(Received for publication, May 10, 1995; and in revised form, July 6, 1995)
From the
An improved method for constructing and screening subtractive cDNA libraries has been used to identify 46 mRNA transcripts that are expressed selectively in neonatal rat dorsal root ganglia (DRG) as judged by Northern blots and in situ hybridization. Sequence analysis demonstrates that both known (e.g. peripherin, calcitonin gene-related peptide, myelin P0) and novel identifiable transcripts (e.g. C-protein-like, synuclein-like, villin-like) are present in the library. Half of the transcripts (23) are undetectable in liver, kidney, heart, spleen, cerebellum, and cerebral cortex. Of the DRG-specific transcripts, 12 contain putative open reading frames that show no identity with known proteins. The construction of such a subtractive library thus provides us with both known and novel markers, and identifies new predicted DRG-specific proteins. In addition, the DRG-specific clones provide probes to define the regulatory elements that specify peripheral nervous-system-specific gene expression.
Tissue-specific gene expression specifies cell fate and function. The transcriptional regulatory events that define the complement of genes expressed by particular cell types are therefore of considerable interest. One approach to understanding the development of specialized cells is to search for vertebrate homologues of key regulators defined by genetic studies of simple organisms such as Caenorhabditis elegans or Drosophila melanogaster (see, e.g., Ghysen and Dambly-Chaudiere(1993) and Jan and Jan (1994)). Thus, homologues of the Drosophilaachaete-scute genes and the C. elegans unc-86 gene that specify peripheral neuron fate are known to be expressed in the peripheral nervous system of vertebrates (Anderson, 1993; Ninkina et al., 1993), where they may have a related function, for example in the specification of sympathetic neuron development (Guillemot et al., 1993). A number of molecules, invariably transcription factors, that specify particular cell fates have been identified in this way (He and Rosenfeld, 1991). A complementary approach to identifying key regulators relies upon the isolation of mRNA transcripts that are selectively expressed in defined cell types, followed by the identification of the regulatory elements that specify the pattern of their expression. Thus, two homeodomain proteins (Lmx-1 and Cdx-3) that bind to the insulin enhancer were isolated by screening expressed proteins that bound to regulatory elements of the insulin gene (German et al., 1992). Similarly, a transcription factor selectively expressed in olfactory neurons (Olf-1) has been identified, because its cognate binding sequence was found upstream of a number of olfactory neuron-specific genes (Wang and Reed, 1993).
We are
studying the mechanisms that specify the development of a peripheral
sensory neuron phenotype and, in particular, the development and
function of small unmyelinated sensory neurons. Subsets of these cells
respond to tissue damage, and thus play a critical role in the
initiation of some forms of pain perception (Scott, 1993). As a first
step toward the definition of critical regulatory steps in sensory
neuron development and function, we have developed a modified method
for subtractive library construction and a novel difference screening
method to isolate a battery of transcripts exclusively or selectively
expressed in rat dorsal root ganglia. By subtracting liver, kidney,
cortex and cerebellum mRNA transcripts from cDNA generated from rat
DRG, ()we have found 23 transcripts that are expressed in
DRG, but not heart, liver, spleen, cortex, or cerebellum. We present a
classification of these transcripts into known, identifiable, and novel
transcripts by DNA sequencing and demonstrate the selectivity of their
expression by Northern blotting and in situ hybridization.
Hybridization was carried out at 58 °C for 40 h in 20% formamide, 50 mM MOPS, pH 7.6, 0.2% SDS, 0.5 M NaCl, 5 mM EDTA. The total volume of the reaction was 5 µl, and the reaction was carried out under mineral oil, after an initial denaturation step of 2 min at 95 °C. 100 µl of 50 mM MOPS, pH 7.4, 0.5 M NaCl, 5 mM EDTA containing 20 units of streptavidin (Life Technologies, Inc.) was then added to the reaction mixture at room temperature, and the aqueous phase retained after two phenol/chloroform extraction steps. After sequential hybridization with biotinylated mRNA from liver and kidney, followed by cortex and cerebellum, a 80-fold concentration of DRG-specific transcripts was achieved.
