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Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain
Journal of Neuroscience Methods 89 (1999) 1 – 8
Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain W.F. Silverman a,*, A. Alfahel-Kakunda a, A. Dori a, J.L. Barker b a
Department of Morphology, Zlotowski Center for Neuroscience, Ben-Gurion Uni6ersity of the Nege6, Beer She6a, 84105 Israel b Laboratory of Neurophysiology, NINDS, NIH, Bethesda, MD 20892, USA Received 7 July 1998; received in revised form 5 February 1999; accepted 7 February 1999
Abstract The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral ‘tiers’ with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Development; Cell separation; Cell culture; Substantia nigra; Dopamine; Percoll; Pattern
1. Introduction Although poorly understood, regional ‘patterning’ or compartmentalization is known to reflect intrinsic differences in efferent and afferent connections, as well as developmental priorities. Both the medium-spiny neurons of the caudate-putamen, or striatum, and the dopaminergic (DA) cells of the mesencephalon which project to them, are arranged in separate and complementary compartments in their respective areas. Unlike the immediately evident pattern observed in the hippocampus or cerebellum, the division of the mesencephalic and striatal neurons into compartments requires artificial manipulation, e.g. immunohistochemistry for phenotypic markers, to visualize. The significance of compartmentalization in the meso-stri* Corresponding author. Fax: +972-7-277655. E-mail address: [email protected] (W.F. Silverman)
atal pathway appears to lie in the mutually reciprocal, though asymmetric nature of its interconnections, and the essential role each component plays in the etiology of several of the most common and intractable CNS disorders. The DA neurons of the ventral mesencephalon, including the substantia nigra pars compacta (SNc), substantia nigra pars reticulata (SNr) and ventral tegmental area (VTA), are generated in two waves of neurogenesis: Between E12 and E15 and then from E18 until soon after birth. On this basis and with regard to their final topographical positions, efferent and afferent connections (Gerfen, 1984; Gerfen et al., 1987a) and dendritic morphology (Bjo¨rklund and Lindvall, 1975; Fallon and Moore, 1978) the two groups of DA neurons are said to occupy distinct ventral and dorsal compartments or ‘tiers’ (Fallon and Moore, 1978; Veening et al., 1980). Ventral and dorsal tier DA neurons differ as well with regard to expression of
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calcium binding proteins (Gerfen et al., 1987b; Rogers, 1992; Alfahel-Kakunda and Silverman, 1997) and sensitivity to neurotrophic factors (Lindsay et al., 1991; Korsching, 1993; Seroogy et al., 1994). Despite the marked differences in their ontogeny and connectivity, understanding of the function of compartmentalization in this system has been complicated by the inability to study the individual elements. In the present study, we evaluate an approach to isolating the anatomically-mixed, developmentally distinct sub-populations of neurons featuring the fractionation of cell suspensions from the area of interest according to their natural specific buoyant densities. Cell separation by buoyant density fractionation is based on the principle that buoyant densities primarily reflect differences in the ratio of nucleus to cytoplasm (Pertoft and Laurent, 1982). Proliferative cells synthesize DNA and possess less cytoplasm relative to the size of the nucleus than differentiated cells, and hence, accumulate in the lower, i.e. less buoyant fractions of the gradient. Employing this approach, we have succeeded in obtaining fractions enriched in DAergic neurons corresponding phenotypically to ventral and dorsal tiers.
2. Methods
2.1. Animals Timed-pregnant Sprague-Dawley rats were injected i.p. with the halogenated thymidine analog 5-bromo-2%deoxyuridine (BrdU) (5 mg/ml per 100 g body weight) and euthanized 2 h later with CO2 at gestational day 19, i.e. at the time of dorsal tier neurogenesis, and embryos delivered by cesarean section. Embryonic age was calculated from insemination, i.e. the presence of a vaginal plug (day 0) and correlated with crown-rump length (CRL) at delivery.
2.2. Preparation of cell suspensions For each experiment, between 15 and 20 [E18] fetuses (CRL= 23 mm) were removed from the dams and placed into ice-cold Earle’s Balanced Salt Solution (EBSS). The mesencephalic flexure, containing the nascent ventral mesencephalon was rapidly microdissected from each embryo, transferred to fresh, ice-cold EBSS, minced with forceps, and gently triturated. The pooled tissue was then dissociated in 50 units of papain (Worthington Biochemical Corp., Freehold, NJ) in EBSS with 1 mM L-cysteine (Sigma), 0.5 mM EDTA, and 0.01% DNAse (Boehringer Mannheim) at 37°C with gentle agitation for 45 min. The partially dissociated tissue was triturated a second time briefly by repeated passage through a 10-ml pipette. The cells were then spun in an ultracentrifuge
at 1600 rpm for 5 min at room temperature. The pellet thus obtained was resuspended in 2.5 ml of 1 mg/ml trypsin inhibitor and a 1 mg/ml bovine serum albumin solution (‘1/1’). To remove non-viable cells and cellular debris, the suspensions were lightly triturated and the supernatant layered onto 5 ml of a solution containing 10 mg/ml trypsin inhibitor and 10 mg/ml albumin in a 15 ml centrifuge tube. This mix was spun at 800 rpm at room temperature, and the pellet resuspended again in ‘1/1’.
