Teleost FTZ-F1 Homolog and Its Splicing Variant Determine the Expression of the Salmon Gonadotropin IIß Subunit Gene

Dong Liu1, Yves Le Drean, Marc Ekker, Fei Xiong and Choy L. Hew

Research Institute, Hospital for Sick Children, Departments of Clinical Biochemistry and Biochemistry, University of Toronto (D.L., Y.L.D., F.X., C.L.H.), Toronto, Ontario, Canada,
Loeb Institute for Medical Research, Ottawa Civic Hospital and University of Ottawa (M.E.), Ottawa, Ontario, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenic factor 1, a member of the fushi tarazu factor 1 (FTZ-F1) subfamily of nuclear receptors, is a key regulator in mammalian reproduction. From an embryonic complementary DNA library, the zebrafish homolog of FTZ-F1 (zFF1A) and an alternatively spliced variant (zFF1B) were isolated. zFF1B represented a C-terminally truncated version of zFF1A. Whole mount in situ hybridization and reverse transcriptase-PCR analysis revealed that both zFF1A and B transcripts were present in the developing pituitaries, adult fish brain, gonads, and liver, albeit zFF1B messenger RNA was absent in testis. Comparison of the primary sequences of zFF1 with those of other FTZ-F1 subfamily members showed a close structural relationship between the mouse liver receptor homolog, which activated the {alpha}1-fetoprotein gene in rodent liver. However, similar to mouse steroidogenic factor 1, zFF1A regulated chinook salmon gonadotropin IIß subunit gene expression. On the contrary, zFF1B, which could bind a consensus gonadotrope-specific element with an affinity similar to that of zFF1A, lacked both the trans-activation function and synergistic interaction with the estrogen receptor. Furthermore, cotransfection studies in HeLa cells showed that zFF1B was a strong competitor for the action of zFF1A on the chinook salmon gonadotropin IIß subunit gene promoter. Our investigation suggests that 1) zFF1 represents an ancestor protein of the vertebrate FTZ-F1 homologs; 2) the antagonistic relationship between zFF1A and -B may dictate the expression of the FTZ-F1 target genes in a variety of tissues, including the pituitary; and 3) the naturally occurring zFF1B provides evidence that the C-terminal portion of zFF1A (80 amino acid residues) contains a major trans-activation function and a protein-protein interface.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Drosophila fushi tarazu factor 1{alpha} (dFTZ-F1{alpha}), a member of the nuclear receptor superfamily, is crucial for activation of the homeobox segmentation gene fushi tarazu in early embryogenesis (1, 2). Various dFTZ-F1{alpha} homologs have been discovered in other insects and vertebrate species (3, 4, 5, 6, 7, 8), and collectively, these FTZ-F1 homologs constitute a distinct subgroup of the orphan receptors (9, 10). In particular, they share high identities in their DNA-binding domains (DBD) and conserved FTZ-F1 box (11), whereas other regions are either less conserved or quite divergent. Recent evidence reveals that, in contrast to the majority of nuclear receptors binding as dimer to direct or inverted repeat of 5'-AAGGTCA-3' (12), FTZ-F1 binds as monomer to its cognate 5'-PyCAAGGPyCPu-3' site. The FTZ-F1 box recognizes the first 3 bp (5') of the DNA sequence and determines the specificity of the monomeric binding (7, 9, 10, 11).

Mammalian steroidogenic factor 1 (SF-1) is a homolog of dFTZ-F1{alpha} (4). SF-1 is a key regulator of the hypothalamus-pituitary-gonadal axis (13, 14, 15, 16). It is expressed in all primary steroidogenic tissues and acts as a crucial transcription factor of enzymes involved in steroid production (including the sex hormones) (17, 18, 19, 20, 21, 22, 23). As demonstrated in the SF-1 gene-disrupted mice, along with the disappearance of steroidogenic organs, such as the adrenal cortex and gonads (13), the loss of gonadotrope-specific markers, including LH ß-subunit (LHß) and GnRH receptors, has been observed (15, 16). Furthermore, the development of the ventromedial hypothalamus in FTZ-F1 null mice was defective (14). In addition to its determinant role in the steroidogenic events and gonadotrope function, SF-1 is strongly suggested to be involved in sex determination events and gonadal differentiation (24). Specifically, SF-1 is likely to be a regulator of the anti-Mullerian hormone, as anti-Mullerian hormone gene promoter activity in Sertoli cells is supported by a promoter region containing the SF-1-binding site. Although SF-1 expression appears in the urogenital ridge of both sexes at 9–9.5 days postconception, the persistence of SF-1 is only found in males beyond 12.5 postconception, at which stage the first differences in sexes develop. Furthermore, all SF-1-disrupted mice are born with developed female internal genitalia (13, 16, 25, 26).