One-third of the 1-2 ng
of residual cDNA was then G-tailed with terminal deoxynucleotide
transferase at 37 °C for 30 min. PCR was used to amplify the cDNA
using an oligo(dT)/NotI primer adapter and oligo(dC) primers
starting with the sequence AATTCCGA(C). Amplification was
carried out using two cycles of 95 °C for 1 min, 45 °C for 1
min, 72 °C for 5 min, followed by two cycles of 95 °C for 1
min, 58 °C for 1 min, 72 °C for 5 min. The resulting product
was then separated on a 2% Nu-sieve agarose gel, and material running
at a size of greater than 0.5 kb eluted and further amplified with 6
cycles of 95 °C for 1 min, 58 °C for 1 min, and 72 °C for 5
min. This material was further separated on a 2% Nu-sieve agarose gel,
and the material running from 6 kb on the gel was eluted and further
amplified using the same PCR conditions for 27 cycles. The amplified
DNA derived from this high molecular region was than further
fractionated on a 2% Nu-Sieve gel, and cDNA from 0.5 to 1.5 kb, and
from 1.5 to 5 kb, were pooled.
The subtracted and amplified double-stranded DRG cDNA
was random-prime labeled with [P]dATP (Life
Technologies, Inc. Multiprime kit). Replica filters were then
prehybridized for 4 h at 68 °C in hybridization buffer (see below).
Hybridization was carried out for 20 h at 68 °C in 4
SSC, 5
Denhardt's solution containing 150 µg/ml salmon sperm
DNA, 20 µg/ml poly(U), 20 µg/ml poly(C), 0.5% SDS, 5 mM EDTA. The filters were briefly washed in 2
SSC at room
temperature, then twice with 2
SSC with 0.5% SDS at 68 °C
for 15 min, followed by a 20-min wash in 0.5% SDS, 0.2
SSC at
68 °C. The filters were autoradiographed for up to 1 week on Kodak
X-Omat film.
Clones that hybridized with DRG probes but not cortex and cerebellum probes were picked and excised into Bluescript according to the maker's instructions. The plasmids were plaque-purified and finally cross-hybridized with each other. Unique clones were further analyzed by Northern blotting and sequencing.
Figure 1: Diagrammatic representation of the difference cloning strategy used to identify DRG-enriched clones.
The
products of this multi-step PCR protocol were then directionally cloned
into -Zap II. The resulting library was screened with radiolabeled
probes derived from DRG mRNA and a mixture of cortex and cerebellum
mRNA, using reverse transcription, and the final cDNA derived from the
PCR amplification. By isolating only those clones that tested positive
for homology with DRG poly(A)
probes, a subset of
clones was isolated. This method of differential screening allowed us
to identify 91 clones that were likely to be expressed selectively in
DRG.
The 91 putative DRG-specific clones were rescued into
Bluescript, plaque-purified, and their insert sizes analyzed. Inserts
ranged in size from 0.3 to 4 kb (Table 1). In order to eliminate
redundant clones, the inserts were random-prime labeled with
[P]dCTP and cross-hybridized with each other.
This analysis showed that there were, in fact, 46 distinct clones
present in the library.
Figure 2: Northern blots of RNA from neonatal rat DRG (lane 1), cortex (lane 2), cerebellum (lane 3), spleen (lane 4), liver (lane 5), kidney (lane 6), and heart (lane 7) probed with DRG-specific clones (size in kb to the left of each Northern blot). Panel A, clone C1 (myelin P0); panelB, clone F7; panelC, clone i3; panelD, clone B4; panelE, clone E0; panelF, clone A4; panelG, clone E2; panelH, clone H7 (villin-like). Northern blots were stripped and reprobed with L27 probes (LeBeau et al., 1990) to confirm that equivalent amounts of mRNA were present in each lane.
In all 46 different transcripts were analyzed by Northern blotting. Of these, 23 (50%) were apparently DRG-specific, while essentially all the clones are substantially enriched in DRG RNA compared to RNA from other tested tissues. A number of clones (13) of size up to 3.5 kb were either full-length or almost complete, suggesting that the entire coding region of the transcripts would be present. The Northern analysis obviously gives no information on the cell types expressing the various DRG-specific transcripts. In some cases, therefore, cRNA transcripts labeled with digoxygenin-UTP were synthesized and used to analyze the distribution of expression of mRNA transcripts in sections of neonatal rats. Fig. 3shows a number of examples (e.g. clone H7, a villin-like molecule) of transcripts that are clearly expressed in large diameter cell bodies corresponding to DRG sensory neurons, but not other cell types. Interestingly, some transcripts (e.g. clone G7) were expressed in subsets of sensory neurons (Fig. 3). The analysis of distribution of expression suggested that many clones were indeed selectively expressed in DRG and were suitable for further analysis.
