Characterization of the 5′ End of the Mouse L1 Gene
We previously characterized the structures of the genes for two avian neural CAMs that are closely related to mammalian L1: Ng-CAM (19
) and Nr-CAM (unpublished data). Comparison of the 5′ sequences of the Ng-CAM and Nr-CAM genes with those reported for the mouse L1 gene sequence (22
) revealed a close correspondence in intron/exon structure (see Fig. A
). However, in both the Ng-CAM and Nr-CAM genes, the ATG codon is situated in the second exon, whereas in the L1 gene the ATG codon was located in a region proposed to be the 5′-most exon, called exon A (22
). These contrasting comparisons suggested to us that there may be an additional exon and a promoter region upstream of exon A in the L1 gene. Primer extension analyses of mouse L1 mRNA transcripts in the original study (22
) were in accord with this supposition.
Figure 1 (A) Schematic representations of the mouse and human L1 genes. (Top) Diagram of the 5′-most sequences established in previous studies (22). The nomenclature for the exons are those given by Kohl et al. in their study (22). The extended portion (more ...)
To obtain cDNA sequences corresponding to the full-length 5′ end of the L1 messenger RNA, we performed a RACE experiment (11
) on RNA isolated from N2A cells. DNA sequencing of RACE clones revealed an additional segment of 5′ untranslated sequences not detected in previous studies (22
). No RACE products terminated at the 5′ end of exon A, suggesting that, contrary to previous conclusions (22
), the 5′ end of exon A is not a major site for initiation of L1 gene transcription in N2A cells.
To characterize the 5′ end of the L1 gene, we cloned and sequenced a 13-kb region of genomic DNA immediately upstream of the 5′-most sequences previously reported (22
). These sequence data are available from GenBank/ EMBL/DDBJ under accession number U91929
. They include a 2,943-bp segment of 5′ flanking sequence, the 119-bp first exon, and 9,429 bp of the first intron. The location of this gene segment relative to the previously characterized portion of the L1 gene (22
) is shown in Fig. A.
The additional 5′ untranslated sequences found in RACE experiments mapped the first exon ~10 kb upstream of exon A (Fig. A
). Given these new findings, we have redesignated exon A as exon 2.
Using RNAse protection assays, multiple transcription initiation sites were mapped to the 5′ end of the first exon. No transcripts were detected initiating at exon 2. Exon 1 was defined to be the 119 bp between the 5′-most RNA start site and the beginning of the 3′ splice junction. When combined with the results from our RACE experiments, these data provide additional support for the conclusion that exon 2 (previously exon A) is not a site of transcription initiation.
The sequences of 97 bp of the proximal promoter, the first exon, and a portion of the first intron are shown in Fig. B.
The RNA start sites initiated within a region containing several trinucleotide repeats of GCC and CAG (Fig. B
). Searches of the promoter and first exon for elements known to regulate gene expression revealed a binding site for the transcription factor SP1 (8
) that overlaps the 5′-most transcription initiation site. In previous studies of the L1 gene (22
), a binding site for homeodomain proteins was also identified 170 bp upstream of exon A (3
). Our analysis, however, now places this DNA element within the 3′ end of the first intron. Most significant for the present experiments, the mouse L1 gene sequence contains a single NRSE immediately downstream of exon 2 (Fig. A
Comparison of the Mouse and Human L1 Promoter Regions
A portion of the human L1 gene sequence is available from GenBank/EMBL/DDBJ under accession number U52112 and is part of a larger sequence from the X chromosome. We compared the sequence of the human X chromosome upstream of the existing human L1 gene with our sequence of the mouse L1 promoter and first exon and found that there was a high degree of similarity between the 5′ end of the mouse and human L1 genes (Fig. A). As is found in the mouse gene, the first exon of the human L1 gene is ~10 kb upstream of exon 2. As shown in Fig. , B and C, the sequences of the first exon, the 3′ splice junctions, and the proximal promoters containing the SP1 sites are highly conserved between the mouse and human L1 genes. A region of the L1 promoter further upstream, containing a putative binding site for transcription factors of the NF-1 and CBP families, is also highly conserved between the two species (Fig. C). The single NRSE in the second intron of the mouse L1 gene is also found in a comparable position in the human L1 gene. No other NRSEs were found in searches of the mouse and human L1 gene sequences.
