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A TRANSGENIC MOUSE CLASS-III β TUBULIN REPORTER USING YELLOW FLUORESCENT PROTEIN

. Author manuscript; available in PMC: 2010 Jan 30.

Published in final edited form as: Genesis. 2007 Sep;45(9):560. doi: 10.1002/dvg.20325

Abstract

A yellow fluorescence protein (YFP) reporter construct was cloned downstream of the β-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, noteably in regions of the brain undergoing adult neurogenesis, i.e., the rostral migratory stream and sub-granular layer of the dentate gyrus. This reporter-based staining was compared with anti-class-III β-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.

Keywords: beta-tubulin III, YFP, transgenic mouse, axonal outgrowth, neuronal differentiation, TuJ1

INTRODUCTION

β-tubulins are encoded in vertebrate genomes by a family of seven to eight functional genes (Luduena, 1998; Sullivan, 1988). Their protein products have been assigned to six distinct isotypic classes (I–VI) that differ primarily, but not exclusively, by the approximately last 15 carboxy-terminal amino acids. In cells of normal, late embryonic, postnatal, and adult mammalian tissues, the Class III β-tubulin isotype (gene symbol: Tubb3) is detectable in only two cell types, terminally differentiated neurons of the central and peripheral nervous systems and the Sertoli cells of the testes (Lee et al., 1990a). In neurons, its expression is coincident with the terminal mitosis of neuronal precursors (Easter et al., 1993; Lee et al., 1990a; Lee et al., 1990b; Moody et al., 1989) . Accordingly, β-tubulin III has been used extensively as a marker for neuronal differentiation. In contrast, β-tubulin III is aberrantly expressed in high-grade gliomas, and a number of other cancers, where its presence is associated with drug resistance and is predictive of aggressiveness and unfavorable prognosis (Ferrandina et al., 2006; Kamath et al., 2005; Katsetos et al., 2001; Mozzetti et al., 2005; Seve et al., 2005) . Currently, no published resources in the mouse have been constructed to exploit this interesting gene.

Herein, we present a characterization of two transgenic mouse lines in which yellow fluorescent protein (YFP) accumulation is under the control of the Class III β-tubulin gene promoter. We demonstrate that YFP accumulation mirrors to a remarkable extent the accumulation of β-tubulin III. The establishment of this transgenic mouse line provides a very useful experimental tool for studying specifically β-tubulin III expression, and more generally neuronal development and differentiation.

RESULTS

Using the construct illustrated in supplementary material, two transgenic founder lines, beta tubulin line 1 (BTL1) and 2 (BTL2), were produced and made homozygous for the transgene for this analysis. In general, the regional expression patterns of YFP in the nervous system at all ages were similar in these two lines with BTL2 yielding much stronger signals than BTL1 (Fig. 1a, b).. Homozygous transgenic mice showed marked increase in YFP expression compared to heterozygous transgenics in both lines (Fig. 1c, d). YFP expression was mainly observed in the peripheral and central nervous system during the embryonic and the early postnatal times. Then as the animals reached adult age the expression levels decreased, but many neurons still demonstrated YFP expression noteably in regions of the brain undergoing adult neurogenesis, i.e., olfactory bulb, the rostral migratory stream and sub-granular zone of the dentate gyrus. YFP was clearly observed in the neuronal cell bodies and dendrites and axons in the central and peripheral nervous system traveling toward their targets. This labeling pattern of cell bodies, axons and dendrites persisted in YFP-positive cells in the adult, although to a more limited degree. The co-labeling of tissue for immunocytochemistry for β-tubulin III and YFP demonstrates that the distribution pattern of YFP positive cells coincided with β-tubulin III immunostaining. This coincidence of YFP expression and β-tubulin III was exclusive to neurons and there was no co-localization of YFP with glial cell markers such as glial fibrillary acidic protein (GFAP), Rip, 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase (CNPase) and MAC1 (Fig.2) during the embryonic stage as well as in the adult.

Fig.1.

Fig.1

YFP expression in homozygous and heterozygous mice of the two transgenic lines BTL1 and BTL2 at postnatal day 0. (a–b): BTL2 shows greater numbers of YFP-positive cells compared to the BTL1 cortex (a vs b); YFP positive cells are largely relegated to layers III, IV and V in BTL1 transgenic mouse (a), while all layers of cortex demonstrate YFP positive cells in the BTL2 transgenic (b). (c–d): Heterozygous transgenic mice show much weaker YFP expression in the cortex compared to the homozygous transgenic mice. Bar= 100µm.

