(Received for publication, November 1, 1995; and in revised form, December 28, 1995)
From the
Nuclear factor I (NFI) family members are transcription factors that are believed to also participate in DNA replication. We have cloned two Xenopus laevis NFIs that are up-regulated by thyroid hormone. They are 84-95% identical to their counterparts in birds and mammals. In contrast, the two Xenopus NFIs are much less homologous to each other, sharing only 58% homology, which largely resides in the DNA binding domain at the amino terminus. However, both NFIs can bind to a consensus NFI binding site and activate the transcription of a promoter bearing the site. Northern blot reveals that both NFI genes are regulated in tissue- and developmental stage-dependent manners. They are first activated, independently of thyroid hormone, to low levels at stages 23/24, around the onset of larval organogenesis. After stage 54, their mRNA levels are dramatically up-regulated by endogenous thyroid hormone, and high levels of their expression correlate with organ-specific metamorphosis. Furthermore, gel mobility shift assay indicates that the NFI proteins are present in different organs and that their levels are regulated similarly to the mRNA levels. These results strongly suggest that NFIs play important roles during postembryonic organ development, in contrast to the general belief that NFIs are ubiquitous factors.
The proteins of nuclear factor I (NFI) ()family are
transcription factors encoded by multiple genes in birds and mammals
(Gil et al., 1988; Santoro et al., 1988; Meisterernst et al., 1988; Paonessa et al., 1988; Inoue et
al., 1990; Rupp et al., 1990). In addition, different
forms of these factors can be generated by multiple alternative
splicing of individual NFI genes (Santoro et al., 1988; Inoue et al., 1990; Apt et al., 1994), although the
functional difference among these various forms is still unclear. NFIs
are sequence-specific DNA binding proteins that recognize a consensus
NFI binding site made of TGGCA(N)
TGCCA (Nowock et
al., 1985; Gronostajski, 1986; Nilsson et al., 1989).
Upon binding to NFI binding sites, these NFIs can activate the
transcription of the corresponding promoters (Jones et al.,
1987; Cereghini et al., 1987; Santoro et al., 1988).
While the mechanism of this transcriptional activation is still
unknown, NFI binding sites have been found in a wide variety of genes
(Raymondjean et al., 1988; Zorbas et al., 1992; Inoue et al., 1990), and the NFI genes are expressed in many
different tissues (Cereghini et al., 1987; Paonessa et
al., 1988; Apt et al., 1994), suggesting that NFIs are
crucial for cell function in many organs. In addition, NFIs have also
been found to be required for the initiation of adenovirus replication
both in vitro and in vivo (Nagata et al.,
1982; Leegwater et al., 1985; Hay, 1985; Wang and Pearson,
1985; Bernstein et al., 1986; Gronostajski et al.,
1988). This raises the possibility that NFIs may also participate in
cellular DNA replication. However, it remains to be seen whether NFIs
play specific roles during development.
We have identified two NFI
genes that are up-regulated during the metamorphic transition in Xenopus laevis. Amphibian metamorphosis is an ideal model
system to study postembryonic development (Tata, 1993). It
systematically transforms every single organ/tissue of a tadpole, for
example the total resorption of the tail, de novo development
of the limb, and the remodeling of the simple tubular tadpole intestine
into a complex, multiply folded adult organ (Dodd and Dodd, 1976;
Gilbert and Frieden, 1986; Yoshizato, 1989). While different tissues
undergo drastically different changes at distinct developmental stages,
all are under the control of thyroid hormone (T) (Dodd and
Dodd, 1976; Galton, 1983; Kikuyama et al., 1993). T
is believed to affect amphibian metamorphosis by regulating the
transcription of specific target genes in different tissues through its
nuclear receptors (Tata, 1993; Shi, 1994). The two NFI genes were
isolated as two such T
- regulated genes during intestinal
remodeling, a process that involves both apoptosis of the larval
epithelial cells and proliferation and differentiation of the adult
epithelial cells (McAvoy and Dixon, 1977; Ishizuya-Oka and Shimozawa,
1987, 1992).
We demonstrate here that the two Xenopus NFIs bind DNA specifically and activate transcription in an oocyte transcription system. More importantly, we show that the expression of the NFI mRNAs as well as the NFI or closely related proteins is up-regulated in the intestine during metamorphosis as the larval organ degenerate and adult intestine develops. Furthermore, the mRNA and protein levels are also high during both limb morphogenesis and tail resorption while very low in premetamorphic tadpoles or embryos. These results strongly implicate the participation of NFIs in frog organogenesis.