Only recently, however, has a direct link between SF-1 and pituitary LHß gene been identified both in vitro and in vivo (27, 28, 29, 30). Our recent in vitro studies on the salmon gonadotropin gene showed that mouse SF-1 (mSF-1) is an essential transcription factor controlling salmon gonadotropin IIß subunit (sGTHIIß) gene expression (30). A dramatic enhancement of the sGTHIIß gene promoter activity was exhibited by the synergy of mSF-1 with ligand-activated rainbow trout estrogen receptor (rtER). Our data suggest that these two nuclear receptors ultimately lead to the GTHII surge in the later phase of salmon reproduction. Similarly, a synergistic effect is necessary for the mammalian LH surge, i.e. NGFI-A, a Cys-His zinc finger transcription factor. SF-1 could induce a dramatic increase in the activity of rodent LHß gene promoter in heterologous cell lines (31). In each case, two transcription factors of limited tissue specificity were needed to trigger gonadotropin ß-subunit (GTHß) gene expression, indicating a common mechanism of GTHß gene regulation.

To better understand reproductive regulation in teleost and explore the mechanisms of sex determination in fish, we cloned the zebrafish FTZ-F1 homolog (zFF1A) and a novel splicing variant (zFF1B) from a zebrafish embryonic complementary DNA (cDNA) library. We showed that zFF1B is a C-terminally truncated zFF1A, and zFF1 gene transcripts exhibit a wider tissue distribution than the mammalian SF-1. Cotransfection studies in HeLa cells indicate that zFF1A resembles mSF-1 functionally, whereas zFF1B lacks the trans-activation function. However, zFF1B is a strong competitor for the action of zFF1A on sGTHIIß promoter. Our results suggest a novel mechanism to control target gene(s) expression in salmon pituitary.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular Cloning of Zebrafish FTZ-F1 Homolog Gene
The zebrafish embryonic cDNA library was constructed from embryos pooled between 6 and 72 h stages. The complementary DNA fragment of the zFF1 gene transcript was obtained by reverse transcription-PCR (RT-PCR) using total RNA isolated from both embryos and adult fishes. Due to the high conservation of P box and FTZ-F1 box within the known FTZ-F1 subfamily members (2, 3, 4, 6, 7, 8, 32), two primers (P and A), overlapping parts of DBD and the FTZ-F1 box encoding DNA sequences, respectively, were synthesized. One DNA fragment of the expected size (273 bp) was obtained and used as a probe to screen the library.

Two types of positive cDNA clones were identified (Fig. 1AGo). The majority of them (zFF1A) were identical to each other, with a 3126-bp insert, whereas the minor one (zFF1B) contained an insert, 2286 bp in length, that was different from zFF1A at both its 5'- and 3'-untranslated regions (UTR). Except for an extra 118 bp at the 5'-end of the zFF1B cDNA insert, both zFF1A and zFF1B cDNAs were identical in their 5'-UTR, and the identity extended to the translation start site and another 1292 bp. In their 3' UTRs, there was no significant similarity between these two types of cDNAs (Fig. 1BGo).



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Figure 1. Zebrafish dFTZ-F1{alpha} Homolog and an Alternative Splicing Variant Were Isolated from an Embryonic cDNA Library

A, Schematic map of both zFF1A and -B cDNAs. ORFs are represented as shadowed boxes with black frames. The thick black lines are indicative of the identical UTR sequences between the zFF1A and -B cDNAs, and the hatched lines represent sequences unique to the zFF1B cDNA. The PstI restriction sites (and their locations), which were employed to release either zFF1A- or zFF1B-specific fragments to be used in probe labeling for in situ hybridization, are indicated in italic. The arrowheads represent three primers (I, II, and III) used in the RT-PCR experiment shown in Fig. 4Go. An AATAAA site is shown in the zFF1B cDNA 3'-end. The 5'-flanking region obtained by inverse PCR using primer designed from the zFF1A cDNA is also shown. The broken line represents the sequence that was not shown in either A or B cDNA. The scheme is not to scale. B, The complete sequences of zFF1A cDNA and the deduced amino acid residues in ORF. Both C and E regions are boxed. Two zinc fingers and the FTZ-F1 box are shadowed by light dots, and the FTZ-F1 box is framed. Region II, region III, and AF-2 core are highlighted with heavier dots, and the AF-2 core is boxed. Several putative polyadenylation signals are underlined.