The NRSE and the First Intron Are Required for Silencing of the L1 Gene Expression by REST/NRSF in Cellular Transfection Experiments
To examine whether the DNA sequences upstream of exon 1 in the mouse L1 gene had promoter activity, we prepared six luciferase reporter constructs containing exon 1 together with varying lengths of 5′ flanking sequence from the gene and examined their activity in either NIH3T3 fibroblasts or N2A neuroblastoma cells. All six L1 gene fragments (containing as few as 70 and as many as 2,943 bp upstream from the transcription initiation sites) showed promoter activity (Fig. ). L1-5 and L1-1 were the most active promoter constructs in NIH3T3 and N2A cells, respectively. In general, the luciferase activities of L1-1 through L1-6 were comparable or greater than the activity of the SV-40 early promoter and on average were between 13- and 8-fold greater than those produced by the promoterless luciferase vector in NIH3T3 cells and N2A cells, respectively. These findings indicate that there is a promoter upstream of exon 1 that is constitutively active in both NIH3T3 and N2A cells.
Tests of NRSE Function.
The fact that reporter constructs containing the L1 promoter and the first exon were equally active in neural (N2A) and nonneural (NIH3T3) cells indicated that cell type–specific expression required other regions of the gene. To assess whether the NRSE within the second intron of the L1 gene played a role in the silencing of L1 promoter in nonneural cells, we prepared a number of L1 gene constructs containing or lacking the NRSE and examined their activity in cellular transfection experiments.
Two constructs, L1-5N and L1-5Nr, were prepared by inserting two copies of the NRSE from the L1 gene in either forward or reverse orientation upstream of the most active L1 promoter, L1-5. These constructs were transfected into NIH3T3 or N2A cells and examined for activity. The presence of NRSEs in either orientation led to a significant reduction in L1 promoter activity in NIH3T3 cells, but not in N2A cells (Fig. A). These data indicate that the NRSEs are sufficient for silencing L1 promoter activity in a cell type–specific manner.
To assess the role of the NRSE in the context of the native L1 gene, six constructs containing up to 18 kb of the mouse L1 gene (L1-7 through L1-12; Fig. B) were prepared and examined for their activity in NIH3T3 and N2A cells. To compare the relative level of silencing by downstream segments of the gene, the activity of L1-7 was set at 100%. In NIH3T3 cells, addition to the promoter of an L1 gene segment containing exons 2–4 including introns and the NRSE (L1-8) reduced the activity to 24% of control (L1-7). Deletion of the NRSE in L1-8 (L1-9) resulted in 50% of the control activity, indicating that the NRSE was responsible for only a portion of the silencing found in this DNA fragment. Addition of the first intron and second exon of the L1 gene (L1-10) showed a reduction in activity (21% of L1-7) that was approximately equal to inserting the L1 genomic segment containing the NRSE (L1-8). This result suggests that the first intron contains a silencer that is as effective as the NRSE. Addition of the entire L1 genomic region between intron 1 through exon 4 (including the NRSE) (L1-11) resulted in the most significant reduction in activity (3% of L1-7). Deletion of the NRSE in L1-11 yielded L1-12. L1-12 had reduced activity (19% of L1-7) that was approximately equal to L1-10, further indicating that a silencing activity within the first intron was still effective even in the absence of the NRSE.
In N2A cells, the activities of L1-8 through L1-12 were indistinguishable from that of L1-7 (Fig. B). Overall, the results from the transfection experiments indicate that the NRSE and an additional silencer present in the first intron, both of which function in NIH3T3 cells, are not active in N2A cells.
Effects of Dominant Negative and Native Forms of REST.
To demonstrate that the NRSE in the L1 gene was a target for silencing via REST/NRSF, we performed two types of cellular cotransfection experiments. In the first experiment, a plasmid directing the expression of a truncated REST/NRSF protein (called D-REST) containing the DNA-binding zinc fingers but not the silencer domain (4
) was cotransfected into NIH3T3 cells together with various L1 gene constructs to examine whether a dominant negative form of the REST/NRSF protein could prevent silencing of L1 promoter activity. In the second experiment, N2A cells were cotransfected with L1 constructs and a plasmid expressing the fully active REST/NRSF protein to determine whether ectopic expression of REST/NRSF in cells that normally contain low levels of REST/NRSF activity could reduce the activity from the L1 constructs.