Fig. 2.

Fig. 2

Double immunostaining shows that YFP expressing cells are not co-localized with glia cells. (a, c and e): YFP and anti GFAP immunostaining, to label astroglia, demonstrate that YFP expressing cells are not co-stained with GFAP in spinal cord (c, arrows) and CA3 area of hippocampus (e, arrows) at P0. (b, d and f): YFP and anti Rip or anti-CNPase immunostaining, to label oligodendroglia, show that oligodendroglia are also not YFP-positive. b and d show anti Rip immunostaining in spinal cord at P0 (arrows in d point Rip-positive elements), f shows anti-CNPase positive cells in the sub-ventricular zone of adult mouse (arrows). (g and h): YFP and anti-MAC1, to mark microglia, also show no co-staining (g, sub-ventricular zone and h, hypothalamus with arrows pointing to MAC1 positive cells). Bar=200µm in a and b, 50µm in c–h. These images are from the BTL line 2.

Embryonic Days 9.0–9.5(E9.0–E9.5)

Numerous YFP expressing cells were found throughout the peripheral and certain central nervous system at E9.0–E9.5 (Fig.3). Dorsal root ganglion (DRG) cells and their processes show strong YFP expression (Fig. 3a, c and ciii). In the CNS, YFP expression was observed in radial cohorts of midbrain neurons that extend from near the ventricular surface to the pia and from midline out laterally (Fig. 3a, c, ci and d). TuJ1 immunostaining was observed in the neuronal cell bodies and processes (Fig. 3b, d and e), while YFP expression was detected shortly thereafter (within 12 hours), first in the cell body and then in the dendrite and axon as well (Fig. 3d and e). Compact YFP-positive cell bodies were also located in the hindbrain, with the greatest number of cells located medially. Strong YFP expression is found in the trigeminal ganglion (V), facio-acoustic ganglia (VII–VIII), glossopharyngeal, vagal and spinal accessory (IX–XI) ganglia cell bodies as well as their axons that travel in the central and peripheral directions (Fig.3a, c). A sagittal view of the spinal cord (Fig.3cii) shows dorsal and ventral neurons separated by axon bundles traveling up and down the spinal cord. There were also positive cells located in the olfactory placode and optic cups (Fig.3a and c).

Fig.3.

Fig.3

YFP and anti- β-tubulin III immunocytochemistry demonstrate co-labeling of early neural structures in the E9.5 whole mount embryo (a, b and c). These low magnification images provide an overview of the cells and processes in mid- and hindbrain, spinal cord and peripheral nervous system that have YFP expression (a) and are stained by TuJ1 (b). There is extensive YFP expression observed in midbrain (mb) and hindbrain (hb), trigeminal (ganglion V), facio-acoustic (ganglia VII–VIII), and glossopharyngeal-vagal (IX–X) ganglia cell bodies as well as their axons that travel in the central and peripheral directions. Positive cells are found in the olfactory placode (op) and the optic cup (oc). These areas largely overlap with anti-TuJ1 immunocytochemistry (c). (ci–iii): Double labeling in the boxed areas in (c) is shown at higher magnification in panels ci–ciii. Beta tubulin III is localized in the cytoplasm with strong expression in cell processes while most YFP is found largely in the cell body. Images of double-labeled cells are shown from midbrain (ci), upper dorsal part of the cervical spinal cord (cii), and dorsal root ganglion (ciii). In early developing systems, YFP is also present in axons as illustrated in the spinal accessory nerve (cii, arrows). (d–e): Low and high magnification images from the E9.0 midbrain show that TuJ1 is in the cytoplasm and neuronal processes while YFP expression is first seen mainly in the cell body (arrows in e). In more mature neurons, YFP expression is seen in neuritic processes (arrowheads in e). (f): TuJ1 and YFP expression is shown in the ventral part of the spinal cord in an E9.0 embryo to further demonstrate the developmental evolution of YFP expression relative to Tuj1 immunolabeling. While there are less mature cells that only stain for Tuj1 (dorsally, to the right in the figure), in more mature neurons (ventral spinal cord, to the left), YFP expression is found in most TuJ1 positive cells (arrows) and dendrites (arrowheads). Bar=100 µm in ci–iii and d, 50µm in e and f. These images are from BTL line 2

Embryonic Day 10.5–12.5 (E10.5–E12.5)

Compared to E9.0–E9.5, strong YFP expression was found in the forming neocortex and retina while the midbrain, pons, medulla oblongata, spinal cord and DRG continued to have many YFP-positive cells (Fig.4a–e). Some cells in the follicles of vibrissae were YFP positive as well (Fig.4a). In the spinal cord, YFP positive cells were largely localized in the ventral horn but an emerging population of dorsal horn neurons was apparent (Fig.4b, c and d). YFP was clearly observed in cell bodies and their cell processes (Fig.4c and d).