 
The open reading frames (ORF) deduced from these cDNAs indicate that both cDNAs encode proteins of 516 and 436 amino acid (aa) residues, respectively. The ORFs of zFF1A and B cDNAs (ORF-A and -B) are identical from their first Met up to the 434th Leu, which is followed immediately by two additional aa residues and a stop codon in ORF-B (Fig. 1BGo). Therefore, zFF1B represents a C-terminally truncated zFF1A.

Both zFF1A and B Transcripts Are from the Same Gene Locus
Southern blot analysis of genomic DNA using a probe restricted from zFF1B cDNA covering nucleotides 1–306 suggested that there was a single gene copy responsible for both zFF1A and -B (data not shown). When a primer based on the 5'-end of zFF1A cDNA was used in the inverse PCR, a 500-bp DNA fragment was generated (Chung, B. C., personal communication). In the 500-bp fragment, a 118-bp sequence unique to zFF1B cDNA was found directly adjacent to the start site of zFF1A cDNA (Fig. 1AGo), providing further evidence that both zFF1A and -B transcripts are derived from a single genomic locus.

Polyadenylation signal and poly(A) tail have been found near or at the 3'-ends of all cloned cDNAs, arguing against the possibility that zFF1B cDNA was a cloned artifact. It is likely that both zFF1A and -B transcripts were derived from the same gene by differential splicing. The divergence between them is reminiscent of the site where intron 6 was found in the mSF-1 genomic gene (Fig. 2BGo). Furthermore, a nearly perfect intron donor site, GG/GTGAGT (the vertebrate consensus is AG/GTRAGT), was present at the point of divergence, strongly suggesting the involvement of an intron or alternative exon during the zFF1 gene heterogeneous nuclear RNA process (33). Therefore, alternative splicing results in the production of zFF1A and -B messenger RNAs (mRNAs).



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Figure 2. Both zFF1A and -B Transcripts Come from the Same Gene Locus

A, The divergent point between zFF1A and -B cDNA is reminiscent of the site where intron 6 was found in the SF-1/ELP genomic gene. B, The C-terminal truncation in zFF1B is due to an early stop codon introduced by an intron-like sequence or alternative exon. The putative intron donor site is underlined, and the three asterisks represent the stop sign.

 
The zFF1A Is an Ancestral Protein of Vertebrate dFTZ-F1{alpha} Homologs
A comparison of the primary structures in the FTZ-F1 subfamily revealed that in both the DBD and FTZ-F1 box regions, the overall identity within the group was high. Notably, zFF1A was almost identical to Xenopus fushi tarazu factor 1-related protein A (xFF1rA) and mouse liver receptor homolog (mLRH-1) (7, 32). Furthermore, the identity between zFF1A and mSF-1/Ad4BP was among the highest. In other conserved regions, such as R-II and R-III (which are located in the putative ligand-binding domain of the nuclear receptors), the highest identity was found among zFF1A, xFF1rA, and LRH-1. Even in the divergent A/B region, although mLRH-1 has a longer A/B domain than zFF1A and xFF1rA, high identity of these three proteins was observed. These data indicate that zFF1A, xFF1rA, and mLRH-1 are the closest relatives in the FTZ-F1 subfamily. In addition to xFF1rA and mLRH-1, zFF1A also shows a strong similarity to mSF-1, particularly in the R-II, R-III, and AF-2 regions (Fig. 3Go).



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Figure 3. The zFF1 Structurally Resembles mLRH-1 and Xenopus FF1r

Schematic alignment of all known members in the FTZ-F1 subfamily. Only regions of the two zinc fingers, FTZ-F1 boxes II and III, are compared, and the overall identity of each region is shown as a percentage compared with the zFF1 protein. The AF-2 core is LLIEML.