Specific DNA binding by NFI was analyzed by
the gel mobility shift assay. 15 µl of the buffer containing 20
mM HEPES, pH 7.5, 5 mM MgCl, 100 mM NaCl, 5 mM dithiothreitol, 10% glycerol, 0.1% Triton
X-100, and proteinase inhibitors (5 µg/ml aprotinin, 5 µg/ml
pepstatin A, 5 µg/ml leupeptin, and 5 mM phenylmethylsulfonyl fluoride) was mixed with 2.5 µl of
poly(dI-dC) (500 ng) and
P-labeled double-stranded
oligonucleotide (5 ng) containing the consensus palindromic binding
site for NFI (ds-NFI, see below). The binding reaction was initiated by
adding the above protein extracts to the mixture (2.5 µl of control
or NFI-B1 extract, or 0.5 µl of NFI-C1 extract supplemented with
2.0 µl of control extract; less NFI-C1 extract was used due to more
efficient translation of NFI-C1 mRNA (see ``Results'')).
Samples were incubated for 20 min at room temperature and analyzed on a
6%, 0.5
TBE native polyacrylamide gel. As a nonspecific
competitor in DNA binding, a double-stranded oligonucleotide (ds-NS)
was used, which contained a binding site for thyroid hormone receptors
(Ranjan et al. (1994), where it was named xTRE).
For antibody supershift experiments, the oocyte extract and poly(dI-dC) were first incubated with 1 µl of either preimmune or anti-NFI serum for 45 min at room temperature. The labeled ds-NFI was then added, and the mixture was incubated for another 20 min. Alternatively, the labeled ds-NFI was added 20 min before the addition of the serum, and the incubation was continued for 45 min after the serum addition. The resulting complexes were analyzed as above.
Twenty-five ng of NFI mRNA/oocyte were injected into the cytoplasm of stage 6 oocytes. After 6 h of incubation at 18 °C, 5 ng/oocyte of the CAT promoter vector with or without the ds-NFI insert were injected into the nucleus. After incubation at 18 °C for 16-18 h, RNA and the plasmid DNA were isolated from the oocytes as described (Wong and Shi, 1995). The RNA was analyzed by primer extension using an antisense CAT primer (Wong and Shi, 1995), and the recovered plasmid DNA was analyzed by Southern hybridization (Ranjan et al., 1994).
Figure 1: X. laevis NFIs (xNFI) share strong homology with mammalian and avian NFIs. A, Xenopus NFI-B1 (xNFI-B1) is the homolog of the chicken NFI-B subfamily (cNFI-B) (Rupp et al., 1990). B, Xenopus NFI-C1 (xNFI-C1) is the homolog of human CTF/NFI (hCTF/NFI) gene (Santoro et al., 1988) and chicken NFI-C subfamily (Rupp et al., 1990). The DNA binding domains are bracketed. The sites of sequence divergence among different NFIs are putative alternative splicing sites and are indicated by arrows. Dots represent amino acid deletions, and dashes indicate identical amino acids. The boldface italic letters are amino acids that are conserved between Xenopus NFI-B1 and Xenopus NFI-C1 (58%), which concentrate in the DNA binding domain (86%).
To clone the missing coding regions, the anchor PCR method of Frohman et al.(1988) was used. The anchor PCR clones of NFI-B fell into three groups, NFI-B1, NFI-B2, and NFI-B3, respectively (Fig. 2). The NFI-B1 group of clones had completely identical DNA sequences in the region overlapping with the original cDNA clone, and their initiation codon lay 180 bp upstream of the 5`-end of the original cDNA. The NFI-B2 class of clones contained a deletion of 135 bp near the amino terminus and a few nucleotide sequence changes, resulting in 2 or 3 amino acid substitutions in the region overlapping the original clone. The last class, NFI-B3, had a deletion of 162 bp immediately after the initiation methionine of NFI-B1 and nucleotide sequence changes that produced two amino acid substitutions. Anchor PCR cloning of the 5`-end of NFI-C1 identified an in-frame stop codon upstream of the first methionine codon of the original cDNA, indicating that this cDNA clone contained the entire amino-terminal coding region. In addition, another clone (NFI-C2) was isolated that had a 27-bp insertion immediately after the methionine codon of the original NFI-C1 clone (Fig. 2). The anchor PCR cloning of the 3`-end of NFI-C1 resulted in four clones, one completely identical with the original cDNA clone and three other clones containing nucleotide sequence changes that resulted in only a single amino acid substitution in the region overlapping the original cDNA clone (Fig. 2B and data not shown). All anchor PCR clones were otherwise identical and encoded the carboxyl terminus of NFI-C1.
