Frank Hoover

Retinoid X receptor γ gene transcripts are expressed by a subset of early generated retinal cells and eventually restricted to photoreceptors

We have examined the distribution of the retinoid X receptor γ (RXRγ) in the developing chicken retina by using in situ hybridization and RNase protection assays. We detected RXRγ transcripts as early as 4 days of embryonic development (d4) in central regions of the retina, spreading to more peripheral regions by d8. The first few RXRγ‐positive cells were scattered within the depth of the retinal neuroepithelium, but as they increased in number they became localized predominantly to the apical (outer, ventricular) layer. The identity of the RXRγ‐positive cells at these stages is unknown, due to the lack of cell type‐specific markers. By d10, when photoreceptors and ganglion cells have been generated and begun to establish their definitive layers, RXRγ‐positive cells were virtually restricted to the photoreceptor layer, and maintained this distribution to posthatch stages. RNase protection assays were performed on whole retinae to verify the temporal pattern of in situ hybridization results and showed that between d5 and d16 there was a significant increase in the mRNA levels of the RXRγ2 isoform. Between d16 and early posthatch stages the level of RXRγ2 mRNA did not change significantly. Consistent with previous studies, mRNA levels of the RXRγ1 isoform were substantially lower than mRNA levels of the RXRγ2 isoform at all time points examined. These results demonstrate that RXRγ mRNA is expressed in photoreceptors in the developing chicken retina and implicate RXRγ as the earliest marker of photoreceptor differentiation documented to date. J. Comp. Neurol. 391:204–213, 1998.

Regional pattern of retinoid X receptor-? gene expression in the central nervous system of the chicken embryo and its up-regulation by exposure to 9-cis retinoic acid

We have investigated the expression of the retinoid X receptor-alpha (RXRalpha) gene in the developing chicken embryo by using nonradioactive wholemount in situ hybridization. At the earliest stage of development examined (stage 9; Hamburger and Hamilton [1951] J. Morphol. 88:49-92), we detect RXRalpha transcripts in a stretch of neuroepithelium corresponding roughly to the presumptive caudal hindbrain. Upon formation of the rhombomeres at stage 12, a strongly RXRalpha-positive region extends from a sharp rostral limit at the boundary between rhombomeres 6 and 7 caudad to at least the level of somite 9. This pattern of highest expression continues at least until stage 22 but with some variability in the caudal extent. A lower level of expression extends throughout the spinal cord. Transverse sections show that RXRalpha transcripts are expressed in a gradient, with the highest levels near the roof plate and decreasing toward the floor plate. At later stages, the level of expression is highest in the proliferative ventricular zone. However, at reduced levels, RXRalpha transcripts are also detectable in the mantle zone as well as outside the developing central nervous system, for example, in the neural crest and the limb buds. Nine-cis-retinoic acid up-regulates RXRalpha transcripts at stages 19.5-22.0 within a few hours, augmenting but not expanding the expression pattern. Northern blots demonstrate the potential expression of multiple RXRalpha isoforms in the central nervous system at posthatch stages. These results implicate the RXRalpha receptor in both rostrocaudal and transverse patterning of the neural tube.

Inducible expression of the GLT-1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake

Inducible expression of the mammalian glial cell glutamate transporter GLT‐1 has been established in a CHO cell line selected for low endogenous Na+‐dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline‐containing medium, to activate GLT‐1 expression via the Tet‐On system, increased uptake of the GLT‐1 substrate d‐aspartate 280‐fold, and increased cell size. Applying glutamate to whole‐cell clamped, doxycycline‐treated cells evoked a transporter‐mediated current with characteristics appropriate for GLT‐1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT‐1, the most abundant glutamate transporter in the brain.

Differential expression and regulation of the PKA signalling pathway in fast and slow skeletal muscle.

To identify intracellular signalling pathways that transduce muscle electrical activity, we have investigated the Protein Kinase A (PKA) pathway in fast and slow skeletal muscle. The slow soleus muscle (SOL) displayed approximately twice as much PKA catalytic activity and cAMP-binding compared to the fast Extensor Digitorum Longus (EDL) muscle. These results were confirmed by Western blot analysis using antibodies directed against the catalytic or regulatory subunits of PKA. PKA subunits were concentrated at the neuromuscular junction in innervated and denervated muscle fibers demonstrating that PKA is expressed post-synaptically. In addition, we also detected PKA subunits outside the junctional area, suggesting that PKA functions outside of the synaptic regions. Following denervation, levels of cyclic AMP, PKA C activity, R cAMP-binding and RI alpha protein levels increased significantly in the SOL, in contrast to the EDL where only elevated levels of RI alpha protein were observed. These observations demonstrate that PKA levels in skeletal muscle are subject to control at several levels and suggest that some of the differences may be in the pattern of electrical activity that motoneurons impose on the SOL and EDL.

