NOMA-GAP or a mutant of NOMA-GAP that lacks the RhoGAP domain, suggesting that other signaling functions are involved. Indeed, we show that NOMA-GAP directly interacts with several MAGUK (membrane-associated guanylate kinase) proteins, and that this modulates NOMA-GAP activity toward Cdc42. Moreover, we demonstrate that NOMA-GAP is a major regulator of PSD-95 in the neocortex. Loss of NOMA-GAP leads to strong upregulation of serine 295 phosphorylation of PSD-95 and moreover to its subcellular mislocalization. This is associated with marked loss of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and defective synaptic transmission, thereby providing a molecular basis for autism-like social behavior in the absence of NOMA-GAP.

Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization.

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.

Expression of PTTG and prc1 genes during telencephalic neurogenesis.

We present the first time/space analysis using in situ hybridization for PTTG and prc1 genes during development of the mouse telencephalon. During the stages E11.5-E13.5 PTTG and prc1 are expressed in most tissues of the embryo. Within the telencephalon, PTTG and prc1 are found exclusively inside of the ventricular zone (VZ). The intensity of the expression of both genes in the ventricular zone reaches its peak by E15.5. Expression starts to decrease by E18.5, it is still visible at least up to P2 and not detectable in the adult brains. Expression of the prc1 gene, but not that of the PTTG, is also found in the mitoticaly active cells outside of the VZ within the telencephalon. Most of the cells expressing the PTTG gene were found in the lower part of the ventricular zone suggesting that the level of PTTG mRNA is regulated during different phases of the mitotic cycle.

Detection of planarian Antennapedia-like homeobox genes expressed during regeneration.

Using the polymerase chain reaction with degenerate oligo primers we identified six new Antennapedia-like homeobox sequences expressed in a regenerating planarian. cDNA fragments containing the entire homeobox sequences for four of these genes were obtained. Two of them, Dutarh-3 and Dutarh-4, belong to the classical Antennapedia type and display extensive identity with Antennapedia- and Deformed-type homeodomains respectively. The third homeodomain, Dutarh-1, exhibits some similarity to the Hox1.5-, AbdB- and Dfd-type homeodomains and a fourth gene, Dutarh-6, is very similar to Mox2 in the mouse. Our results suggest that, in spite of their simple body plan, planarians contain a number of Antennapedia-like homeobox genes, probably enough to fill a cluster such as found in higher animals.

Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.

An efficient cDNA amplification procedure is described for determining of the 5' and 3' ends of mRNAs and cloning full-length cDNAs. In this approach, a double-stranded (ds) adaptor is ligated to both ends of a library of ds cDNA by T4 DNA ligase. This adaptor-ligated ds cDNA is then used to selectively amplify 5'- or 3'-cDNA fragments by PCR with a combination of gene-specific and adaptor-specific primers. This is a unified method for 5' and 3' rapid amplification of cDNA ends (RACE) from the same adaptor-ligated ds cDNA template. A specially designed adaptor combines features of "vectorette PCR" and "suppression PCR" technologies that significantly reduce background during amplification. The application of "long and accurate PCR" (LA PCR) technology makes possible the amplification of large RACE products and full-length cDNAs with high fidelity to the original mRNA. We investigated efficacy and limitations of this PCR-based approach for cDNA cloning by amplification of 5'- and 3'-RACE fragments and full-length cDNAs of three members of the abundant human actin gene family (1.3-1.9 kb), the medium abundance transferrin receptor mRNA (5.0 kb) and the low-medium abundance insulin-like growth factor II receptor mRNA (9.1 kb).

The novel PCR-based technique of genotyping applied to identification of scrambler mutation in mice.

For a wide range of research purposes it is necessary to perform genotyping, i.e. to test which alleles, each corresponding to a particular locus, are present in the individual genome. Here we suggest a protocol of genotyping for mice with scrambler (scm) mutation. This mutation results in the aberrant splicing of the corresponding mRNA and affects the expression of mdab1 protein. Traditional approaches using genomic Southern hybridization or PCR with specific primers are not suitable for the genotyping of scm because of the lack of comprehensive information on the organization of the gene and on the presence of repetitive sequences in the known region. Here we propose a quick and highly reproducible method for genotyping scm mutant mice. The protocol consists of the following steps: isolation of genomic DNA, digestion with the restriction endonuclease, anchoring of resulting fragments with the adapter, and PCR amplification using adapter-specific primers. The final product of PCR amplification has a characteristic length which is different for the wt (wild type) and scm alleles. Thus, the characteristic pattern of bands obtained for each individual mouse specimen serves as criteria for the presence of wild type and/or scm allele. We believe that this approach could have wider application. The protocol can be easily modified and used as a convenient tool for identification of other genomic defects including those artificially introduced into genome by knockout or gene-trap techniques.