Figure 3: In situ hybridization using DIG-cRNA probes to detect the cell type distribution of DRG-enriched clones in transverse frozen sections (10 µM) of rat dorsal root ganglia. PanelA, clone H7 (villin-like); panelB, clone D3 (synuclein-like); panelC, unidentified clone G7; panelD, higher power picture of clone G7 to show positive and negative stained cell bodies; panelE, control (sense G7); panelF, clone B4. Neuronal cell bodies are stained positive with each of the three clones shown.
The results of the sequencing analysis are summarized in Table 2. The clones analyzed fall into three classes; known proteins, proteins that are homologous to known proteins, and unknown transcripts. Clone E0 is a rat homologue of the skeletal muscle myosin-associated immunoglobulin superfamily molecule C-protein (Kojima et al., 1990; Einheber and Fischman, 1990). Clone E8 encodes a protein with a similar but not identical sequence to carbonic anhydrase III (Mayeux et al., 1983). Clone H7 is a close homologue of the actin-bundling protein villin, found in secretory epithelial cells, and associated with cell shape determination (Otto, 1994). Clone D3 is a homologue of the major central nervous system phosphoprotein synuclein, which is associated with presynaptic membranes, but whose function remains undiscovered (Maroteaux et al., 1988, 1991; Nakajo et al., 1993; Jakes et al., 1994). Interestingly, clone D3 is also homologous to synuclein-like proteins associated with amyloid deposits found in Alzheimer's disease (Ueda et al., 1993). We further analyzed the distribution of expression of this novel transcript with a variety of other tissues ( Fig. 4and Table 2). This analysis confirmed an association between this transcript and sensory neurons, as the transcript is highly enriched in DRG compared with other tissues.
Figure 4: Tissue-specific expression of clone D3 (DRG-enriched synuclein-like clone) Northern blot carried out using total rat RNA extracted from skeletal muscle (1), hypothalamus (2), hippocampus (3), DRG (4), kidney (5), spleen (6), cerebellum (7), ileum (8), spinal cord (9), lung (10), liver (11), cortex (12), and heart (13).
The synuclein family thus comprises both central and peripheral nervous system proteins. The complete sequence of the synuclein-like clone and a partial sequence of the rat C-protein-like clone are shown in Fig. 5.
Figure 5: Protein sequence of novel identified clones encoding synuclein and C-protein-like transcripts; comparison with human homologues. A, synuclein-like protein. Panel shows amino acid homology between the predicted protein encoded by DRG-specific clone D3 and the synuclein-like molecule found associated with human amyloid deposits (Ueda etal., 1993). The novel clone is printed in boldface. B, C-protein-like molecule. Panel shows sequence comparison of predicted protein encoded by clone E0 (boldface) and the sequence of human C-protein (Furst et al., 1992).
Subtractive library production provides a powerful approach to defining tissue-specific transcripts that are likely to play a role in the specialized function of the cell-types targeted. In addition, an analysis of the genomic organization of tissue-specific genes should define the motifs that play a critical role in the regulation of gene expression of the tissue of interest. Here we describe the subtractive cloning and differential screening of a number of new DRG-specific transcripts, with the eventual aim of defining the critical steps in the specification of sensory neuron cell fate.