D-REST released silencing of all L1 gene constructs that showed silencer activity in NIH3T3 cells (L1-5N, L1-5Nr, L1-8, L1-9, L1-10, L1-11, and L1-12) (Fig. , A and B). Expression of the full-length REST protein in N2A cells led to partial silencing of the activities of L1-5N, L1-5Nr, L1-8, L1-10, L1-11, and L1-12 but did not affect the activities of L1-7 and L1-9 (Fig. , A and B). Thus, REST reduced the activity of all L1 gene constructs containing the NRSE, the first intron alone, or the two elements in combination.
We conclude that (a) the NRSE in the L1 gene responds to REST/NRSF; (b) the first intron contains a silencer that can function without the NRSE, but nonetheless acts in response to REST/NRSF; and (c) optimal silencing of the L1 gene by REST/NRSF is achieved when both the first intron and the NRSE are combined. These in vitro findings prompted an analysis in vivo of the modulation of L1 gene expression by the NRSE.
Production of Transgenic Mice Containing L1 Gene Constructs
To determine whether the 5′ end of the L1 gene was sufficient to direct expression of the gene to the nervous system in vivo and to examine the effect of NRSE removal on L1 expression, transgenic mice were generated containing two different L1-lacZ reporter constructs (Fig. ). The two transgenes, designated L1lacZ and L1lacZΔN, were identical to L1-11 and L1-12 (Fig. B), except that the luciferase gene was replaced by a lacZ gene cassette containing a nuclear localization signal. The only difference between these L1 transgenes was that L1lacZ contained the NRSE, whereas L1lacZΔN did not.
Figure 4 (Top) Diagram of the L1lacZ and L1lacZΔN constructs that were used to generate transgenic mice. A detailed description of the construction of these transgenes is provided in Materials and Methods. (Bottom) β-galactosidase expression (more ...)
33 transgenic lines were established for the L1lacZ transgene. 15 showed no expression of β-galactosidase, suggesting that in these cases, the transgene most likely integrated into genomic regions that completely silenced its expression. Of the 18 expressing lines, 12 showed neurally restricted β-galactosidase expression patterns that were identical (Fig. , pattern I). The remaining six lines showed the same neural pattern but in addition displayed some staining outside of the nervous system. For example, the line shown in Fig. called Ia has the same neural pattern as I but has additional staining in the cephalic mesenchyme. The other lines were similar to Ia but differed slightly in the locations in which the nonneural staining was seen. In all cases, the nonneural staining appeared in locations that were a subset of those carrying the L1lacZΔN transgene (see below). Such patterns might arise from a partial release of silencing of L1 gene expression upon integration of the transgene in a particularly active region of the genome.
For the L1lacZΔN transgene that lacked the NRSE, seven transgenic lines were obtained, and four of these showed β-galactosidase expression. All four lines had an identical expression pattern that included the neural pattern characteristic of the L1lacZ lines, but in addition had intense and expansive β-galactosidase staining outside of the nervous system (Fig. , pattern II).
To examine in detail the differences between neurally restricted and unsilenced patterns of L1 gene expression, one L1lacZ line showing the neurally restricted pattern I and one L1lacZΔN line showing the extensive nonneural staining pattern II were selected. The expression patterns of both transgenes in whole mounts were first compared at each day of embryonic development between E8.5 and E12.5 (Fig. ).
Figure 5 Analyses of the β-galactosidase staining patterns in whole mount from mouse embryos carrying either L1lacZ (A–E) or L1lacZΔN (F–J) transgenes. The stages of embryonic development are as follows: E8.0–8.5 (A and (more ...)