 
It is now clear that in mammals, there are two FTZ-F1 homologs, i.e. mSF-1 and LRH-1. LRH-1 was recently identified to be expressed mainly in liver and pancreas and is involved in activation of {alpha}1-fetoprotein gene expression (32). To distinguish to which category the cloned zFF1 gene belongs, a documentation of the tissue distribution of zFF1A and -B messenger RNA is necessary. Except for the absence of zFF1B mRNA in testis, both zFF1A and -B transcripts were detected in brain, liver/pancreas, and gonads by RT-PCR, using primers either common or specific to zFF1A and -B cDNAs (Fig. 4AGo).



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Figure 4. Tissue Distribution of Both zFF1A and -B Transcripts in Adult Organs and Embryos

A, Both transcripts are present in adult brain (B), liver (L), and ovary (O), whereas zFF1B is absent in testis (T). Primers I, II, and III are illustrated in Fig. 1AGo and described in Materials and Methods. The left panel is from the zFF1A-specific reaction, and the right panel is for zFF1B. Fragments from both reactions are predicted to be 475 and 701 bp in length, respectively. PCR products were fractionated, blotted, and probed with the labeled zFF1A cDNA. Shown are the results after exposure to the x-ray film for 12 h (room temperature), and no band was observed in right T lane by a longer exposure (48 h at -70 C). B, Whole mount in situ hybridization. An 1.5-kb fragment (specific to A) was released by PstI digestion (nucleotides 1582 and 3105) from the zFF1A cDNA, whereas an 1.1-kb portion was released by PstI (nucleotide 1144) and XhoI (near the 3'-end of insert on pBluescript vector) double restriction from the zFF1B cDNA (see Fig. 1AGo). These two DNA fragments were then used as the templates to generate antisense RNA probes for the in situ hybridization. The thick arrows point to the signals in the developing pituitaries, and the thin arrow indicates a subset of cells in the mandibular arch that expresses zFF1A.

 
To further examine the localization of these two transcripts in the pituitary, whole mount in situ hybridization of zebrafish embryos was performed using antisense RNA probes specific to 3'-UTR of either zFF1A or -B. The anterior and intermediate lobes of fetal rat pituitary were developed from a pocket of oral ectoderm known as Rathke’s pouch (34). In fish, the complete development of the pituitary is at its larvae stage, and the developmental pattern of this organ is similar to that of its higher vertebrate counterpart (35).

Cells in positions corresponding to that of the developing pituitary expressed both the zFF1A and -B transcripts starting around 27 h (36), and the highest intensity of the in situ hybridization signals in the developing pituitary cells was obtained between 36–48 h (data not shown, or Fig. 4Go, bottom panel). In addition to cells of the developing pituitary, a subset of cells in the mandibular arch expressed both zFF1A and -B transcripts. Mandibular arch expression of zFF1 persisted well into the third day of development and diminished gradually (data not shown). RT-PCR using primers specific to either zFF1A or -B cDNA confirmed the appearance of both transcripts before 48 h (data not shown).

Recently, it has been proposed that mSF-1 and LRH-1 loci are the result of duplication of an ancestral FTZ-F1 gene in vertebrates (7). Our data suggest that the zebrafish gene might represent an ancestor for the mammalian FTZ-F1 homolog genes before gene duplication.

Although zFF1A Resembles mSF-1 Functionally, zFF1B Is Devoid of Its Trans-Activation Function
Both full-length cDNAs and the ORF-A fragment were introduced into the pCDNA3 expression vector, and the resulting constructs were used in the cotransfection study in HeLa cells. The sGTHIIß -39/+42 promoter was demonstrated as a strong basal promoter in HeLa cells (37). This promoter, linked with a consensus GSE upstream (consGSE/CAT-39), was chosen as the reporter construct. As shown in Fig. 5AGo, zFF1A, similarly to mSF-1, was sufficient to up-regulate the GSE-containing reporter gene. Moreover, no significant variation was found between zFF1A and ORF-A expression constructs in their trans-activation abilities, suggesting that the UTR sequences of zFF1A cDNA make a limited contribution to the expression efficiency of the gene in the in vitro system. On the other hand, with the truncation of 80 aa residues, including the AF-2 domain in zFF1B, the reporter activity mediated by zFF1B is minimal. Neither zFF1A nor zFF1-B affected sGTHIIß -39/+42 promoter in the absence of consGSE, indicating that this DNA sequence was necessary to mediate the trans-activation of the orphan receptor. Other non-GSE sequences in front of the basal promoter could not be activated by either zFF1A or -B (see Fig. 7BGo).