Fig.4.

Fig.4

YFP expression in the mid-embryonic stage of development of the nervous system. (a): Whole mount embryo at E12.5, to show YFP expression (green, with the general cell stain TOTO3 in blue). Strong YFP expression is found in the forebrain (fb), midbrain (mb), pons (p), medulla oblongata (mo), spinal cord and DRG (arrows). YFP labeled cells are observed in the optic cup ( oc) and follicles of vibrissae (f) as well. (b–d): In the spinal cord, YFP positive cells are mainly located in the large motor neurons of the ventral horn (vh, arrows in c). The inset in d shows a region of the ventral horn where neurons are co-labeled with YFP (green) and β-tubulin III immunocytochemistry (red). (e): Sagittal sections showing that most neurons in DRG are most YFP positive cells are co-labeled with anti- β-tubulin III and shown at higher magnification in the inset. (f–g): Sagittal view of cortex shows labeling in the E15.5 developing forebrain as well as cells in the forming cortical plate (cp). These neurons are shown at higher magnification in g. dh=dorsal horn, drg=dorsal root ganglia, t=Thalamus, ac=anterior commissure, mfb=medial forbrain bundle. Bar=100µm. These images are from BTL line 2.

Embryonic Day 15.5 (E15.5)

YFP expressing neurons and the tracts of nerve fibers were increased in number and intensity in the midbrain, diencephalon and forebrain (Fig.4f and g). More neurons in the dorsal horn of the spinal cord were expressing YFP . In the retina, most YFP expressing cells were found in the inner neuroblastic layer of retina (ganglion cells and inner nuclear layer) with no YFP expression found in the outer neuroblastic layer (Fig.7a–c).

Fig.7.

Fig.7

YFP expression in the eye at E15.5, P0.5 and adult. (a–c): At E15.5 YFP positive cells are located in the inner neuroblastic layer of retina (ganglion cells and inner nuclear layer, white dots indicated the division of inner and out nuclear layers), no YFP expression is seen in the outer neuroblastic layer at this age. (d and e): At P0.5 the retinal ganglion cells are most heavily labeled with YFP and β-tubulin III immunostaining. In addition cells of the inner nuclear layer are also YFP-positive and lightly β-tubulin III immunopositive. White dashed line marks the outer neuroblastic layer (onl). (f): In the adult, YFP expression is found in the ganglion cells of the retina and show prominent double labeling with the β-tubulin III antibody. Note: In the transgenic lines that were made, the background genotype (FVB) of the host mice carries the gene for retina degeneration (Gimenez and Montoliu, 2001), and the outer nuclear layer of the retina undergoes degeneration during the postnatal period and is therefore absent in the adult. Currently, we are backcrossing these mice onto the C57BL/6J genetic background to eliminate the retinal degeneration allele. v = vitreous. gc= Layer of ganglion cells. ipl= Inner plexiform layer. inl= Inner nuclear layer, onl= outer neuroblastic layer, ib= Inner border of outer neuroblastic layer. Bar=50 µm. These images are from BTL line 2

Postnatal Day 0.5(P0.5)

In the cortex, YFP positive neurons and their dendrites and axons were clearly observed in the layer II–VI (Fig.1b, Fig.5a). In the cerebellum, strong YFP positive cells were observed in the inner granular cell layer, Purkinje cells were YFP-positive as well (Fig.5b). Some cells in the medullary region of cerebellum also expressed YFP (Fig.5b). In the spinal cord, motor neurons in the ventral horn had decreased YFP expression compared to earlier developmental stages and more YFP positive cells were observed in the dorsal horn (Fig.5c). DRG neurons and their central and peripheral processes were still YFP positive (Fig.5d).

Fig.5.

Fig.5

YFP expression at postnatal day 0.5. (a): In the cortex, strong YFP positive cells are still observed in layers II, III. IV, V and VI. Nearly all cells are also immunopositive for β-tubulin III (inset, double labeled cells are yellow). (b): In the cerebellum, YFP cells are observed in the inner portion of the external granular cell layer (egl), in Purkinje cells (p, arrowheads) and some cells in the medullary region (m). The inset shows Purkinje cells that are positive for YFP and β-tubulin III. (c): In the spinal cord, YFP cells are observed in the ventral and dorsal horns. (d): DRG cells still demonstrate strong YFP expression. dh=dorsal horn, vh= ventral horn. CTX= cortex, CB= cerebellum, SC= spinal cord, DRG= dorsal root ganglia. Bar=100 µm. These images are from BTL line 2.