2.3. Buoyant density fractionation Percoll was prepared as a stock solution by mixing nine parts of Percoll to one part of a 10 × physiological salt solution. A 5-step, discontinuous gradient of 10, 35, 40, 45 and 50% Percoll was made by diluting the Percoll stock solution with decreasing concentrations of EBSS, and overlaying one atop the other in a 15-ml centrifuge tube (Fig. 1). Dissociated cells in ‘1/1’ were applied on the top of the gradient and spun in a centrifuge at 1600 rpm for 15 min at 18°C. Cells accumulated in the interface between fractions and were collected with a fire-polished Pasteur pipette. The fractions were washed twice with EBSS, and resuspended in minimum essential medium (MEM) with glutamine, 5% horse serum, 5% fetal calf serum, BSA 0.001%, transferrin 200 mg, putrescine 200 mM, sodium selenite 60 nM, triodothyronine 20 ng/ml, insulin 10 mg/ml, progesterone 40 ng/ml. Following fractionation, a 10-ml sample from each recovered fraction was transferred to a hemocytometer and the number of cells counted from the central square. The number of cells was multiplied by 10 000 (the dilution factor) to obtain the cell concentration (cell/ml). Approximately 3–7× 104 cells/well were plated onto glass microwell tissue culture chamber slides (Lab-Tek, Miles laboratories Inc., Naperville, IL) precoated with 150–300 ml Lpolylysine (20 mg/ml) in the fresh MEM containing 5% fetal calf serum. Cells from each fraction were seeded onto glass chamber slides. Half of the medium was changed every second day. The cultures were incubated at 37°C under 5% CO2. Some of the cultures were fixed with 4% paraformaldehyde 2–4 h, 3 days and 1 week after seeding. Cultures were fixed with 4% paraformaldehyde, and immunohistochemistry and in situ hybridization carried out singly or sequentially on the same culture (see below) to compare expression of each marker in the different fractions, and at different times. The following antisera were used in the study: Mouse anti-rat neurofilament 200 (monoclonal, Sigma, 1:12 000), BrdU (monoclonal, Sigma, 1:200), Mouse anti-rat parvalbumin (monoclonal, Sigma, 1:1000), nestin (Rat-401; monoclonal, Developmental Studies Hybridoma Laboratory, 1:1000), TH (tyrosine hydroxy-
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lase; rabbit polyclonal, Eugene Tech, 1:2000), Rabbit anti-goat calbindin-D28 (monoclonal, Sigma, 1:2000), Rabbit anti-goat calretinin (rabbit polyclonal, Gift of David Jacobowitz, NIMH, NIH, 1:5000), neuron-specific enolase (NSE; rabbit polyclonal, Polyscience, 1:2000). Immunocytochemistry was carried out simultaneously for all of the cultures from each experiment and using the same antisera, incubation materials and conditions (Silverman, 1992). All procedures were carried out at room temperature unless otherwise noted. Control cultures were processed without the primary antiserum. Prior to BrdU immunohistochemical processing, the cultures were fixed for 10 min in 4% paraformaldehyde, washed two times for 1 min with PBS, incubated in 70% ethanol for 10 min, washed two times for 5 min with PBS, and incubated in 2N HCl/ 0.5% Triton-X for 30 min at 37°C. The avidin-biotinHRP protocol used follows Hsu et al. (1981).
2.4. Characterization of dopaminergic components A modification of the method of Seroogy et al. (1994) for combined immunocytochemical/in situ hybridization was employed to characterize the DA cells in the putative ventral and dorsal tier cultures. Oligonucleotide antisense and sense (control) probes were commercially prepared for identification of TH mRNA. The probe, a 45 mer fragment complementary to nucleo-
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tides 1238–1283 of the TH gene sequence (Grima et al., 1985), was 3%-end-labeled with [35S]ATP using terminal deoxytransferase. The sections, which had previously been processed for immunocytochemistry (see above), were hydrated in sodium citrate buffer (1×SSC) and then covered with the hybridization buffer which consisted of the 35S-labeled probe (average concentration= 1.0× 106 cpm/ml) with the following additives: 50% formamide (Fluka), salmon sperm DNA (Sigma) 100 mg/ml, yeast transferase RNA (Sigma) 250 mg/ml, H2O, 1×Denhardt’s solution (Sigma), 10% dextran sulfate (Sigma) and 100 mM dithiothreitol (DTT). Glass coverslips were applied to the sections to limit evaporation and the slides incubated overnight at 37°C in a humid chamber. After rinsing twice in 1×SSC at 37°C and twice more at room temperature, the slides were dried, coated with nuclear track emulsion for autoradiography and exposed in the dark for 10–14 days. The autoradiographs were developed in D-19 developer, fixed in Kodak fixer and lightly post-stained with cresyl violet.
2.5. Data analysis Images for cell counting were digitized from the chamber slides via an RBG video camera mounted on a BX-50 microscope (Olympus) to a Macintosh Quadra 800 computer equipped with an LG-3 digitizing card
Fig. 1. Schematic illustration of the experimental protocol employed. At top left, the pregnant dam is injected with BrdU. The fetuses are removed 2 h later and the mesencephalic flexure dissected and dissociated. The cell suspension is applied to the discontinuous Percoll gradient which is then spun in a refrigerated centrifuge. At lower right, the cell bands are present between the Percoll dilutions. The top fraction consists entirely of cellular debris, and subsequent bands contain cells of an increasingly immature and undifferentiated nature. The lowest fractions, i.e. 45 and 50% Percoll, contain mostly proliferating cells. The cells from the various bands are recovered with a Pasteur pipette and seeded onto poly-lysinecoated chamber slides for characterization.
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Fig. 2. Videomicrographs of Percoll-fractionated cells from the E19 striatum against the backdrop of the hemocytometer. The 35% cells (HBF) are clearly largest on average, while the 50% band (LBF) was composed of the smallest cells.
(Scion Inc., Rockville, MD). The images were analyzed using the public domain software NIH Image, 1.54 (Wayne Rasband, NINDS, NIH). Immunoreactive cells to be quantified were highlighted after an arbitrarily assigned threshold value was chosen which excluded clearly non-immunoreactive elements in the region of interest. The cell profiles were then counted and the percentage of immunoreactive (IR) cells determined by dividing the number of positively stained cells (i.e. above threshold) in a field by the total number of cells in the same field. Between 200 and 500 cells were counted in cultures prepared from a given fraction. At least three separate fields were sampled per chamber, and at least three chambers. The percentage of labeled cells was calculated out of the total number of cells sampled. The percentage of labeled cells in the different fractions was compared by ANOVA using InStat 2.0 (GraphPad Inc., San Diego, CA).