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Figure 5. Although zFF1A Resembles mSF-1 Functionally, zFF1B Loses Its Trans-Activation Function

A, In HeLa cells, both zFF1A and mSF-1 trans-activated a consGSE-containing reporter construct similarly, whereas zFF1B did not. Ten micrograms of DNA, including 5 µg reporter and 1 µg expression vector, were used in each transfection. Data represent the mean ± SEM in triplicate of at least three independent experiments. B, Both zFF1A and -B could bind consGSE with similar affinities. About 10 µg extracted protein were used in each binding reaction. Either a 25-fold excess of cold DNA fragment or the same volume of water was used in each line. Competitors used in this experiment are detailed in Materials and Methods. consGSE, Consensus SF-1-binding sites; sGSE2, GSE presenting sGTHIIß promoter.

 


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Figure 7. The zFF1B Is a Strong Competitor for the Action of zFF1A on the sGTHIIß Promoter

A, In the presence of ER, zFF1B alone did not affect the action of E2 on the promoter. However, the dramatic stimulation of the promoter triggered by the synergistic interaction between ER and either zFF1A or mSF-1 was inhibited by zFF1B. The ratios shown on top of the histograms indicate the proportion of zFF1A or mSF-1 to zFF1B. B.S. represents pBluescript plasmid, which was used to adjust the total DNA amount; a total of 12 µg of DNA were used in each transfection. Data were pooled from four independent experiments (performed in triplicate) and are shown as the mean ± SEM. B, The zFF1B is a constitutive competitor. In the absence of ER, zFF1B specifically decreases the action of zFF1A on the GSE-containing constructs. Data were collected from two independent experiments (performed in triplicate). The constructs used in these experiments are detailed in Materials and Methods.

 
As reported in the study of embryonal long terminalrepeat-binding protein (ELP) and xFF1rAshort (23, 38, 39), the splicing variants of mSF-1 and xFF1rA, respectively, the DNA binding abilities of these C-terminally deleted variants were drastically reduced. To verify the nature of the loss of trans-activation function of zFF1B, a gel mobility shift assay was conducted using the whole cell extract from various FTZ-F1 gene-transfected HeLa cells. Fig 5BGo shows that cell extract from either zFF1A or B expression vector transfected cells were able to retard the labeled consGSE with similar affinity. This binding was comparable to the mSF-1 transfected cell extract. Competition experiments further confirmed the specificity of the binding. The shifted band was abolished with the addition of a 25-fold excess of consGSE, while the same amount of unlabeled sGSE2 (the GSE sequence found in sGTHIIß promoter) was less effective (30). When other probes were used, such as the non-specific Homo oligo or the pERE (proximal estrogen receptor element) palindrome (40), there was no significant competition. Therefore, the failure in trans-activation function of zFF1B is not due to its lack of direct GSE binding.

Another distinct feature of mSF-1 is its ability to synergize with rtER to stimulate sGTHIIß promoter activity (30). To assess the functional similarity between mSF-1 and zFF1, cotransfection studies were performed in HeLa cells, with the sGTHIIß-289-CAT (chloramphenicol acetyltransferase) promoter containing pERE and sGSE2 sites necessary for the mSF-1/ER synergistic interaction. There was no significant variation in the trans-activation of either zFF1A or mSF-1 alone, whereas zFF1B had no effect on the sGTHIIß -289/+42 promoter. When rtER/estradiol (E2) was introduced, only zFF1A and mSF-1 synergized with rtER to trigger a dramatic enhancement of the promoter activity, whereas zFF1B failed to do so (Fig. 6Go). Similar results were obtained when longer promoters were used (see Fig. 7Go). These data clearly demonstrate that zFF1A resembles mSF-1 functionally in terms of its ability to regulate sGTHIIß gene expression. However, zFF1B lost not only its trans-activation function, but also its ability to interact with ER.



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Figure 6. Both zFF1A and mSF-1 Exerted Synergistic Interaction with ER to Trans-Activate the sGTHIIß Gene

Thymidine kinase (TK)-CAT, sGTHIIß-39/CAT, and sGTHII-289/CAT reporters were transfected in HeLa cells, either individually or with the expression vector for ER, mSF-1, z.FF1.A, z.FF1.B, z.FF1.A/ER, or z.FF1.B/ER. Ten micrograms of DNA were used in each transfection. Cells cotransfected with ER were treated with E2 (1 µM). Histograms represent the mean ± SEM of at least three independent experiments (in duplicate or triplicate).