Expression of the L1lacZ Transgene Is Coincident with Neural Differentiation and Is Restricted to the Nervous System
Expression of the L1lacZ transgene was not detected before neural differentiation at E8.5 (Fig. A) and was first observed at E9.5 in the central nervous system (CNS) within the midbrain and in the peripheral nervous system (PNS) within the trigeminal ganglion (Fig. B; mb). At E10.5, the punctate β-galactosidase expression in the midbrain became more intense, showing a distinct posterior boundary at the mesometencephalic fold (Fig. C; mf). At E11.5, expression of the L1lacZ transgene extended to more rostral locations of the CNS and was observed in the telencephalon (Fig. D; t). In the PNS starting at E10.5, expression of β-galactosidase was observed in cranial and dorsal root ganglia and along the nerves emanating from these ganglia (Fig. C; cg and drg). Between E11.5 and 12.5, the cranial and dorsal root ganglia and the sympathetic chain showed strong L1lacZ transgene expression (Fig. , D and E; cg, drg, and sc).
Expression of the L1lacZΔN Transgene Occurs before Neural Differentiation and Appears at Several Nonneural Sites
Expression of L1lacZΔN transgene was observed first at E8.5, a full day earlier than in embryos carrying the L1lacZ transgene (compare Fig. , F and G, with A and B). This initial expression of the L1lacZΔN transgene was found within the first branchial arch and in the prosencephalon (Fig. F; b1 and p). These areas contain migratory neural crest cells from the regions of the developing hindbrain and mesencephalon that differentiate into both PNS and craniofacial mesenchymal tissues.
The most dramatic differences in the intensity of β-galactosidase expression patterns between the L1lacZ and L1lacZΔN transgenes were apparent at E9.5. While L1lacZ embryos showed faint expression of β-galactosidase in the midbrain and trigeminal ganglion (Fig. B), L1lacZΔN embryos showed intense β-galactosidase expression in the head and trunk (Fig. G). In the head, the L1lacZΔN transgene was expressed in the prosencephalic region surrounding the eye and in the frontonasal mass. Expression was widespread in the cephalic mesenchyme, branchial arches 1 and 2, and in the circumpharyngeal tract (Fig. G; cm, b1, b2, and ct). In the trunk, expression of L1lacZΔN was also evident in the posterior mesoderm (Fig G; pm). In the PNS, expression of the L1lacZΔN was observed earlier than that of the L1lacZ transgene appearing at E9.5–10.5, when neural crest cells were condensing into the primordia of the cranial and dorsal root ganglia (Fig. , G and H). In the CNS, expression of the L1lacZΔN transgene appeared in the midbrain, telencephalon, and spinal cord in a pattern that was very similar to that of the L1lacZ transgene (compare Fig. , H–J, to C–E).
Between E10.5 and E12.5 the L1lacZΔN transgene continued to be expressed during the differentiation of neural crest cells into both neural and mesenchymal tissues. Expression of the transgene persisted in the periocular region, the snout, and in dorsal cranial regions, particularly within an area overlapping the telencephalon and midbrain, and over the hindbrain (Fig. , H–J). In the PNS between E11.5 and E12.5, the L1lacZΔN transgene was expressed in the fully differentiated cranial and dorsal root ganglia, the sympathetic chain, and in peripheral nerves (Fig. , I and J; drg, sc, and pn). Between E10.5 and E12.5, expression of L1lacZΔN was prominent in the dorsal ectoderm, particularly over the hindbrain and cervical spinal cord, and extended over the entire ventral body wall and into the limbs (Fig. , H–J; de and bw).
Expression of the L1lacZΔN Transgene during Limb Development
As an example of ectopic extraneural expression, the pattern of appearance of the L1lacZΔN transgene was followed in more detail in limbs that were isolated from embryos between E10.5 and E13.5 (Fig. , A–D). In the anterior limb bud at E10.5, expression was seen in the apical ectodermal ridge (Fig. A; aer). At E11.5, expression became more prominent in the anterior portion of the limb and also along the nerves (Fig. B; am, pn). At E12.5, expression of β-galactosidase was observed in both anterior and posterior portions of the limb (Fig. C; am, pnz). At this time, extensive staining was observed in the nuclei of cells along peripheral nerves that penetrate and ramify within the limb. Since the lacZ reporter gene contained the nuclear localization signal, much of the staining along the nerves is likely to be in the nuclei of the precursors of peripheral glial cells ensheathing the nerves. By E13.5, expression of the L1lacZΔN transgene was prominent in the interdigital mesenchyme and persisted in cells ensheathing the nerves (Fig. D; im). Such apical ectodermal ridge and interdigital β-galactosidase staining was not observed in embryos carrying the L1lacZ transgene (data not shown).