3. Results
3.1. Buoyant density fractionation Greater than 80% of the cells that were applied to the gradients were recovered in four discrete cell-fractions; a 5th band at the surface of the 10% Percoll component contained cellular debris (Fig. 1). The 50% cell band contained the fewest cells, approximately 1% of the total number in the four fractions. The remaining bands contained roughly similar numbers of cells. Visual inspection of the isolated fractions at seeding
revealed distinct differences in cell size and morphology (Fig. 2). The cells harvested from the highest (i.e. most buoyant) cell band (i.e. 35%) were typically the largest (average diameter = 8.1 mm). In contrast, the lowest bands (i.e. 45% and 50%) were characterized by smaller cells, with a high nucleus to cytoplasm ratio (average diameter = 7.2 mm). The cells of the middle fractions ranged in size from 6.4 to 8.1 mm with an average diameter of 7.65 mm. On average, 7.4% of the neurons present in the 45% and 6.5% of cells in the 35% fractions expressed TH mRNA. Cultures hybridized with the sense probe exhibited no discernible signal. Incorporation of BrdU differed significantly between the 35% and 45% fractions at 2 h (Fig. 3C–D). The 35% band contained less than a tenth of the BrdU-incorporating cells observed in the 45% fraction (1.44 profiles vs. 16.5 profiles/sampled area). Other maturation markers assessed, e.g. neurofilament protein (NFP) and NSE (Fig. 3E), indicated a similarly clearcut, albeit opposite compartment preference. For instance, the 200 kD NFP was present exclusively in differentiated neurons, and though observed in all fractions, was seen in more than 90% of the cells in the high buoyancy (i.e. 35%) fraction at 2 h post seeding. By comparison, relatively few cells (i.e.B 10% of the total) from the 50% band were immunopositive for this protein at 2 h. Similarly, NSE-IR was observed in almost all of the cells from the 35% band but only 60% of the cells from the 50% band at 2 h. Cells immuno reactive to another developmental marker, nestin (Hockfield and McKay, 1985), were concentrated in the 45 (44.3 profiles vs. 5.7 profiles) and 50% frac-
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tions (data not shown), initially restricted to small undifferentiated cells, but by day 3 were much more frequently observed in process-bearing, glia-like profiles.
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3.2. Compartment markers in buoyant-density fractionated mesencephalon Co-localization of calcium-binding proteins and TH
Fig. 3. Videomicrographs showing cultured mesencephalic neurons from Percoll fractions at various times post-seeding following in situ hybridization with and without immunohistochemistry. (A) Autoradiographic silver grains over a TH mRNA-expressing profile from a 45% culture at 2 h. Insert: two neurons from the 35% fraction at 2 h. Note the fine process (arrow) emanating from the cells (arrow). (B) DA neurons positive for TH mRNA (arrows) in a 45% culture. The culture was co-stained with cressyl violate and all cells appear gray. Labeled cells are also immunoreactive for calbindin-D28K. (C, D) Darkfield micrographs from 45 and 35% Percoll fractions, respectively, following immunocyctochemistry for BrdU. Few BrdU-incorporating cell profiles (thick arrow) are seen among the mostly differentiated cells (thin arrow) in the 35% fraction. In contrast, numerous BrdU-IR profiles are seen in (D) (e.g. thick arrow). Some profiles appeared partially immunopositive (thin arrow), possibly indicating that the cell exited or entered S-phase at the beginning or end respectively of the waiting period between the injection of BrdU into the pregnant dam and removal of the embryos. (E) Cultured 45% cells at 2 h. The cell at lower right is immunoreactive for NSE. Insert: NSE-IR cell (on right) appears to be in the final stages of mitosis. A ruffled border appears on the right side of the NSE-IR cell, suggesting a nascent growth cone. (F) Double-labeled calbindin-DA neuron in 45% culture. Because of the intensity of the immunolabeling, autoradiographic silver grains are largely obscured over the perikaryon, though scatter of the emitted radioactivity causes silver grain development beyond the cell borders, producing a ‘halo’ effect, and enabling identification of dual-labeled cells. (G) Calretinin-IR neuron (arrow) adjacent to two immunonegative TH mRNA-expressing neurons (borders between the cells are indistinct in this focal plane). (H) DA neurons co-expressing calretinin in a 35% culture at 14 DIV. A glial cell extends a process towards the dual-labeled neuron (arrow).
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dual-labeled cells in the 35% cultures remained static. Parvalbumin expression in DA cells did not differ between presumptive ventral and dorsal tier cells at 2 h and 3 DIV. By 7 DIV, however, a significantly higher percentage of DA cells in the 35% cultures were parvalbumin-IR compared to those from 45 and 50% fractions. Calretinin-IR was present in large numbers of cultured cells originating in both high- and low-buoyancy fractions. In DA cells, calretinin was consistently co-localized more often in 35% cultures.
4. Discussion
Fig. 4. Graphic demonstration of marker expression in 35% (putative ventral tier) and 45% (putative dorsal tier) Percoll fractions. Calbindin exhibited the most unambiguous compartment specificity of the three markers examined, though calretinin also favored one compartment (ventral tier). Error bars represent SEM. *PB 0.01 (calbindin), P B0.05, (parvalbumin), w PB 0.02, F PB 0.05.