Adult

The level of YFP expression decreases as the mice age with the obvious exception of the regions of CNS where adult neurogenesis occurs, such as cells in the olfactory bulb (Fig.6a) and dentate gyrus (Fig.6b) which arise from the rostral migratory stream (RMS) and subventricular zone, respectively (Fig.6e, f and g). Neurons throughout the rest of the brain still showed YFP expression, but levels were weaker compared to developing stages. (Fig.6c and d). Many YFP positive cells were found in the retinal ganglion cell layer (Fig.7f).

Fig.6.

Fig.6

YFP expression in regions of the adult brain. (a): YFP-positive cells and dendrites are found in the Mitral cell layer (m, arrows point to positive cells in this region) and external plexiform layer (ep) of the olfactory bulb. (b):YFP-positive cells in the dentate gyrus (DG) are still observed in smaller cells in the hilus (h, arrows). (c): Many YFP-positive neurons are still observed in cortex. (d): In cerebellum, Purkinje cell bodies show YFP expression (arrows). (e): YFP-positive cells are seen in the rostral migratory stream denoted by arrows that travel from anterior to posterior. (f and g): Higher magnification of the boxed areas in (e) are shown. The migrating neurons in the RMS are obvious. OB=olfactory bulb. DG=dentate gyrus, h=hilus, m=Mitral layer of olfactory bulb, ep=external plexiform layer. RMS=rostral migratory stream. Bar=50 µm. These images are from BTL line 2

DISCUSSION

We find expression of YFP in developing and differentiating neurons of the CNS and PNS of our transgenic mice that largely reproduce immunocytochemical methods that tag endogenous β-tubulin III expression (Easter et al., 1993) . By observing YFP expression at various timepoints, we are able to track neuronal development and differentiation in neuronal cell bodies, axons and dendrites from early times in development through early stages of postnatal life. In the adult, neurons in many regions of the brain still show YFP expression with less intensity compared to developing stages. Of our two transgenic lines, YFP expression in Line 2 was found to be most robust in differentiated neurons. The reason why one line should have higher expression than the other is likely due to the nature of the integration of the transgene into the host genome. This has been discussed by Feng et al to account for the rather extreme variability in reporting Thy1 expression in twenty-five transgenic lines (Feng et al., 2000). Transgene expression can also be affected by the number of copies of the transgene that have become integrated, the site of chromosomal integration or other factors such as position effect variegation (see: Dorer and Henikoff, 1997; Garrick et al., 1998; Robertson et al., 1995; Sabl and Henikoff, 1996) . The difference in expression is not accompanied by any aberration in development as both transgenic lines develop normally and no YFP-induced toxicities are found. Both lines also reproduce very well and there have been no problems in maintaining the colonies. Thus, these transgenic lines of mice allow rapid identification and direct visualization of differentiating neurons that can have utility in studying nervous system development.

There are both minor spatial and temporal differences between endogenous β-tubulin III expression and the YFP reporter. In early development (E9.0), β-tubulin III immunocytochemistry shows that the protein is found in neurites and cytoplasm while YFP is mainly found in the cell bodies with limited expression in neuronal processes. This difference in early localization of YFP is only for a short period of time and may be due to the fact that YFP is a foreign, non cytoskeleton protein compared to β-tubulin III which is an integral cytoskeletal component of the cell. In addition to possible differences in the transport of these proteins, the differences in the quantities of YFP and β-tubulin III might also result in a disparity between the intracellular localization of these two proteins. At later developmental times and in the adult, YFP expression is clearly observed in the cell bodies and dendrites as well (Fig5a and d, Fig.6a,c, f and g). In addition, although cells are found to largely co-express YFP and β-tubulin III, it is notable that the time frame of expression is slightly offset between the immunocytochemical detection of β-tubulin III and YFP expression; that is, immunocytochemical staining for β-tubulin III was found to precede YFP expression while YFP expression is still present for a short period when anti- β-tubulin III staining is less obvious. For example, anti- β-tubulin III immunopositive neurons are present in the dorsal horn of the spinal cord before we find YFP positive neurons in the same area. The mechanisms causing the timing differences at the earliest embryonic stage, and in adulthood , are unknown. The most obvious possibility is that the YFP construct may not contain all of the regulatory elements present in the endogenous β-tubulin III promoter and introns. Second, a less likely cause are differences in protein turnover effecting the kinetics of YFP and β-tubulin III accumulation.