Figure 2:
Anchor PCR cloning revealed a multimember
family of NFI proteins in X. laevis. Only the deduced amino
acid sequences are shown. A, anchor PCR clones of the
amino-terminal end of NFI-B. NFI-B2 class contains three different
anchor PCR clones. All contain two amino acid substitutions compared
with NFI-B1 (positions 68 and 108). In addition, one of them contains
one additional amino acid change (position 72). Only a single clone was
isolated for NFI-B3. B, anchor PCR clones of the amino and
carboxyl terminus of NFI-C1. The clone NFI-C2 is identical to the
original clone of NFI-C1 except for a different N-terminal end.
Anchor PCR cloning of the carboxyl terminus identified four clones, one
as shown in the figure and the other three with only a single
amino acid substitution at position 376 (Ser or Ala for Pro) or
position 400 (Asn for Lys), respectively. The protein sequences derived
from original
cDNA clones are in boldface type, while
the new sequences obtained by anchor cloning are represented by lightface characters. Dashes indicate identical amino
acids.
The anchor PCR cloning, therefore, revealed the existence of a family of NFI proteins in X. laevis, which can be divided into two subfamilies based on sequence homology (Fig. 1). The strong homology among subfamily members suggests that three members of the NFI-B subfamily are most likely derived from alternative splicing of a single gene, and the subtle differences in their sequences are probably due to polymorphism. Similarly, NFI-C1 and NFI-C2 are most likely encoded by a single gene that is alternatively spliced. Such a prediction is supported by the previous reports of different forms of avian and mammalian NFI proteins (Santoro et al., 1988; Rupp et al., 1990), where the sites of sequence divergence match exactly with what we have found for the Xenopus NFI proteins (Fig. 1).
Sequence comparison among Xenopus, chicken, and human NFI proteins showed a strong homology among the various NFIs (Fig. 1). In particular, the predicted Xenopus NFI-B1 protein is over 94% identical to the chicken NFI-B subfamily members with the DNA binding domain being essentially identical (Rupp et al., 1990). Similarly, the Xenopus NFI-C1 is most homologous to the chicken NFI-C subfamily members and human NFI/CTF (Santoro et al., 1988; Rupp et al., 1990). Overall, about 84% identity exists among the different NFI-C proteins, and again the DNA binding domain is the most conserved region.
In contrast to the extremely high degree of sequence conservation among the members of a given subfamily, members of different subfamilies are more divergent. Thus, Xenopus NFI-B1 shares only 58% identity with Xenopus NFI-C1 (Fig. 1, boldface letters). While the carboxyl terminus has only a low level of homology between NFI-B1 and NFI-C1 (42%), the DNA binding domains share over 86% identity.
Figure 3:
Expression of NFI-B1 and NFI-C1 proteins
in X. laevis oocytes. A,
[S]methionine was coinjected with water
(-) or NFI mRNAs into oocytes. Protein extracts were analyzed on
a 10% gel. Dots indicate the positions of the overexpressed
proteins. B and C, Western blot analysis of the same
protein extracts electrophoresed on 7.5% gels with anti-NFI-B1 (B) or NFI-C1 (C) antibody. Note that both antibodies
were specific to their antigens. The two bands of very similar sizes
detected by the anti-NFI-C1 antibody that were also present in the
water-injected oocytes (-) are probably non-NFI peptides. Dashes on the left indicate the positions of the size
markers: 30, 46, 66, 97, and 220 kDa,
respectively.