mRNA in the cultures from the 35% and 45% Percoll fractions is demonstrated in Fig. 3 and summarized graphically in Fig. 4. At 2 h, only calbindin-D28K was differentially distributed, appearing most frequently in DA cells of the 35% fraction (Fig. 4a). By 3 DIV, calbindin-D28K expression in DA cells had increased 5-fold in the 45% cultures, while the percentage of
The use of buoyant density sedimentation for separation of neural cells into primary cell culture is not new. Hatten (1985), for instance, demonstrated the utility of this approach for separation of cerebellar cell suspensions into granule and glial cell fractions. Similarly, Guillemin et al. (1997) produced enriched microglial cultures on Percoll gradients. Most recently, buoyant density has been used to define spatiotemporal patterns of neuronal differentiation during development (Maric et al., 1997). Here we report on our use of this technique to isolate the sequentially assembled elements from discrete regions in the developing ventral mesencephalon. Previously, efforts to study these elements were limited. In the ventral mesencephalon, labeling or destruction of the early-generated ventral tier cells was possible via retrograde transport of markers or the DA-specific neurotoxin 6OHDA (Fishell and van der Kooy, 1987), respectively, injected into the striatum before, or just after birth, i.e. before the arrival there of the projection from the late-born dorsal tier DA neurons. In this way, preexisting and already connected ventral tier DA neurons are labeled on the one hand, or removed on the other, leaving the late-developing dorsal tier cells. Neither of these techniques, however, permitted the establishment of cultures containing primarily ventral tier neurons. Because of the relatively low number of 50% band cells obtained from the Percoll gradients, comparisons provided are restricted to the 35 and 45% fractions except where specified. Thus, the degree to which the cultures represented pure ventral or dorsal compartments was undoubtedly reduced. Nevertheless, the differences, e.g. between BrdU incorporation in 45 and 35% cultures were typically impressive. It is important to note that the number of cells in S-phase, as demonstrated by a 2-h BrdU pulse is only a fraction of the actual proliferating cell population. Assuming a cell cycle time of 12–17 h (Waechter and Jaensch, 1972) and an S-phase lasting approximately 4 h (Takahashi et al., 1993), the percent of BrdU-incorporating, i.e. proliferating cells in the 45% fraction can be extrapolated to 55%. Similarly, the low buoyancy fraction at 2 h was
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characterized by a high percentage of nestin-IR cells relative to the high buoyancy (putative ventral tier) cells. These cells represent the dorsal tier progenitors, which in the rat are born between E18 and P0. In support of this thesis, cells immunoreactive for calbindin-D28K, the best known marker for dorsal tier DA cells (Gerfen et al., 1987b), also concentrated in the 45 and 50% Percoll bands. It isn’t clear from the present study how many of these represent DA cell precursors, though it can be, at most, a small number, since the average percentage of TH mRNA-expressing neurons in the cultures produced from low buoyancy cells was only about 6% at 2 h post seeding. Single cell cultures featuring dual BrdU and TH immunocytochemistry can address this question.
4.1. Markers of compartment identity in fractionated cultures By E18, the vast majority of DA cells born during the first wave of neurogenesis are already well differentiated. These differentiated cells, corresponding to the 35% Percoll cells, most often expressed calretinin and parvalbumin, while calbindin-D28K-IR neurons were most often found in the cultures produced from 45 to 50% Percoll fractions. This result is largely in agreement with previous, in vivo studies showing a differential pattern of calcium binding protein distribution in the DAergic ventral mesencephalon (Gerfen et al., 1987b; Rogers, 1992; Isaacs and Jacobowitz, 1994; Alfahel-Kakunda and Silverman, 1997). These patterns were maintained with regard to DA cells expressing calbindin-D28K and calretinin, though not for parvalbumin which failed to show a clear preference for DA neurons from one fraction. In fact, unlike parvalbumin, which rarely co-localized with TH mRNA-expressing cells in the cultures, DA neurons often contained calretinin immunoreactivity, though these exhibited only a slight, though statistically significant preference for the low buoyancy fraction. Thus, the characterization of DA neurons from the high-buoyancy fraction(s) as putative ventral tier cells is supported more in a negative fashion, i.e. due to their low co-localization with calbindin-D28K than by their expression of a ‘ventral tier marker’ per se. Although the differences observed between the fractions with regard to CaBP’s were often statistically significant, the segregation of cells in this regard was far from complete. Several factors must be considered to understand why. Firstly, just as in the striatum, compartmentalization itself is probably imprecise; it seems likely that there is overlap of ventral and dorsal tier neurogenesis, with late-born ventral tier cells mixing with precocious dorsal tier cells. In sections through the embryonic and postnatal midbrain, we have previously reported that the three CaBP’s studied are present
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within substantial numbers of neurons in both the SN and the VTA (Alfahel-Kakunda and Silverman, 1997). In these studies as well as in primary cultures, calbindin-D28K was preferentially localized to dorsal tier (45 and 50% fractions) more than to ventral tier cells (35% fraction), whereas parvalbumin, and to a lesser degree, calretinin, tended to localize to ventral tier DA cells. A second factor to be considered is the method itself. The buoyant density gradients employed in the present study were step, or discontinuous gradients, i.e. consisting of a series of Percoll dilutions added stepwise one to the other, and each sufficiently different to capture a range of buoyant densities in the interfaces between dilutions. While the results point to a rather high degree of homogeneity of developmental stages from particular Percoll fractions, some contamination by cells of different stages is inevitable. We frequently noted clusters of incompletely dissociated cells for example, which might have physically interfered with the sedimentation of smaller 45 and 50% cells. Similarly, large neural profiles were occasionally found in the 45 or 50% Percoll bands. We speculate that such cells were pulled downwards during centrifugation as a result of adhesion to smaller, dense cells. With experience and perhaps more aggressive dissociation measures, the occurrence of such artifacts would undoubtedly be reduced. In addition, increasing the number of steps in the gradients or the use of continuous Percoll gradients, which produce up to 20 discrete cell bands (Pertoft and Laurent, 1982; Maric et al., 1996) also yield much more homogeneous fractions (Maric and Barker, unpublished observations).
4.2. Potential applications A difficulty frequently encountered in determining whether developmental events and processes are, in fact, specified/programmed or are rather influenced by extrinsic or so-called epigenetic factors, is that development typically occurs in a phased manner. That is to say that later-appearing cells become intermixed with earlier-born cells and make assessment of their inherent capabilities difficult. By preventing the obscuring of the initial phases by biophysical or chemical manipulation and separating these cell-generating phases in vitro, the opportunity is created to study the development of each phase on its own. This should also expedite the search for specific compartment markers. Furthermore, with the advent of methods for identification of differentially expressed gene sequences, e.g. arbitrarily-primed PCR/ differential display (Welsh and McClelland, 1990) and differential screening of cDNA libraries (Nedivi et al., 1993), rapid, large-scale comparisons of genes expressed in the separated compartments, including genes not previously known, should be possible. For example, we envision comparison by differential display RT-PCR of
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precursor and post-differentiation cell populations produced from ventral and dorsal tier-generating periods. Such an approach may provide the means to identify molecular determinants of specific developmental events.