The construction of a representative library from relatively small amounts of tissue is problematic, and sensitive screening protocols are necessary to isolate interesting clones. The development of the photobiotin/streptavidin subtractive hybridization technique (Sive and St. John, 1988) is the basis for the construction of the subtractive library described here. Recently this approach has been successfully extended to identify transcripts specifically expressed even in very small numbers of cells (Wieland et al., 1990; Klar et al., 1994; Korneev et al., 1994). The protocols described here address the two problems of representative library production and sensitive screening by means of a number of technical innovations. First, a novel multi-step PCR amplification procedure was used to generate a library from picogram quantities of subtracted DRG mRNA. The major problem with the use of PCR to generate libraries is the over-representation of small transcripts, because of their more efficient amplification. One approach to overcoming this difficulty is to amplify larger transcripts in the pool of cDNA for more PCR cycles (Belyavsky et al., 1989). The approach described here was arrived at empirically and uses three separate PCR amplification steps. First, all the subtracted cDNA was amplified, and the fraction of a size >0.5 kb isolated and further amplified, in order to discard small transcripts containing unsubtracted material. The resulting products were separated on an agarose gel, and material running with an apparent size of 6-9 kb was isolated and further amplified. By isolating and amplifying this apparently high molecular weight fraction, which also contains relatively small amounts of cDNA of a size 0-6 kb, cDNA encoding a truly representative spectrum of transcripts was generated. This material was used for the production of a representative DRG-specific library and encoded a number of large DRG-specific partial transcripts of size up to 4 kb. Another important factor in the production of a representative library is the choice of the ``driver'' RNA that is used to remove irrelevant transcripts. Both the spectrum of transcripts as well as their abundance in the subtracted library are influenced by the driver RNA, because the relative proportion of various DRG-specific genes may be altered by different levels of subtraction. Thus if skeletal muscle RNA had been included in place of one of the components of the driver RNA used here (cerebellum, cortex, liver, and kidney), then a different repertoire of DRG-specific genes would be identified.
Screening of the library also involved
technical modifications. The subtracted amplified DRG cDNA, as well as
cDNA derived from DRG RNA, was used to screen the library. Because the
subtracted DRG cDNA contained a relatively high proportion of
DRG-specific clones, it provided sensitive probes for the
identification of cognate clones in the library. In fact, all those
clones that were detected both by DRG cDNA and the subtracted DRG DNA
were effectively DRG-specific. Of the 19 clones that were detected only
with the DRG poly(A)-derived probes, all were
relatively DRG-specific. Essentially all the clones isolated showed a
DRG-enriched pattern of expression. The differential screening and the
confirmatory screening using both DRG-derived probes and subtracted DRG
probes thus avoided the isolation of any artifactual clones.
Several of the clones that show relatively selective expression in DRG are also expressed in skeletal muscle. It is known that troponin and myosin are expressed at high levels in DRG neurons (e.g. Roisen et al.(1983)). Although such genes are clearly not useful as probes for the definition of regulatory sequences that specify expression solely in DRG, their regulatory elements must include positive signals for DRG expression. Increasing evidence suggests that developing skeletal muscle shows overlapping programs of gene expression with neuronal cell types. Thus myogenic cells have been identified in the developing neural tube (Tajbaksh et al., 1994), and the myogenic gene Mef-2 has been identified in neural crest cells (Edmondson et al., 1994) Given the physical interactions among sensory neurons, motor neurons, and skeletal muscle, these overlapping repertoires of gene expression may underlie aspects of cell-cell recognition.
The analysis of tissue-specific cDNA libraries as described here provides complementary information to that obtained by the random sequencing of genomic DNA or expressed messages The transcripts that are expressed are likely to play a role in the function of the tissue used, because of their selective expression. For example, the peripheral nervous system-specific synuclein, the central nervous system homologues of which are known to be abundantly expressed at presynaptic terminals (Maroteaux et al., 1988), could be associated with aspects of neurotransmitter release that are characteristic of sensory neurons such as neuropeptide release.
In summary, we have successfully developed differential cloning and screening approaches to identify DRG-specific transcripts. The isolation of two markers associated with proprioreceptive neurons, carbonic anhydrase (Mayeux et al., 1993) and parvalbumin (Zhang et al., 1993), both of which are ablated in trk-c null mutant animals that have lost large number of proprioreceptive neurons (Ernforth et al., 1993), shows that the difference library encodes markers for specific subpopulations of sensory neurons, as well as for non-neuronal cells present in DRG cell types (e.g. myelin P0, a Schwann cell marker) (Lemke and Axel, 1985). The precise cell-type distribution of the majority of DRG-neuron-specific clones remains to be established. It will be particularly interesting to define those transcripts that arise in the early stages of neural crest cell commitment and that are expressed in sensory, but not sympathetic, neurons.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and X86789[GenBank].