To study the DNA binding activity of the NFIs, a double-stranded oligonucleotide containing a consensus NFI binding site (ds-NFI) for avian and mammalian NFIs (Nilsson et al., 1989) was end-labeled and mixed with extracts isolated from uninjected or mRNA-injected oocytes. The resulting complex was analyzed by the gel mobility shift assay. While the uninjected oocyte extract gave no detectable complex (Fig. 4, lanes 1-4), extracts from the oocytes preinjected with NFI-C1 or NFI-B1 mRNA formed a strong complex with ds-NFI (Fig. 4, lanes 5 and 12). The complex formed with NFI-C1 migrated faster than that with NFI-B1 (Fig. 4, compare lanes 5-11 with lanes 12-18), consistent with the smaller size of NFI-C1 (Fig. 1). The complexes could be efficiently competed out by the unlabeled ds-NFI itself. In contrast, even a 50-fold excess of a nonspecific double-stranded oligonucleotide (ds-NS) had no effect on the binding by either NFI-B1 or NFI-C1, demonstrating the specificity of the binding.
Figure 4:
Specific DNA binding by X. laevis NFIs. Extract from control(-), NFI-B1 (NFI-B1), or
NFI-C1 (NFI-C1) mRNA-injected oocytes were used in the gel
mobility shift assay with 5 ng of P-labeled ds-NFI and the
indicated amount of unlabeled ds-NFI or a nonspecific DNA (ds-NS)
competitor.
We next investigated whether the Xenopus NFIs were able to activate the transcription from a promoter bearing the NFI binding site. For this purpose, we inserted two copies of ds-NFI about 140 bp upstream of the major transcription start site of the SV40 early promoter in the CAT promoter vector. The original (CAT) or modified (CAT/NFI) vector was injected into Xenopus oocytes that had or had not been preinjected with the NFI mRNA. After overnight incubation, the transcribed RNA was analyzed by the primer extension assay. No signal was detected in the absence of injected promoter vector (Fig. 5, lanes 1, 4, and 7), demonstrating the specificity of the primer extension. Injection of both the CAT and CAT/NFI vectors gave low levels of transcription in oocytes uninjected with any NFI mRNA (Fig. 5, lanes 2 and 3). The levels of transcription from both vectors were comparable, and both vectors used the same expected major transcription start site.
Figure 5: Transcriptional activation by X. laevis NFIs in a reconstituted oocyte system. Control oocytes(-) or oocytes preinjected with the mRNA for NFI-B1 or NFI-C1 were injected with either one of two promoter vectors. The first vector (CAT) was a vector containing the SV40 promoter upstream of the CAT gene, and the second one (CAT/NFI) had two copies of the NFI binding site inserted into the CAT vector. Half of the oocyte homogenate was used for RNA analysis by primer extension (upper panel). The other half was used to quantify the injected DNA by slot blot analysis (lower panel). The relative promoter activity was determined by normalizing the primer extension signal with the DNA signal.
When the CAT and CAT/NFI vectors were
injected into oocytes that had been preinjected with either NFI-B1 (Fig. 5, lanes 5 and 6) or NFI-C1 (lanes 8 and 9) mRNA, they produced very different levels of
transcription. The preinjection of NFI mRNAs did not alter the
transcriptional activity of the CAT vector (compare lanes 5 and 8 with lane 2) but activated the
transcription of the CAT/NFI vector by about 10-fold (compare lanes
6 and 9 to lane 3). As controls for the
injection of the mRNA, our DNA binding,
[S]methionine-labeling, and Western blot
analysis had consistently shown that both NFI-B1 and NFI-C1 were
efficiently translated when their mRNAs were injected into the oocyte
cytoplasm. However, we consistently observed more efficient translation
of NFI-C1 mRNA, which might explain the slightly higher level of
transcriptional activation by NFI-C1. In addition, when the injected
promoter DNA was recovered after overnight incubation from the same
oocytes used to assay the transcriptional activity and analyzed by
hybridization (Fig. 5, lower panel), the results
clearly demonstrated that equal amounts of promoter DNA were present in
the nuclei of different samples. Thus, like their homologs in other
vertebrates, both NFI-B1 and NFI-C1 can activate a promoter containing
the consensus NFI binding site.