 
The zFF1B Is a Strong Competitor for zFF1A Action on the sGTHIIß Gene Promoter
Alternative splicing is a mechanism that regulates gene expression and generates protein diversity (41). Due to the premature termination of the reading frame of the constitutively spliced protein or transcription factor, the splicing variant usually shows deletion or modulation of a function(s). Based on the functional analysis shown above and the colocalization of both zFF1A and -B transcripts in the developing pituitaries, it is possible that there is antagonism between zFF1A and -B to control FTZ-F1-regulated genes in vivo.

To verify this hypothesis, cotransfection studies were conducted by including both zFF1A and -B with the reporters. As shown in Fig. 7AGo, trans-activation of sGTHIIß-1260-CAT by either zFF1A or mSF-1 was indistinguishable, whereas zFF1B was ineffective. Similarly, zFF1A could stimulate sGTHIIß-3358-CAT, but zFF1B could not (Fig. 7BGo). Interestingly, when an equal amount of zFF1B was added to the cotransfection, the promoter activities activated by zFF1A decreased (Fig. 7BGo). The inhibitory effect of zFF1B was GSE dependent. There was no significant interference in the ER/E2-induced activity when the same amount of zFF1B was incorporated into the pERE/sGTHIIß-39/ER cotransfection. Such an antagonistic relationship between zFF1A and -B was more dramatic when the sGTHIIß promoter was tested for FTZ-F1 and ER synergy (Fig. 7AGo). The inhibitory effect is dependent on the quantity of zFF1B present in cells. When the ratio of A to B was less than 10:1, a significantly reduced CAT activity was found. Similarly, stimulation of the target promoter triggered by mSF-1 and rtER was impaired by the addition of an equal amount of zFF1B, confirming that zFF1B is a strong competitor for either mSF-1 or zFF1A in sGTHIIß gene regulation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The zFF1 Protein Is an Ancestor of the Vertebrate FTZ-F1 Homologs
In addition to their presence in the adult reproductive axis, both zFF1A and -B transcripts were detected in liver and possibly in pancreas or kidney as well. In Xenopus, there was also only one type of FTZ-F1 homolog closer to mLRH-1 identified in its tetraploid genome (7), and both xFF1rA and xFF1rAshort are transcribed in liver, kidney, and oocytes (38). However, there is no evidence to indicate that both full-length and truncated transcripts are found in frog brain and pituitary. We currently do not know whether the functions of mSF-1 and mLRH-1 are exchangeable, even though both proteins recognize the same DNA-binding site indistinguishably (32). Evolutionarily, both proteins should share a common ancestor. By RT-PCR and in situ hybridization studies, we have provided evidence of such an ancestral protein. The activity of liver in lower vertebrates is closely related to reproductive activities. For instance, vitellogenins and steroid-binding proteins are produced in liver, then secreted out of the liver, and finally transferred to either ovary or steroid hormone-regulated tissues, such as hypothalamus and pituitary.

The C-Terminal 80 aa Residues of zFF1A Contain a Trans-Activation Domain and a Protein-Protein Interface Domain
The nuclear receptor superfamily is characterized by the presence of six regions (A–F) that provide the receptor with multiple functions necessary for its DNA-binding ability and transcriptional activation. Some recent studies revealed that in addition to the ligand-binding domain, region E of the nuclear receptor contained a ligand-dependent trans-activation function activating factor-2 (AF-2) (42, 43). The core of the AF-2 domain has been mapped in the C-terminal part of the E region and shown to form an amphipathic {alpha}-helix (43). This {alpha}-helical motif is well conserved among all known transcriptionally active members of the superfamily. In the presence of its putative ligand or coactivator, conformational changes in the E region could reorientate the AF-2 surface to interface with the basal transcriptional machinery.

The zFF1A and other transcriptionally active FTZ-F1 homologs in vertebrate showed conserved AF-2 domain at their C-terminal ends (Fig. 3Go), whereas the complete loss of trans-activation of zFF1B indicated that the AF-2 domain in zFF1A is solely responsible for its transcriptional activity. Due to the loss of their DNA-binding abilities, ELP and xFF1rAshort no long act as transcriptional activators. In another case, complete removal of the AF-2 motif LLIEML from the C-terminus of rat LRH-1 also caused the loss of its trans-activation function (32). Taken together, these results suggest that the transcriptional activities of the members of the FTZ-F1 subfamily are mainly due to their C-terminal regions.