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Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain W.F. Silverman a,*, A. Alfahel-Kakunda a, A. Dori a, J.L. Barker b a
Department of Morphology, Zlotowski Center for Neuroscience, Ben-Gurion Uni6ersity of the Nege6, Beer She6a, 84105 Israel b Laboratory of Neurophysiology, NINDS, NIH, Bethesda, MD 20892, USA Received 7 July 1998; received in revised form 5 February 1999; accepted 7 February 1999
Abstract The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral ‘tiers’ with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Development; Cell separation; Cell culture; Substantia nigra; Dopamine; Percoll; Pattern
1. Introduction Although poorly understood, regional ‘patterning’ or compartmentalization is known to reflect intrinsic differences in efferent and afferent connections, as well as developmental priorities. Both the medium-spiny neurons of the caudate-putamen, or striatum, and the dopaminergic (DA) cells of the mesencephalon which project to them, are arranged in separate and complementary compartments in their respective areas. Unlike the immediately evident pattern observed in the hippocampus or cerebellum, the division of the mesencephalic and striatal neurons into compartments requires artificial manipulation, e.g. immunohistochemistry for phenotypic markers, to visualize. The significance of compartmentalization in the meso-stri* Corresponding author. Fax: +972-7-277655. E-mail address: [email protected] (W.F. Silverman)
atal pathway appears to lie in the mutually reciprocal, though asymmetric nature of its interconnections, and the essential role each component plays in the etiology of several of the most common and intractable CNS disorders. The DA neurons of the ventral mesencephalon, including the substantia nigra pars compacta (SNc), substantia nigra pars reticulata (SNr) and ventral tegmental area (VTA), are generated in two waves of neurogenesis: Between E12 and E15 and then from E18 until soon after birth. On this basis and with regard to their final topographical positions, efferent and afferent connections (Gerfen, 1984; Gerfen et al., 1987a) and dendritic morphology (Bjo¨rklund and Lindvall, 1975; Fallon and Moore, 1978) the two groups of DA neurons are said to occupy distinct ventral and dorsal compartments or ‘tiers’ (Fallon and Moore, 1978; Veening et al., 1980). Ventral and dorsal tier DA neurons differ as well with regard to expression of
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calcium binding proteins (Gerfen et al., 1987b; Rogers, 1992; Alfahel-Kakunda and Silverman, 1997) and sensitivity to neurotrophic factors (Lindsay et al., 1991; Korsching, 1993; Seroogy et al., 1994). Despite the marked differences in their ontogeny and connectivity, understanding of the function of compartmentalization in this system has been complicated by the inability to study the individual elements. In the present study, we evaluate an approach to isolating the anatomically-mixed, developmentally distinct sub-populations of neurons featuring the fractionation of cell suspensions from the area of interest according to their natural specific buoyant densities. Cell separation by buoyant density fractionation is based on the principle that buoyant densities primarily reflect differences in the ratio of nucleus to cytoplasm (Pertoft and Laurent, 1982). Proliferative cells synthesize DNA and possess less cytoplasm relative to the size of the nucleus than differentiated cells, and hence, accumulate in the lower, i.e. less buoyant fractions of the gradient. Employing this approach, we have succeeded in obtaining fractions enriched in DAergic neurons corresponding phenotypically to ventral and dorsal tiers.
2. Methods
2.1. Animals Timed-pregnant Sprague-Dawley rats were injected i.p. with the halogenated thymidine analog 5-bromo-2%deoxyuridine (BrdU) (5 mg/ml per 100 g body weight) and euthanized 2 h later with CO2 at gestational day 19, i.e. at the time of dorsal tier neurogenesis, and embryos delivered by cesarean section. Embryonic age was calculated from insemination, i.e. the presence of a vaginal plug (day 0) and correlated with crown-rump length (CRL) at delivery.
2.2. Preparation of cell suspensions For each experiment, between 15 and 20 [E18] fetuses (CRL= 23 mm) were removed from the dams and placed into ice-cold Earle’s Balanced Salt Solution (EBSS). The mesencephalic flexure, containing the nascent ventral mesencephalon was rapidly microdissected from each embryo, transferred to fresh, ice-cold EBSS, minced with forceps, and gently triturated. The pooled tissue was then dissociated in 50 units of papain (Worthington Biochemical Corp., Freehold, NJ) in EBSS with 1 mM L-cysteine (Sigma), 0.5 mM EDTA, and 0.01% DNAse (Boehringer Mannheim) at 37°C with gentle agitation for 45 min. The partially dissociated tissue was triturated a second time briefly by repeated passage through a 10-ml pipette. The cells were then spun in an ultracentrifuge
at 1600 rpm for 5 min at room temperature. The pellet thus obtained was resuspended in 2.5 ml of 1 mg/ml trypsin inhibitor and a 1 mg/ml bovine serum albumin solution (‘1/1’). To remove non-viable cells and cellular debris, the suspensions were lightly triturated and the supernatant layered onto 5 ml of a solution containing 10 mg/ml trypsin inhibitor and 10 mg/ml albumin in a 15 ml centrifuge tube. This mix was spun at 800 rpm at room temperature, and the pellet resuspended again in ‘1/1’.