Figure 6: Northern blot analysis showing differential regulation of X. laevis NF1 genes in the intestine, tail, and hind limb during metamorphosis. Ten µg of RNA were used per lane except for the tail at stage 64 and the hind limb at stage 56, which had only 5 µg RNA. Duplicate blots were probed with the coding regions of NFI-B1 and NFI-C1. After boiling off the probes, the filters were hybridized with rpL8 as a control for loading (Shi and Liang, 1994). The blots containing limb RNA were exposed for a longer period. The positions of 28 and 18 S rRNA are indicated. Note that both genes had similar expression profiles. High levels of their mRNAs were present in the intestine during remodeling (stages 60-66), in the tail during resorption (stages 62-64), and in hind limb during and immediately after limb morphogenesis (stages 56-60; note that only half as much RNA was used for stage 56). The smeary signals for both genes were most likely due to partial degradation of the mRNAs because of their large sizes, about 10 and 8 kilobases for NFI-B and NFI-C mRNA, respectively. In addition, some size heterogeneity might be due to alternative splicing.
Figure 7:
T activation of NFI genes in
premetamorphic tadpoles. 10 µg of total RNA from intestine and tail
of stage 56 tadpoles treated with 5 nM of T
for
the indicated number of days were electrophoresed on 1%
agarose/formaldehyde gels. Duplicate blots were probed with the coding
regions of NFI-B1 and NFI-C1 cDNA. After boiling off the probes, the
same filters were probed with rpL8 as control of loading. The positions
of 28 and 18 S rRNA are indicated.
Figure 8:
Xenopus NFI genes are activated
during late embryogenesis and further up-regulated during
metamorphosis. Ten µg of total RNA from ovary, whole embryos, or
tadpoles up to stage 66 (the end of metamorphosis) were analyzed by
Northern blot hybridization. The hybridization signals were quantified
using a PhosphorImager. Note that both NFI-B and NFI-C genes were
activated around stage 23/24 (the early tailbud stages). Relatively low
levels of their expression were present throughout late embryogenesis
(stages 23-45; tadpole hatches around stage 35/36 and feeding
begins around stage 45). The mRNA levels were then up-regulated after
stage 54 when endogenous T levels began to increase (Leloup
and Buscaglia, 1977).
Figure 9: Anti-NFI-B1 antibody supershifts NFI-DNA complexes. DNA binding was performed as in Fig. 4with control (-) or NFI mRNA-injected oocyte extracts. Anti-NFI antibodies were added either before (lanes 3, 5, 8, 10, 13, and 15) or after (lanes 2, 4, 7, 9, 12, and 14) the addition of labeled ds-NFI. Note that anti-NFI-B1 antibody could supershift both the NFI-B1-DNA complexes (arrowhead) and less efficiently the NFI-C1-DNA complexes (asterisk) while anti-NFI-C1 antibody had no effect, independently of the order of antibody addition.
Tissue extracts from the intestine, limb, and tail of tadpoles at different stages were prepared and subjected to DNA binding analysis. The binding activity for ds-NFI was found to be regulated identically as the NFI mRNA levels in all three organs during metamorphosis ( Fig. 10and data not shown). Thus in both the intestine (Fig. 10A) and tail (Fig. 10C), the NFI binding activity was low in tadpoles before stage 58 and was up-regulated during metamorphosis (stages 62 and 64). On the other hand, the ds-NFI binding activity was high in the limb at stage 56 when morphogenesis took place. Subsequently, the activity decreased as the hind limb underwent growth with little morphological changes (Nieuwkoop and Faber, 1956; Fig. 10B).
Figure 10: NFI binding activity is regulated similarly as the NFI mRNAs during development. Whole cell extracts were isolated from the intestine (A), hind limb (B), and tail (C) of tadpoles at different developmental stages and analyzed for binding to labeled ds-NFI. Specific complexes were formed in the absence (lanes 1-4) or presence of a 20-fold excess of a nonspecific competitor (lanes 5-8) but not in the presence of a 20-fold excess of the unlabeled ds-NFI (lanes 9-12). The addition of the anti-NFI-B1 antibody could supershift most of the complexes formed (lanes 13-16). The arrowheads and asterisks indicate complexes of similar mobilities as the supershifted NFI-B1-DNA and NFI-C1-DNA complexes, respectively, shown in Fig. 9. Note that longer exposure was necessary for the tail samples (C) due to weaker binding activity and that more smear was present in stage 62 and 64 samples. This smear was likely due to protein degradation even though proteinase inhibitors were present in the samples. This is probably because proteinases were more abundant in the tail at these stages as the tail resorbs (Nieuwkoop and Faber, 1956). The protein degradation might be also responsible for the inefficient antibody supershifting.