[Highly-effective subtractive hybridization of cDNA]. / Vysokoéffektivnaia vychitaiushchaia gibridizatsiia kDNK.

A scheme for subtractive hybridization is described allowing for a 500-1000 fold enrichment of low abundant cDNA. The scheme is based on the previously described principle of normalization of an initial mixture of differently represented cDNAs in the single-stranded portion of a tracer after the first round of subtraction and the principle of a trapper excluding the fraction of the double-stranded cDNAs formed during the first round from the subsequent PCR-amplification. The technique is simple and makes unnecessary the separation of the tracer, driver and hybrids formed after the subtraction.

Simple method for cDNA amplification starting from small amount of total RNA.

We describe a novel simple PCR-based technique for construction of cDNA libraries starting from the small samples of cells or tissues. This technique is based on the insertion of inverted terminal repeats (ITR) into amplified cDNA which causes a part of molecules to generate "pan"-type structures at each cycle of PCR amplification. This allows to avoid generation of the primer dimmer and makes possible the regulation of an average length of amplified sequences varying in concentration of primers.

[Cloning and analysis of a new neurogene in the mouse]. / Klonirovanie i analiz novogo neirogena myshi.

The previously described gene sip1 belongs to transcription factors of the zinc finger family. It has been ascertained recently that this gene is involved in TGF signaling cascade. Mutations in human gene sip1 cause Hirschprung syndrome. The expression of gene sip1 during embryonic mouse development was studied by in situ hybridization and immunostaining. Starting at E12.5, sip1 transcripts are present in a number of tissues: in the cortical plate, ventricular zone of the basal ganglion, thalamus, pons and midbrain, in specific nuclei of the brain stem and in the dorsal part of the spinal cord. In the developing cerebral cortex, sip1 expression is region-specific. In the brain of adult mice, sip1 expression is mostly detected in hippocampus, dentate gyrus, and white matter of the neocortex. Sip1 protein expression in the cerebral cortex is mostly confined to glutamatergic neurons.

[Differential gene expression during the reparative regeneration of differing polarities in planarians]. / Differentsial'naia ékspressiia genov pri reparativnoi regeneratsii razlichnoi poliarnosti u planarii.

We identified two new genes (scarf and collar) of planarians using subtracting hybridization. mR-NAs of these genes are distributed in different ways in regeneration blastemas of different polarity. Zone-specific expression of these genes in non-regenerating planarians has been demonstrated. The level of expression of the identified genes in the head regeneration buds of the first-third days of regeneration was lower than that in the corresponding intact tissues and tail regeneration buds.

[The cloning of fragments of homeobox genes expressed during planarian regeneration]. / Klonirovanie fragmentov gomeobokssoderzhashchikh genov, ékspressiruiushchikhsia v khode regeneratsii planarii.

The polymerase chain reaction with degenerate primers corresponding to the most conservative amino acids 16-21 (ELEKEF) and 49-54 (WFQNRR) of the Antennapedia class homeodomain was used for the amplification of cDNA from regenerating planarians (asexual race of Dugesia tigrina). A total of six new Antennapedia-like homeobox sequences, designated Dutarh-1-Dutarh-6 (Dugesia tigrina asexual race homeobox gene), were obtained. Their comparison with other homeobox genes using a "Genebee" software (the EMBL Data Library) showed that all sequences except Dutarh-6 belong to the Antennapedia class. Dutarh-6 is closely related to a recently described novel homeobox gene subfamily that includes mouse mesodermal homeobox genes Mox-1 and Mox-2 and rat homeobox gene Gax.

[Identification of new genes and analysis of their expression during lens and retinal regeneration in adult newts]. / Identifikatsiia novykh genov i analiz ikh ékspressii v protsessse regeneratsii khrustalika i setchatki u vzroslykh tritonov.