2.3. Buoyant density fractionation Percoll was prepared as a stock solution by mixing nine parts of Percoll to one part of a 10 × physiological salt solution. A 5-step, discontinuous gradient of 10, 35, 40, 45 and 50% Percoll was made by diluting the Percoll stock solution with decreasing concentrations of EBSS, and overlaying one atop the other in a 15-ml centrifuge tube (Fig. 1). Dissociated cells in ‘1/1’ were applied on the top of the gradient and spun in a centrifuge at 1600 rpm for 15 min at 18°C. Cells accumulated in the interface between fractions and were collected with a fire-polished Pasteur pipette. The fractions were washed twice with EBSS, and resuspended in minimum essential medium (MEM) with glutamine, 5% horse serum, 5% fetal calf serum, BSA 0.001%, transferrin 200 mg, putrescine 200 mM, sodium selenite 60 nM, triodothyronine 20 ng/ml, insulin 10 mg/ml, progesterone 40 ng/ml. Following fractionation, a 10-ml sample from each recovered fraction was transferred to a hemocytometer and the number of cells counted from the central square. The number of cells was multiplied by 10 000 (the dilution factor) to obtain the cell concentration (cell/ml). Approximately 3–7× 104 cells/well were plated onto glass microwell tissue culture chamber slides (Lab-Tek, Miles laboratories Inc., Naperville, IL) precoated with 150–300 ml Lpolylysine (20 mg/ml) in the fresh MEM containing 5% fetal calf serum. Cells from each fraction were seeded onto glass chamber slides. Half of the medium was changed every second day. The cultures were incubated at 37°C under 5% CO2. Some of the cultures were fixed with 4% paraformaldehyde 2–4 h, 3 days and 1 week after seeding. Cultures were fixed with 4% paraformaldehyde, and immunohistochemistry and in situ hybridization carried out singly or sequentially on the same culture (see below) to compare expression of each marker in the different fractions, and at different times. The following antisera were used in the study: Mouse anti-rat neurofilament 200 (monoclonal, Sigma, 1:12 000), BrdU (monoclonal, Sigma, 1:200), Mouse anti-rat parvalbumin (monoclonal, Sigma, 1:1000), nestin (Rat-401; monoclonal, Developmental Studies Hybridoma Laboratory, 1:1000), TH (tyrosine hydroxy-
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lase; rabbit polyclonal, Eugene Tech, 1:2000), Rabbit anti-goat calbindin-D28 (monoclonal, Sigma, 1:2000), Rabbit anti-goat calretinin (rabbit polyclonal, Gift of David Jacobowitz, NIMH, NIH, 1:5000), neuron-specific enolase (NSE; rabbit polyclonal, Polyscience, 1:2000). Immunocytochemistry was carried out simultaneously for all of the cultures from each experiment and using the same antisera, incubation materials and conditions (Silverman, 1992). All procedures were carried out at room temperature unless otherwise noted. Control cultures were processed without the primary antiserum. Prior to BrdU immunohistochemical processing, the cultures were fixed for 10 min in 4% paraformaldehyde, washed two times for 1 min with PBS, incubated in 70% ethanol for 10 min, washed two times for 5 min with PBS, and incubated in 2N HCl/ 0.5% Triton-X for 30 min at 37°C. The avidin-biotinHRP protocol used follows Hsu et al. (1981).
2.4. Characterization of dopaminergic components A modification of the method of Seroogy et al. (1994) for combined immunocytochemical/in situ hybridization was employed to characterize the DA cells in the putative ventral and dorsal tier cultures. Oligonucleotide antisense and sense (control) probes were commercially prepared for identification of TH mRNA. The probe, a 45 mer fragment complementary to nucleo-
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tides 1238–1283 of the TH gene sequence (Grima et al., 1985), was 3%-end-labeled with [35S]ATP using terminal deoxytransferase. The sections, which had previously been processed for immunocytochemistry (see above), were hydrated in sodium citrate buffer (1×SSC) and then covered with the hybridization buffer which consisted of the 35S-labeled probe (average concentration= 1.0× 106 cpm/ml) with the following additives: 50% formamide (Fluka), salmon sperm DNA (Sigma) 100 mg/ml, yeast transferase RNA (Sigma) 250 mg/ml, H2O, 1×Denhardt’s solution (Sigma), 10% dextran sulfate (Sigma) and 100 mM dithiothreitol (DTT). Glass coverslips were applied to the sections to limit evaporation and the slides incubated overnight at 37°C in a humid chamber. After rinsing twice in 1×SSC at 37°C and twice more at room temperature, the slides were dried, coated with nuclear track emulsion for autoradiography and exposed in the dark for 10–14 days. The autoradiographs were developed in D-19 developer, fixed in Kodak fixer and lightly post-stained with cresyl violet.
2.5. Data analysis Images for cell counting were digitized from the chamber slides via an RBG video camera mounted on a BX-50 microscope (Olympus) to a Macintosh Quadra 800 computer equipped with an LG-3 digitizing card
Fig. 1. Schematic illustration of the experimental protocol employed. At top left, the pregnant dam is injected with BrdU. The fetuses are removed 2 h later and the mesencephalic flexure dissected and dissociated. The cell suspension is applied to the discontinuous Percoll gradient which is then spun in a refrigerated centrifuge. At lower right, the cell bands are present between the Percoll dilutions. The top fraction consists entirely of cellular debris, and subsequent bands contain cells of an increasingly immature and undifferentiated nature. The lowest fractions, i.e. 45 and 50% Percoll, contain mostly proliferating cells. The cells from the various bands are recovered with a Pasteur pipette and seeded onto poly-lysinecoated chamber slides for characterization.
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Fig. 2. Videomicrographs of Percoll-fractionated cells from the E19 striatum against the backdrop of the hemocytometer. The 35% cells (HBF) are clearly largest on average, while the 50% band (LBF) was composed of the smallest cells.
(Scion Inc., Rockville, MD). The images were analyzed using the public domain software NIH Image, 1.54 (Wayne Rasband, NINDS, NIH). Immunoreactive cells to be quantified were highlighted after an arbitrarily assigned threshold value was chosen which excluded clearly non-immunoreactive elements in the region of interest. The cell profiles were then counted and the percentage of immunoreactive (IR) cells determined by dividing the number of positively stained cells (i.e. above threshold) in a field by the total number of cells in the same field. Between 200 and 500 cells were counted in cultures prepared from a given fraction. At least three separate fields were sampled per chamber, and at least three chambers. The percentage of labeled cells was calculated out of the total number of cells sampled. The percentage of labeled cells in the different fractions was compared by ANOVA using InStat 2.0 (GraphPad Inc., San Diego, CA).