Two distinct dimerization interfaces have been found in nuclear hormone receptors. Besides the D box located in the second zinc finger of DBD, a helical segment (H9 and 10) of retinoic acid receptor (RAR) was implicated as part of the dimer interface. More recently, the helical segment has been confirmed as a transferable element critical for determining identity in the heterodimeric interaction and for high affinity DNA binding (44, 45). Both interfaces are suggested to differentially modulate target gene specificity of the receptors.

The search of the zFF1A identity box (I box) defined from E regions of retinoid X receptor (RXR), RAR, thyroid hormone receptor (TR), and chicken ovalbumin upstream promoter transcription factor (COUP-TF) (45) revealed that a potential I box is localized at the N-terminus of the extra 80-aa segment in zFF1A. Comparison of all vertebrate FTZ-F1 homologs within this 45-aa stretch indicated that this region is Leu rich and highly conserved within the group. Moreover, the identity among these FTZ-F homologs, hRXR{alpha}, and COUP is significant (Fig. 8AGo). Given that only zFF1A was able to achieve the synergistic effect on the sGTHIIß promoter with ER, it is tempting to speculate that the I box may be directly related to the interaction of the two nuclear receptors. On the other hand, although P box regions of FTZ-F1 subfamily members are identical, D box regions are not well conserved (Fig. 8BGo). The monomeric binding nature of this group of receptors may explain the diversity of D box regions among species.



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Figure 8. The I Box Might be Crucial for the zFF1A and ER Synergistic Interaction

A, High conservation (aa sequences) among the vertebrate FTZ-F1 members in the I box region is shown. H9 and H10 represent helixes 9 and 10 shown in the RXR{alpha} crystal structure, which corresponds to the I box region defined from RAR, RXR, TR, and COUP-TF. B, DNA-binding region of all FTZ-F1 members. Two zinc finger regions are boxed. The P box is responsible for the specific DNA recognition, whereas the D box contributes to the DBD dimerization.

 
The zFF1B Is a Novel Splicing Variant in the FTZ-F1 Subfamily
In most cases, alternative splicing from a single pre-mRNA gives rise to protein isoforms sharing extensive regions of identity and varying only in specific domains, thus allowing for the fine modulation of protein function (41). In the newly emerged FTZ-F1 subfamily, splicing variants of most functional members have been identified, including mELP, xFF1rAshort, DHR39 short (7, 32, 46, 47), and zFF1B, as described in the present report. ELP and xFF1rAshort are apparently due to alternative intron/exon usages (23, 38). Interestingly, the organization of xFF1rAshort cDNA is homologous to that of zFF1B, which also contained an extra 5'-UTR sequence and differs completely from xFF1rA cDNA, downstream of their divergent point (23). Except for the dFTZ-F1{alpha} proteins, all of the differentially splicing events generate the C-terminal truncation in the E region compared with their wild type proteins.

The ligand-binding domain of the E region, in the presence of the receptor’s putative ligand or cofactor (32), is believed to govern allosteric transitions in the receptor structure to facilitate the binding of DBD to DNA. In many circumstances, conserved regions II and III are involved directly in the DNA binding. For instance, a series of deletion mutants with the truncated E region of xFF1rA, in which R-II, R-III, or both are removed, showed impaired DNA-binding abilities (38). Other examples come from TR{alpha}2 and truncated Rev-ErbA{alpha} (38). In both cases, C-terminal truncation modulated their DNA-binding activities. However, in all C-terminally truncated splicing variants of the FTZ-F1 subfamily, only zFF1B retained intact conserved R-II and R-III, whereas all others have their truncations either in the region between R-II and R-III or in R-II (Fig. 3Go). ELP has been indicative of a significantly weaker DNA-binding ability compared with SF-1. Similarly, xFF1rAshort has poor DNA-binding activity, as shown in the gel retardation assay (38).