The present results also provide insight into the regulation of β-tubulin III by the zinc-finger transcription factor NRSF (neuron-restrictive silencing factor), whose function, in part, is to repress the expression of neuron-specific genes in non-neuronal cells (Chong et al. , 1995; Lunyak and Rosenfeld, 2005; Schoenherr and Anderson, 1995). NRSF binds to a conserved ~21 bp element called an NRSE (neuron-restrictive silencer element), and exerts its influence by recruiting enzymes that alter chromatin structure (Lunyak and Rosenfeld, 2005). The mouse β-tubulin III NRSE is located in the first intron (Schoenherr et al., 1996). Anderson and colleagues (Chen et al., 1998; Schoenherr and Anderson, 1995; Schoenherr et al., 1996) have shown that the β-tubulin III gene is regulated in part by the NRSF. It is interesting to note that in this work, the β-tubulin III -YFP construct introduced into the mouse did not include the NRSE, The neuronal accumulation, as well as the lack of non-neuronal accumulation of YFP, provides strong evidence that the control of β-tubulin III expression is not solely dependent on NRSF (Dennis et al., 2002). One possible mechanism by which this may occur has been presented previously (Chen et al., 1998). A further comparison of the rat (Dennis et al., 2002) and mouse β-tubulin III promoters shows that both upstream regions possess similar classes of transcription factor binding sites including AP2, C/EB, E-box, and SP1 sites, although the precise locations upstream from the TATA box are often not identical (Dennis et al., 2002; Uittenbogaard and Chiaramello, 2002). However, the single central nervous system enhancer site is located at a highly similar position upstream of the TATA box. The purine/pyrimidine repeat element (GTTTT)9 is not found in the mouse. Similarly, we did not find identical rat Pit-1 or PEA-3 binding sites in the mouse promoter. A precise role for these elements in neuronal-specific expression is unknown.

Reporter transgenic lines of mice have been generated under the control of different promoters such as beta-actin-GFP mice (Hadjantonakis et al., 1998; Okabe et al., 1997) , thy-1-XFP mice (Feng et al., 2000) and Nestin-GFP transgenic mice (Kawaguchi et al., 2001; Mignone et al., 2004). Nestin-GFP mice have been used in studying neural stem and progenitor cells in the developing and adult nervous systems and those GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain (Kawaguchi et al., 2001; Mignone et al., 2004; Sawamoto et al., 2001) . In this paper we present β-tubulin III - YFP transgenic lines that offer similar promise as experimental tools to mark the conversion of neuronal precursor cell into fully differentiated neurons (Jiang and Oblinger, 1992), the acquisition of populations of differentiating neurons in developing and adult tissues using cell sorting methods, and the prelabeling of specific populations of cells for in vitro analysis to transplant into the adult or developing CNS to examine the behavior of differentiating neurons in a novel environment. Furthermore, because β-tubulin III expression is up-regulated in adult mice after nervous system injury (Geisert and Frankfurter, 1989), the transgenic mice could be used to examine the neuronal response to the injury. In addition, this transgenic line could be used as a reporter line to indicate neuronal responses after genetic or environmental perturbations. In conclusion, these transgenic mice provide an excellent research tool to study neuronal development and differentiation.

MATERIALS AND METHODS

Construction of Vectors

Mouse Class III β-tubulin genomic BAC DNA was ordered from Children’s Hospital Oakland Research Institute http://bacpac.chori.org/. The DNA was digested with Sal I and BamH1 to obtain a ~3516 bp fragment corresponding to a small fragment of the coding region of the β-tubulin III gene, the 5’-UTR, and the promoter region extending 3050 upstream of the initiating ATG. This 3516 bp fragment was cloned into pBluescript to create pBSProm. A 338 bp fragment was amplified from pBSProm using polymerase chain reaction [primers 5’-CAAAATGGGGAGGCAGCTACTG-3’ and 5’-TTTTGGATCCCGGATCTCCCTCATGCTGACTTCACG-3’]. Amplification was performed for 30 cycles 94 °C for 30 sec, 60°C for 15 sec 68°C extension for 60 sec. The 338 bp amplification product was digested with BsmB1 and BamH1, yielding a larger and a smaller fragment. The smaller ~ 37 bp fragment was cloned into Sal I and BamH1 double-digested pBluescript along with a 3,037 bp Sal I/ BsmB1 fragment from pBSProm, to create pM β 6MREI. The coding region for yellow fluorescent protein, EYFP, was obtained by digesting pEYFP-N1 (Clontech) with BamH1 and AFlII. The pMβ6MREI was digested with BamH1 and Not I, and the 985 bp EYGFP fragment was ligated in-frame behind the β-tubulin III promoter by the use of a short annealed adapter (5’-TTAAGAATTC-3’ and 5’GGCCAGAATTC-3’) to yield pM β 6MREIYFP.