3. Results
3.1. Buoyant density fractionation Greater than 80% of the cells that were applied to the gradients were recovered in four discrete cell-fractions; a 5th band at the surface of the 10% Percoll component contained cellular debris (Fig. 1). The 50% cell band contained the fewest cells, approximately 1% of the total number in the four fractions. The remaining bands contained roughly similar numbers of cells. Visual inspection of the isolated fractions at seeding
revealed distinct differences in cell size and morphology (Fig. 2). The cells harvested from the highest (i.e. most buoyant) cell band (i.e. 35%) were typically the largest (average diameter = 8.1 mm). In contrast, the lowest bands (i.e. 45% and 50%) were characterized by smaller cells, with a high nucleus to cytoplasm ratio (average diameter = 7.2 mm). The cells of the middle fractions ranged in size from 6.4 to 8.1 mm with an average diameter of 7.65 mm. On average, 7.4% of the neurons present in the 45% and 6.5% of cells in the 35% fractions expressed TH mRNA. Cultures hybridized with the sense probe exhibited no discernible signal. Incorporation of BrdU differed significantly between the 35% and 45% fractions at 2 h (Fig. 3C–D). The 35% band contained less than a tenth of the BrdU-incorporating cells observed in the 45% fraction (1.44 profiles vs. 16.5 profiles/sampled area). Other maturation markers assessed, e.g. neurofilament protein (NFP) and NSE (Fig. 3E), indicated a similarly clearcut, albeit opposite compartment preference. For instance, the 200 kD NFP was present exclusively in differentiated neurons, and though observed in all fractions, was seen in more than 90% of the cells in the high buoyancy (i.e. 35%) fraction at 2 h post seeding. By comparison, relatively few cells (i.e.B 10% of the total) from the 50% band were immunopositive for this protein at 2 h. Similarly, NSE-IR was observed in almost all of the cells from the 35% band but only 60% of the cells from the 50% band at 2 h. Cells immuno reactive to another developmental marker, nestin (Hockfield and McKay, 1985), were concentrated in the 45 (44.3 profiles vs. 5.7 profiles) and 50% frac-
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tions (data not shown), initially restricted to small undifferentiated cells, but by day 3 were much more frequently observed in process-bearing, glia-like profiles.
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3.2. Compartment markers in buoyant-density fractionated mesencephalon Co-localization of calcium-binding proteins and TH
Fig. 3. Videomicrographs showing cultured mesencephalic neurons from Percoll fractions at various times post-seeding following in situ hybridization with and without immunohistochemistry. (A) Autoradiographic silver grains over a TH mRNA-expressing profile from a 45% culture at 2 h. Insert: two neurons from the 35% fraction at 2 h. Note the fine process (arrow) emanating from the cells (arrow). (B) DA neurons positive for TH mRNA (arrows) in a 45% culture. The culture was co-stained with cressyl violate and all cells appear gray. Labeled cells are also immunoreactive for calbindin-D28K. (C, D) Darkfield micrographs from 45 and 35% Percoll fractions, respectively, following immunocyctochemistry for BrdU. Few BrdU-incorporating cell profiles (thick arrow) are seen among the mostly differentiated cells (thin arrow) in the 35% fraction. In contrast, numerous BrdU-IR profiles are seen in (D) (e.g. thick arrow). Some profiles appeared partially immunopositive (thin arrow), possibly indicating that the cell exited or entered S-phase at the beginning or end respectively of the waiting period between the injection of BrdU into the pregnant dam and removal of the embryos. (E) Cultured 45% cells at 2 h. The cell at lower right is immunoreactive for NSE. Insert: NSE-IR cell (on right) appears to be in the final stages of mitosis. A ruffled border appears on the right side of the NSE-IR cell, suggesting a nascent growth cone. (F) Double-labeled calbindin-DA neuron in 45% culture. Because of the intensity of the immunolabeling, autoradiographic silver grains are largely obscured over the perikaryon, though scatter of the emitted radioactivity causes silver grain development beyond the cell borders, producing a ‘halo’ effect, and enabling identification of dual-labeled cells. (G) Calretinin-IR neuron (arrow) adjacent to two immunonegative TH mRNA-expressing neurons (borders between the cells are indistinct in this focal plane). (H) DA neurons co-expressing calretinin in a 35% culture at 14 DIV. A glial cell extends a process towards the dual-labeled neuron (arrow).
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dual-labeled cells in the 35% cultures remained static. Parvalbumin expression in DA cells did not differ between presumptive ventral and dorsal tier cells at 2 h and 3 DIV. By 7 DIV, however, a significantly higher percentage of DA cells in the 35% cultures were parvalbumin-IR compared to those from 45 and 50% fractions. Calretinin-IR was present in large numbers of cultured cells originating in both high- and low-buoyancy fractions. In DA cells, calretinin was consistently co-localized more often in 35% cultures.
4. Discussion
Fig. 4. Graphic demonstration of marker expression in 35% (putative ventral tier) and 45% (putative dorsal tier) Percoll fractions. Calbindin exhibited the most unambiguous compartment specificity of the three markers examined, though calretinin also favored one compartment (ventral tier). Error bars represent SEM. *PB 0.01 (calbindin), P B0.05, (parvalbumin), w PB 0.02, F PB 0.05.