The specificity of the DNA binding by the extracts was confirmed by the ability of the unlabeled ds-NFI itself (Fig. 10, lanes 9-12) to compete efficiently for the complex formation and the inability of a nonspecific DNA (ds-NS, lanes 5-8) to do so. Furthermore, anti-NFI-B1 antibody could supershift most of the complexes formed (Fig. 10, lanes 13-16). Based on the mobilities of the supershifted complexes (bands labeled by arrowheads and stars; compare them with those in Fig. 9), it appeared that both NFI-B and NFI-C were present in these tissue extracts and regulated similarly. Thus, while the exact identities of the NFI proteins are unknown, these results strongly suggest that NFI-B and NFI-C or closely related proteins are the predominant proteins, if not the only ones, that are responsible for the binding to ds-NFI.
Figure 11: NFI binding activity is present in adult organs. Whole cell extracts were made from different regions of the gastrointestinal tract, hind limb, and liver of young frogs and analyzed for ds-NFI binding activity. The binding activity was present in all tissues, and the binding could be competed out by a 20-fold excess of the unlabeled ds-NFI itself but not by a 20-fold excess of the nonspecific DNA (ds-NS). The adult liver complexes migrated faster, likely due to partial degradation of the NFI proteins. For comparison, stage 56 liver extract contained much less NFI binding activity than the frog liver but produced complexes of similar mobilities as those by the intestinal or limb extracts.
We have identified at least two genes of the NFI
transcription factor family that are regulated by thyroid hormone
during amphibian metamorphosis. Sequence analysis, DNA binding assays,
and transcription activation experiments demonstrate a strong
conservation of the sequence and function among the NFIs from Xenopus, chicken, and human. More importantly, the interesting
regulation of the expression of these genes by T during
metamorphosis provides strong evidence that these transcription factors
are important for postembryonic organ development.
Currently, it is unclear how the transcription activation takes place. It is known that NFIs can bind DNA as homo- and heterodimers (Mermod et al., 1989; Gounari et al., 1990; Kruse and Sippel, 1994). Furthermore, it has been shown that the amino-terminal half, including the DNA binding domain, is sufficient for dimerization, site-specific DNA recognition, and adenovirus DNA replication (Mermod et al., 1989). In contrast, the carboxyl half of protein and the DNA binding domain are required to activate transcription (Mermod et al., 1989; Altmann et al., 1994; Xiao et al., 1994). It is interesting to note that given the sequence divergence between Xenopus NFI-B1 and NFI-C1 in the putative activation domain, which is only 42% conserved, both can activate transcription to a similar extent in the oocyte transcription system. It is known that the oocyte stores large quantities of different factors important for embryogenesis, especially during the period prior to the onset of zygotic transcription. Thus, it is very likely that Xenopus NFI-B1 and NFI-C1 interact with different factors in the transcriptional machinery to activate the promoter. It would be interesting to know the identities of such NFI-interacting factors.
During the premetamorphic stages (before stage 56), the NFI genes are expressed at very low levels in the intestine and tail. They are then drastically activated in the intestine from stage 58 to 66 when larval epithelium undergoes cell death and adult (secondary) epithelial cells as well as the connective tissue and muscle cells proliferate and differentiate (McAvoy and Dixon, 1977; Ishizuya-Oka and Shimozawa, 1987). In the tail, the NFI expression begins to be up-regulated around stage 62. While this appears to be later than that in the intestine, it corresponds exactly to the period when massive tail resorption occurs (Nieuwkoop and Faber, 1956). Finally, highest levels of NFI mRNAs in the hind limb are present between stages 56 and 60, right at or shortly after limb morphogenesis.
The correlation of the NFI-B and NFI-C expression with tissue-specific metamorphosis as described for the mRNAs is also supported by our analysis of the NFI binding activity during development. Although the exact identities of the proteins responsible for the binding to the NFI oligonucleotide remain to be determined, DNA competition shows that the binding is specific. Furthermore, antibody supershift experiments indicate that both NFI-B and NFI-C types of complexes are formed and that most, if not all, of the binding activity can be accounted for by NFI-B and NFI-C or closely related transcription factors. Thus, while it is unknown how the NFI genes are regulated so differently in different organs, the close correlation of their expression with tissue remodeling during metamorphosis argues for a role of these transcription factors in organogenesis.