During lens regeneration in Pleurodeles waltl, the dorsal iris zone is the cell source of the lens regeneration, while the ventral iris zone can serve as the cells' source of lens regeneration only under certain experimental conditions. The method of subtractive hybridization was used for the identification of genes responsible for the different proliferative potential of these zones. Differential screening of the enriched cDNA libraries, which were obtained as a result of subtractive hybridization of the cDNA samples of the ventral and dorsal iris zones 14 days after lens removal, revealed four clones specific to the dorsal iris and six clones specific to the ventral iris. Two of these, LeR-1 and VeR-1, were structurally characterized. Comparison of their primary structure with data from the Gene Bank showed no essential homology with the known sequences. Time-related changes in LeR-1 and VeR-1 expression were shown during lens regeneration. LeR-1 and VeR-1 expression was activated at the early stages of lens regeneration. The peaks of LeR-1 and VeR-1 expression were observed on the 14th day of lens regeneration in the dorsal and ventral iris zones, respectively. Furthermore, LeR-1 is activated during retina regeneration. The results of Southern hybridization suggest the presence of sequences complementary to LeR-1 in the genomes of Pleurodeles waltl and Rana temporaria. We propose that the activation of LeR-1 expression is related to the triggering of lens regeneration, while the activation of VeR-1 expression accompanies the inhibition of proliferative activity in the ventral iris zone.

Cortical upper layer neurons derive from the subventricular zone as indicated by Svet1 gene expression.

The cerebral cortex is composed of a large variety of different neuron types. All cortical neurons, except some interneurons, are born in two proliferative zones, the cortical ventricular (VZ) and subventricular (SVZ) zones. The relative contribution of both proliferative zones to the generation of the diversity of the cortical neurons is not well understood. To further dissect the underlying mechanism, molecular markers specific for the SVZ are required. Towards this end we performed a subtraction of cDNA libraries, generated from E15.5 and E18.5 mouse cerebral cortex. A novel cDNA, Svet1, was cloned which was specifically expressed in the proliferating cells of the SVZ but not the VZ. The VZ is marked by the expression of the Otx1 gene. Later in development, Svet1 and Otx1 were expressed in subsets of cells of upper (II-IV) and deep (V-VI) layers, respectively. In the reeler cortex, where the layers are inverted, Svet1 and Otx1 label precursors of the upper and deeper layers, respectively, in their new location. Interestingly, in the Pax6/small eye mutant, Svet1 activity was abolished in the SVZ and in the upper part of the cortical plate while the Otx1 expression domain remained unchanged. Therefore, using Svet1 and Otx1 as cell-type-specific molecular markers for the upper and deep cortical layers we conclude that the Sey mutation affects predominantly the differentiation of the SVZ cells that fail to migrate into the cortical plate. The abnormality of the SVZ coincides with the absence of upper layer cells in the cortex. Taken together our data suggest that while the specification of deep cortical layers occurs in the ventricular zone, the SVZ is important for the proper specification of upper layers.

Inductive interactions regulating body patterning in planarian, revealed by analysis of expression of novel gene scarf.

Subtractive hybridization was used to search for the early difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. A sequence (named scarf) specific for posteriorly regenerating tissue was isolated, encoding a soluble C-type lectin consisting of two slightly different carbohydrate-recognition domains. Such an unusual bivalent structure allows attribution of the protein to a novel subfamily of C-type lectins. scarf expression in intact planarian is rather abundant and follows a characteristic pattern not linked to any known morphological structure. We performed a series of experiments using scarf as a molecular marker. Its expression was monitored during different types of regeneration by whole-mount in situ hybridization and reverse-transcription polymerase chain reaction. The obtained data suggest that scarf expression is positively regulated by anterior tissues closely adjacent to the scarf-expressing region, so that their surgical removal results in rapid scarf switch-off. In turn, tissues expressing scarf seem to inhibit its activation anteriorly. This indicates that at least some of the body patterning events in planarians are based on a system of reciprocal inductive interactions rather than on a global morphogen gradient.

Inverted terminal repeats permit the average length of amplified DNA fragments to be regulated during preparation of cDNA libraries by polymerase chain reaction.

A simple polymerase chain reaction (PCR)-based technique for construction of cDNA libraries starting with very small amounts of cells or tissues is described. The technique is based on the insertion of inverted terminal repeats into amplified cDNAs which permit short molecules to generate "pan"-type structures at each cycle of PCR amplification and thus to escape annealing with primers. This allows one to avoid amplification of primer dimers and makes it possible to perform oligonucleotide tailing of the first cDNA strands followed by PCR amplification in the same tube. Moreover, the average size of amplified cDNAs can be controlled by varying the primer concentration.