Quantitative assessment of retinoid signaling pathways in the developing eye and retina of the chicken embryo

Retinoid signaling has been implicated as an important regulator of retinal development and differentiation. We have used state of the art high‐pressure liquid chromatography to identify and quantitate biologically active retinoids, immunohistochemistry to localize the retinoic acid synthetic enzyme retinaldehyde dehydrogenase 2 (RALDH2), and nucleic acid assays to quantitate and localize retinoid receptor gene transcripts in the developing eye and retina of the chicken. Our results demonstrate spatial distinctions in retinoid synthesis and signaling that may be related to laminar differentiation in the developing retina. Retinoic acids (RAs) and their precursor retinols (ROHs) are the predominant retinoids in the developing eye. All‐trans‐RA and all‐trans‐3,4‐didehydro‐RA are present in the neuroepithelium in approximately equal amounts from early stages of neurogenesis until shortly before hatching. The retinoid X receptor (RXR) ligand 9‐cis‐RA is undetectable at all stages; if present, it cannot exceed a small percentage of the total RA content. RAs are not detected in the pigment epithelium. All‐trans‐ROH is present in the neuroepithelium and pigment epithelium, whereas all‐trans‐3,4‐didehydro‐ROH is detected only in the pigment epithelium and/or the choroid and sclera. RALDH2 immunoreactivity is intense in the choroid, low or absent in the pigment epithelium, and moderate in the neuroepithelium, where it is highest in the outer layers. Transcripts of all five chicken retinoid receptor genes are present in the neural retina and eye throughout development. During the period of neurogenesis, at least three of the receptors (RARγ, RXRγ, RXRα), exhibit dynamic patterns of differential localization within the depths of the neural retina. J. Comp. Neurol. 436:324–335, 2001.

RXRγ gene is expressed by discrete cell columns within the alar plate of the brainstem of the chicken embryo

With in situ hybridization assays, we mapped the distribution of retinoid X receptor γ (RXRγ) gene transcripts in the central nervous system of the chicken embryo. Previous studies have demonstrated the presence of RXRγ transcripts in migrating neural crest and in neural crest derivatives throughout the peripheral nervous system, implicating RXRγ as an early pan‐neural crest marker (Rowe et al. 1991 . Development 111:771–778), and in the retina (Hoover et al. 1998 . J Comp Neurol 391:204–213). Here we report the presence of RXRγ transcripts in discrete regions of the developing neural tube, within the hindbrain, the cerebellar plate, the optic tectum, and the diencephalon. At stage 10, when migrating neural crest expresses RXRγ transcripts, we detect no transcripts in the neural tube. By stage 13, RXRγ transcripts accumulate to detectable levels along the midline of the posterior optic tectum, where the neural crest‐derived sensory neurons of the mesencephalic trigeminal nucleus are located. By stage 15, RXRγ transcripts also appear in an intermittent longitudinal cell column within the mantle zone of the alar plate of the hindbrain, eventually extending into the cerebellar plate rostrally and into the cervical spinal cord caudally, with a gap at about rhombomere 3. By stage 19, transcripts appear in a discrete population of cells within the diencephalon. Expression in these cell populations continues until at least stage 22.5, when many neuron populations have been generated in the hindbrain. The localization of the RXRγ‐positive cells to the mantle zone suggests that they are postmitotic and are probably neurons. Their specific alar locations indicate that they reside within sensory columns and potential downstream targets, evidently corresponding to some of the central components of the trigeminal system. J. Comp. Neurol. 416:417–428, 2000.

Electrical Muscle Activity Pattern and Transcriptional and Posttranscriptional Mechanisms Regulate PKA Subunit Expression in Rat Skeletal Muscle

We have examined protein kinase A (PKA) subunit expression in adult rat skeletal muscles. Northern blots identified PKA catalytic alpha and regulatory (R) I alpha and RII alpha subunits as the major subunits expressed in slowly contracting soleus (SOL) and rapidly contracting extensor digitorum longus (EDL) muscles. In addition, the steady-state RNA levels of PKA subunit mRNAs and activities of RI alpha and RII alpha promoters are similar in SOL and EDL. These data indicate that posttranscriptional mechanisms account for the twofold differences in PKA subunit protein levels reported earlier. Electrical stimulation of denervated SOL with an EDL-like activity pattern (fast pattern) transformed SOL into an EDL-like muscle with regard to PKA protein levels. These experiments suggest that the posttranscriptional regulation is activity pattern-dependent. Denervation specifically increased RI alpha promoter activity and RI alpha mRNA levels in SOL and EDL. Further experiments indicated that the RI alpha 1a upstream sequences were activated following denervation. Direct electrical stimulation prevented the rise in RI alpha mRNA levels following denervation, demonstrating that electrical muscle activity regulates transcription.