The biphasic development of amphibians, i.e. the embryogenesis and subsequent metamorphosis, serves as a unique model to study gene function during different stages of animal development. Our DNA binding and transcriptional activation experiments as well as Northern blot analysis of the NFI-B and NFI-C expression failed to detect NFI activities in oocytes and early embryos. Thus, if NFIs are required for transcription and/or replication during early embryogenesis, either the very low levels of NFI-B and/or NFI-C that evaded our detection or other NFIs such as the recently cloned Xenopus NFI-X subfamily (Roulet et al., 1995) are sufficient for this early period of development. On the other hand, both NFI-B and NFI-C genes are activated in embryos at early tailbud stages (stages 22/23). The expression during this larval period (up to stage 45, i.e. the feeding stage or the end of larval development), although relatively low, implicates a role of NFIs in larval organogenesis.
More importantly, the expression of the NFI mRNAs and the corresponding DNA binding activities correlate with metamorphosis. Two major events occur during this postembryonic process, i.e. cell death and cell proliferation followed by differentiation. The drastic up-regulation of the NFI genes during tail resorption and intestinal remodeling, both of which involve extensive cell death (Dodd and Dodd, 1976; Gilbert and Frieden, 1981; Yoshizato, 1989), suggest that the NFIs may be involved in the up-regulation of genes that control cell fate and/or encode degradative enzymes that are required for removal of degenerated tissues, such as proteases, nucleases, and extracellular matrix degradation enzymes. In contrast, when Xenopus NFIs are highly expressed in the hind limb at stages 56-60, there is little cell death in this organ except in the interdigital region. In addition, in the intestine, cell death is completed after stage 63 (McAvoy and Dixon, 1977) when NFI mRNA levels remain high. In these two cases, the predominant events are extensive proliferation and differentiation of adult cell types (Dodd and Dodd, 1976; McAvoy and Dixon, 1977; Ishizuya-Oka and Shimozawa, 1987). Thus, the Xenopus NFIs are also involved in the regulation of genes that are crucial for cell growth and/or cell differentiation. Such a function is also consistent with the strong NFI gene expression and presence of NFI binding activities in many organs of postmetamorphic frogs.
Both NFI-B and
NFI-C genes are direct T response genes and, therefore, the
earliest genes activated by T
in the gene regulation
cascade that controls tissue remodeling during metamorphosis. As
transcription factors, they are expected to directly regulate the
expression of downstream genes during metamorphosis. While their target
genes are still unknown, the presence of their binding sites in a wide
variety of promoters in other animal species suggests that the Xenopus NFIs will likely influence the expression of many
genes during metamorphosis. In this regard, it is interesting to note
that several NFI binding sites have been identified in the Xenopus vitellogenin gene (Cardinaux et al., 1994). The
vitellogenin gene is liver-specific and dependent upon estrogen for its
expression (Chang and Shapiro, 1990; Corthésy et al., 1990). The gene becomes competent to respond to
estrogen activation during metamorphosis (Rabelo et al.,
1994). Although T
treatment of tadpoles does not regulate
the vitellogenin gene directly, it can enhance its activation by
estrogen (Rabelo et al., 1994). This enhancement has been
attributed to the up-regulation of the estrogen receptor gene by
T
(Rabelo et al., 1994). Our results here suggest
another possibility. While we have not analyzed in detail the NFI
expression in the liver during metamorphosis, our DNA binding
experiment shows much higher levels of NFI binding activity in
post-metamorphic frog liver compared with that in premetamorphic
tadpoles. Thus, as direct response genes of T
, the
up-regulation of NFI genes in the liver during metamorphosis may enable
the vitellogenin gene to respond to estrogen.
While the vitellogenin gene is a likely target gene regulated by the NFIs in the liver, it will be important to identify other target genes, especially in other tissues. Furthermore, it is still unclear whether NFI-B and NFI-C regulate different target genes. As they can recognize at least some common binding sites, any functional difference is likely to reside in the less conserved carboxyl termini of the proteins. Through cooperative or antagonistic interactions with other transcription factors important for the expression of different promoters, NFI-B and NFI-C can differentially regulate the transcription of different genes. Clearly, the answer to this question waits for the identification of NFI target genes and the characterization of their promoters.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L43146[GenBank]-L43150 [GenBank]for NFI-B1, NFIB2, NFI-B3, NFI-C1, and NFI-C2, respectively.