Figure 6 Analyses of the β-galactosidase staining patterns produced by the L1lacZΔN transgene during the development of the forelimb. The dorsal side of the limb is shown at each stage of development: E10.5 (A), E11.5 (B), E12.5 (C), and E13.5 (more ...)
Deletion of the NRSE Releases Silencing of L1 Gene Expression in Nonneural Derivatives of Neural Crest and in Mesodermal and Ectodermal Cells
To provide further histological analysis of the cell populations within differentiating tissues in which the L1lacZ and L1lacZΔN transgenes showed differences in their expression, transverse sections were taken from embryos at various positions along the anteroposterior axis and examined for cellular expression of β-galactosidase. All tissue sites showing differences in the cellular expression patterns between these two transgenes are illustrated in Fig. . The common and ectopic sites of expression for the two transgenes are also summarized in Table .
Figure 7 Comparison of the patterns of β-galactosidase expression in sections of embryos carrying either the L1lacZ transgene (A–C, E, G, I, K, M, O, and Q) or the L1lacZΔN transgene (D, F, H, J, L, N, P, and R) transgenes. All transverse (more ...)
Cellular Expression of L1lacZ and L1lacZΔN Transgenes
Embryos carrying the L1lacZ transgene showed a neurally restricted pattern of β-galactosidase expression that is consistent with the known pattern of L1 expression in postmitotic neurons and peripheral glia. The L1lacZ transgene was expressed in the marginal zones of the telencephalon and midbrain (Fig. , A and C; mz), in the olfactory nerve (Fig. B; on), and in the intermediate zone of the olfactory bulb (Fig. , B and G; ob). In the spinal cord, L1lacZ was expressed in the mantle layer in motoneurons and in cells surrounding the motor and sensory roots (Fig. M; mn and mnr). The pattern of L1lacZΔN transgene expression was identical to the L1lacZ pattern in all of these neural cells (for examples, see Fig. , D, H, and N; mz, ob, mn, and mnr). In addition to these common regions of expression, the L1lacZΔN transgene showed intense expression in the neuroepithelia within the roof of the midbrain and in Rathke's pouch (Fig. , compare C and D, E and F; rm and rp).
In the PNS, both transgenes were expressed in the trigeminal (Fig. , G and H; tg), facio-acoustic, and glossopharyngeal ganglia. Expression of both transgenes was observed in the sympathetic chain and the vagus nerve, although the expression by the L1lacZΔN was significantly more intense than that of the L1lacZ transgene (data not shown). Both transgenes were also expressed in dorsal root ganglia and in the nuclei of cells ensheathing fiber bundles that project toward the periphery (Fig. , M and N; drg).
In contrast to L1lacZ embryos, mice carrying the L1lacZΔN transgene showed extensive β-galactosidase staining in nonneural tissues. The L1lacZΔN transgene showed intense expression in craniofacial mesenchymal tissues, particularly in the periocular skeleton and the cornea, and in the mandibular and maxillary processes. L1lacZ embryos showed no β-galactosidase expression in these areas (Fig. , compare G and H, and I and J; po, c, man, and max). The heart revealed little if any expression of the L1lacZ transgene (Fig. K) but showed intense expression of the L1lacZΔN transgene in the endocardial cushion tissue and in the wall of the left ventricle (Fig. L; ec and lv). In addition to these sites, the L1lacZΔN transgene was expressed in the dorsal ectoderm overlying the spinal cord (Fig. N; de).
In the abdominal region, both transgenes were expressed in the presumptive enteric ganglia surrounding the esophagus, stomach, midgut, and duodenum. However, the L1lacZΔN transgene showed more intense expression in the cells surrounding these tissues (Fig. , compare O and P; mg). The L1lacZΔN transgene was also expressed in the mesoderm of the abdominal body wall, the genital tubercle, and the hindlimb (Fig. P; bw, gt, and hl). Expression of both transgenes was observed in the abdominal paraganglia and later (at E13.5) in the adrenal medulla (data not shown). At E12.5, the L1lacZΔN transgene was expressed in the kidney within the metanephric tubules and the ureter (Fig. R; k and mt). Such expression was not observed in embryos carrying the L1lacZ transgene (Fig. Q).