Denervation increases protein tyrosine kinase and protein tyrosine phosphatase activities in fast and slow skeletal muscle

We have investigated the expression and regulation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in fast extensor digitorum longus (EDL) and slow soleus (SOL) in adult rat skele-tal muscles. Biochemical assays revealed significantly greater PTK and PTP activities in SOL than in EDL; these results were confirmed and extended by in-gel assays demonstrating that the PTKs and PTPs detected had higher activity levels in SOL compared to EDL extracts. Although phosophotyrosine proteins were concentrated at the neuromuscular junction (NMJ), PTK and PTP activities were observed in extra-junctional regions of the muscle fiber. Following denervation, we observed significant increases in PTK and PTP activities in both SOL and EDL, and gel-based assays showed an increase in the activities of several PTKs and PTPs. These results suggest that the same PTK and PTPs have different activity levels in fast and slow skeletal muscles and are regulated by nerve-dependent mechanisms.

True to Its Roots

S implicity is a virtue in computer programs. But as software developers have expanded the functionality of their offerings, there has often been a price to pay: an increase in the programs size and sometimes a decrease in its speed. Synergys KaleidaGraph (version 3.5) is one application that has resisted these tendencies. With the latest version of KaleidaGraph, a user can rapidly transform data to readily create graphs. Best of all, the program takes up little space and has minimal RAM requirements. For example, under Mac OS 9, KaleidaGraph needs about 5 MB of RAM and less than 10 MB of hard disk space. The software has kept pace with the newer, faster computer processors and has added many new functions while retaining ease of use and maintaining consistency with older versions of the program. Synergy provides KaleidaGraph on both Macintosh- and Windows-compatible platforms. Collaborators on different platforms can trade KaleidaGraph files without confusion or loss of data. The KaleidaGraph program itself and a useful tour of the software both install easily from a CD. The multilingual “quick-start” guide circumvents the bulky manual and provides even novices with almost instant access to the program. Upon opening KaleidaGraph, one is presented with a data window containing columns and rows reminiscent of the Excel spreadsheet. After entering the data into the cells, the user is only a few clicks away from making the first graph. The program has a well-designed, graphic user interface and provides numerous options for modifying graphs. For example, to change the parameters of the abscissa ( y axis) of a plot, one double-clicks on the abscissa and a new window appears showing the various options. Lines can be made thicker, and text and simple objects can be added by selecting the appropriate tool in the tool palette. The figure below shows a sample graph created by the program, complete with the error bars and statistics contained within the graph boundaries. Quickly prepared presentation graphs can easily be reworked into publication-quality documents for printing. The ease of graph generation in KaleidaGraph does not come at the cost of power. KaleidaGraph can work with millions of data points quickly and effortlessly. Veteran users of KaleidaGraph will enjoy the 100 new features in version 3.5. In earlier versions of the program, data entry from other software products into KaleidaGraph occurred mostly by copying and pasting. However, data entry has now been simplified and expanded with the programs ability to open Excel worksheets directly. In addition, four new plot types have been added. KaleidaGraph routinely calculates basic statistics on every data set to include averages, standard deviations, and standard errors. A welcome addition is that of the Students t tests. Synergy has indicated that an expanded repertoire of statistical functions may follow in the future if the demand from users is sufficiently high. KaleidaGraph can also perform basic ( y = m x + b) and complex curve fits from a library of 100 industry-specific algorithms. The programs designers have also increased the range of files that KaleidaGraph can import and export. These include GIF, JPEG, PNG, TIFF, and Windows Bitmap files. There is also a calculator built into the program, if a quick function check is required. KaleidaGraph is a simple program that has remained true to its roots and has not strayed from its original design—namely allowing users to create graphs on a user-friendly software. The range of graphing solutions with KaleidaGraph is impressive in view of its low price. Demo versions and purchase, upgrade, and cross-grade information may be downloaded at the Synergy Web site.