Making transgenic mice

Fertilized eggs were collected from the FVB/N females mated with FVB/N males and maintained in KSOM and M2 media purchased from Special Media (Phillipsburg, NJ). A 4kb fragment cleaved with SalI and EcoRI (New England Biolabs, MA) was purified through agarose and microinjected into pronuclei of one-cell zygotes at a concentration of approximately 3ng/µl. The surviving embryos from the injection procedure were transferred into pseudopregnant ICR females using standard methodologies at the Transgenic Core Facility of UTHSC.

Histological detection of the YFP expression in development and adult mouse brain

The YFP expression was examined in transgenic mice at different developing time points. Noon of the day of the vaginal plug was designated as day 0.5 (E0.5). Females were sacrificed by cervical dislocation, and the embryos from each stage (E9.0, E9.5, E10.5, E11.5, E12.5, E15.5) were taken out and immersed in 4% paraformaldehyde (PF) overnight at 4°C. Postnatal day 0.5 (P0.5) and adult mice (6–8 week old, n=3) were anesthetized with avertin and perfused transcardially with 4% PF in 0.1 M phosphate buffer (pH 7.4) for 10min. The brains were dissected and post-fixed in the same fixation solution for 4 hours. After rinsing with phosphate buffered saline (PBS), E12.5 and E15.5 embryos and brains from P0 and adults were transferred into 30% sucrose – PBS for 24 hours for cryo-protection, and were sectioned in the cryostat. Twenty µm sagittal sections were collected and stored at −20°C until use. E9.0, E9.5, E10.5 and some E11.5 and E12.5 embryos that were used for wholemount preparation were rinsed in PBS prior to staining.

YFP expression in brain, spinal cord or peripheral nervous system of transgenic mice was examined using a BioRad Confocal microscope using excitation wavelength of 495nm and an emission wavelength of 519nm to examine the YFP expression in the whole mount preparations and embryo sections. An antibody to β-tubulin III (rabbit anti β-tubulin III, or mouse TuJ1, Covance Berkeley, California, 1:1000) was used to document endogenous expression. Glial cells markers were also used to examine the specificity of YFP expression. Those markers were anti-GFAP for astrocytes (1:500, rabbit polyclonal, Sigma, USA), anti-MAC1 for microglia (1:100, rat monoclonal, Serotec, UK), anti-RIP for oligodendrocytes (1:500, mouse monoclonal, Chemicon), and anti synaptophysin for neuronal terminals (1:2000, mouse monoclonal, Sigma).

For wholemounts, embryos were pre-incubated in 3% Triton-X 100 (Sigma, USA) for 1 hr, 1% bovine serum albumin (BSA) in PBS for 1hr at room temperature and then placed in primary antisera with pre-incubation buffer for 48 hr at 4°C. For cut material, sections were incubated in 3% Triton-X 100, 1% BSA in PBS for one hour at room temperature, followed by incubation with primary antibody overnight at 4°C. After rinses in PBS, the sections were incubated in secondary antibody goat anti rabbit conjugated with Alexa 594 (1:400, Molecular Probes Inc., for β-tubulin III or GFAP) or donkey anti- mouse antibody (Alexa 594, 1:400, for TuJ1, or RIP) or donkey anti rat (cy3, 1:500 for MAC1) for one hour at room temperature. After rinsing in PBS, the sections were counterstained with a nuclear marker TOTO3 (2µM, Molecular Probes Inc) for 10 min at room temperature to visualize all cells. Coverslips were applied on slides with anti-fade reagent FluroSave Reagent (Calbiochem, San Diego, CA).

Supplementary Material

01

ACKNOWLEDGEMENTS

This work is supported by the RO1 EY012389, UTRCE in Genomics and Bioinformatics , a Methodist Hospital Neuroscience Endowment and Unrestricted Funds from Research to Prevent Blindness, New York, N.Y.

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