mRNA in the cultures from the 35% and 45% Percoll fractions is demonstrated in Fig. 3 and summarized graphically in Fig. 4. At 2 h, only calbindin-D28K was differentially distributed, appearing most frequently in DA cells of the 35% fraction (Fig. 4a). By 3 DIV, calbindin-D28K expression in DA cells had increased 5-fold in the 45% cultures, while the percentage of
The use of buoyant density sedimentation for separation of neural cells into primary cell culture is not new. Hatten (1985), for instance, demonstrated the utility of this approach for separation of cerebellar cell suspensions into granule and glial cell fractions. Similarly, Guillemin et al. (1997) produced enriched microglial cultures on Percoll gradients. Most recently, buoyant density has been used to define spatiotemporal patterns of neuronal differentiation during development (Maric et al., 1997). Here we report on our use of this technique to isolate the sequentially assembled elements from discrete regions in the developing ventral mesencephalon. Previously, efforts to study these elements were limited. In the ventral mesencephalon, labeling or destruction of the early-generated ventral tier cells was possible via retrograde transport of markers or the DA-specific neurotoxin 6OHDA (Fishell and van der Kooy, 1987), respectively, injected into the striatum before, or just after birth, i.e. before the arrival there of the projection from the late-born dorsal tier DA neurons. In this way, preexisting and already connected ventral tier DA neurons are labeled on the one hand, or removed on the other, leaving the late-developing dorsal tier cells. Neither of these techniques, however, permitted the establishment of cultures containing primarily ventral tier neurons. Because of the relatively low number of 50% band cells obtained from the Percoll gradients, comparisons provided are restricted to the 35 and 45% fractions except where specified. Thus, the degree to which the cultures represented pure ventral or dorsal compartments was undoubtedly reduced. Nevertheless, the differences, e.g. between BrdU incorporation in 45 and 35% cultures were typically impressive. It is important to note that the number of cells in S-phase, as demonstrated by a 2-h BrdU pulse is only a fraction of the actual proliferating cell population. Assuming a cell cycle time of 12–17 h (Waechter and Jaensch, 1972) and an S-phase lasting approximately 4 h (Takahashi et al., 1993), the percent of BrdU-incorporating, i.e. proliferating cells in the 45% fraction can be extrapolated to 55%. Similarly, the low buoyancy fraction at 2 h was
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characterized by a high percentage of nestin-IR cells relative to the high buoyancy (putative ventral tier) cells. These cells represent the dorsal tier progenitors, which in the rat are born between E18 and P0. In support of this thesis, cells immunoreactive for calbindin-D28K, the best known marker for dorsal tier DA cells (Gerfen et al., 1987b), also concentrated in the 45 and 50% Percoll bands. It isn’t clear from the present study how many of these represent DA cell precursors, though it can be, at most, a small number, since the average percentage of TH mRNA-expressing neurons in the cultures produced from low buoyancy cells was only about 6% at 2 h post seeding. Single cell cultures featuring dual BrdU and TH immunocytochemistry can address this question.
4.1. Markers of compartment identity in fractionated cultures By E18, the vast majority of DA cells born during the first wave of neurogenesis are already well differentiated. These differentiated cells, corresponding to the 35% Percoll cells, most often expressed calretinin and parvalbumin, while calbindin-D28K-IR neurons were most often found in the cultures produced from 45 to 50% Percoll fractions. This result is largely in agreement with previous, in vivo studies showing a differential pattern of calcium binding protein distribution in the DAergic ventral mesencephalon (Gerfen et al., 1987b; Rogers, 1992; Isaacs and Jacobowitz, 1994; Alfahel-Kakunda and Silverman, 1997). These patterns were maintained with regard to DA cells expressing calbindin-D28K and calretinin, though not for parvalbumin which failed to show a clear preference for DA neurons from one fraction. In fact, unlike parvalbumin, which rarely co-localized with TH mRNA-expressing cells in the cultures, DA neurons often contained calretinin immunoreactivity, though these exhibited only a slight, though statistically significant preference for the low buoyancy fraction. Thus, the characterization of DA neurons from the high-buoyancy fraction(s) as putative ventral tier cells is supported more in a negative fashion, i.e. due to their low co-localization with calbindin-D28K than by their expression of a ‘ventral tier marker’ per se. Although the differences observed between the fractions with regard to CaBP’s were often statistically significant, the segregation of cells in this regard was far from complete. Several factors must be considered to understand why. Firstly, just as in the striatum, compartmentalization itself is probably imprecise; it seems likely that there is overlap of ventral and dorsal tier neurogenesis, with late-born ventral tier cells mixing with precocious dorsal tier cells. In sections through the embryonic and postnatal midbrain, we have previously reported that the three CaBP’s studied are present
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within substantial numbers of neurons in both the SN and the VTA (Alfahel-Kakunda and Silverman, 1997). In these studies as well as in primary cultures, calbindin-D28K was preferentially localized to dorsal tier (45 and 50% fractions) more than to ventral tier cells (35% fraction), whereas parvalbumin, and to a lesser degree, calretinin, tended to localize to ventral tier DA cells. A second factor to be considered is the method itself. The buoyant density gradients employed in the present study were step, or discontinuous gradients, i.e. consisting of a series of Percoll dilutions added stepwise one to the other, and each sufficiently different to capture a range of buoyant densities in the interfaces between dilutions. While the results point to a rather high degree of homogeneity of developmental stages from particular Percoll fractions, some contamination by cells of different stages is inevitable. We frequently noted clusters of incompletely dissociated cells for example, which might have physically interfered with the sedimentation of smaller 45 and 50% cells. Similarly, large neural profiles were occasionally found in the 45 or 50% Percoll bands. We speculate that such cells were pulled downwards during centrifugation as a result of adhesion to smaller, dense cells. With experience and perhaps more aggressive dissociation measures, the occurrence of such artifacts would undoubtedly be reduced. In addition, increasing the number of steps in the gradients or the use of continuous Percoll gradients, which produce up to 20 discrete cell bands (Pertoft and Laurent, 1982; Maric et al., 1996) also yield much more homogeneous fractions (Maric and Barker, unpublished observations).
4.2. Potential applications A difficulty frequently encountered in determining whether developmental events and processes are, in fact, specified/programmed or are rather influenced by extrinsic or so-called epigenetic factors, is that development typically occurs in a phased manner. That is to say that later-appearing cells become intermixed with earlier-born cells and make assessment of their inherent capabilities difficult. By preventing the obscuring of the initial phases by biophysical or chemical manipulation and separating these cell-generating phases in vitro, the opportunity is created to study the development of each phase on its own. This should also expedite the search for specific compartment markers. Furthermore, with the advent of methods for identification of differentially expressed gene sequences, e.g. arbitrarily-primed PCR/ differential display (Welsh and McClelland, 1990) and differential screening of cDNA libraries (Nedivi et al., 1993), rapid, large-scale comparisons of genes expressed in the separated compartments, including genes not previously known, should be possible. For example, we envision comparison by differential display RT-PCR of
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precursor and post-differentiation cell populations produced from ventral and dorsal tier-generating periods. Such an approach may provide the means to identify molecular determinants of specific developmental events.
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