Therefore, zFF1B represents a novel splice variant in the FTZ-F1 subfamily. First, zFF1B is capable of binding to GSE, and the affinity is comparable to that of zFF1A. Secondly, due to the colocalization of both zFF1A and -B transcripts in the developing pituitaries of zebrafish embryos, zFF1B is a naturally occurring competitor to zFF1A in controlling their target genes in pituitary as well as in other tissues involved in reproduction.

Nature of the Transcriptional Competition by dFTZ-F1{alpha} Homolog Splice Variants
We have clearly demonstrated that zFF1B is a strong competitor for the action of zFF1A on the sGTHIIß gene promoter via direct binding to the same DNA site recognized by mSF-1 and zFF1A. The functional antagonism of differentially spliced variants of FTZ-F1 homolog genes have been addressed in mouse and Xenopus. In both cases, excessive amounts of truncated variants were needed in the in vitro testing system despite the expression levels of ELP and xFF1rAshort, which were significantly less than those of the full-length isoforms (SF-1 and xFF1rA, respectively). In fact, ELP exerted no significant inhibitory effect on the SF-1-related trans-activation of the target gene promoter, whereas xFF1rAshort was impaired in its trans-activation and ability to bind to the target DNA sequence (23, 38). Nevertheless, it is interesting to note that all splicing variants found in the FTZ-F1 subfamily are inevitably caused by the truncation of the presumed trans-activation domains, either in the C-terminal AF-2 or in the A/B AF-1 domain (23, 38, 46, 47). This common feature implies that transcriptional antagonism is a shared mechanism in controlling FTZ-F1 target gene expression.

Through RT-PCR experiments, the earliest detectable transcript of the zFF1 gene was at the 12 h stage (Liu, D., Y. Le Drean, and C. L. Hew, unpublished observation). Similarly, in X. laevis, both xFF1rA and xFF1rAshort transcripts could be detected in early embryos (38). ELP mRNA was not found in mouse embryo, but was located in undifferentiated embryonic carcinoma cells. However, the rationale for the early expression of these FTZ-F1 homologs (xFF1r and zFF1) in lower vertebrate embryos is presently unclear. In addition, determination of whether zFF1B is expressed only at particular developmental stages, to keep its target gene dormant or maintain it at a certain expression level, needs further investigation.

The coexpression of both zFF1A and -B transcripts in developing pituitaries would suggest that the expression of their target genes in the pituitary may have been initiated during embryogenesis. Like mammalian SF-1, zFF1 proteins may confer a variety of functions in endocrine tissues. The finding that the zFF1 gene is expressed in adult ovary and testis further proves its commitment in steroidogenesis. However, the lack of the zFF1B transcript in testis is in contrast to the fact that both SF-1 and ELP could be detected in rat gonads (23). Whether the absence of the inhibitory factor in testis is responsible for the different reproductive performances between the sexes will rely on understanding the nature of alternative splicing of zFF1A and zFF1B mRNAs. The antagonistic relationship between zFF1A and -B in the regulation of the sGTHIIß gene promoter provides a novel mechanism in controlling their target genes in the pituitary as well as in other tissues. In addition, the study of zFF1B should provide new insight into the structural and functional domains important for FTZ-F1 homologs’ actions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RNA Isolation, RT-PCR, and Primers
Total RNA was extracted from embryos and adult tissues, either by the method described previously (48) or with the TRIzol reagent supplied by Life Technologies (Gaithersburg, MD) following the manufacturer’s instructions. Complementary DNA was synthesized by incubating 2 µg zebrafish total RNA in 40 µl First Strand Buffer (Life Technologies), which was supplemented with 400 U Moloney murine leukemia virus reverse transcriptase, 0.5 mM of dNTPs (Life Technologies), 20 U RNA Safeguard (Pharmacia, Piscataway, NJ), and 1 µg oligo(deoxythymidine) primers at 37 C for 1.5 h. A total of 2.5 or 5 µl of the first strand mixture was used to perform the PCR reaction in 50-µl volume with 0.5 U Taq polymerase (Promega). The amplification procedure consisted of 3 min of 95 C followed by 30 cycles at 92 C (1 min), 60 C (1 min), and 72 C (2 min), ending with 10 min of extension at 72 C.

For amplification of the probe used for the library screening, one set of primers, P (overlaps the P box) and A (covers the A box), was designed as follows: P, 5'-TGCAAGGGCTTCTTCAAGCGC-3'; and A, 5'-SCGRTCYCKYTTGTACATGGG-3'; S represents G/C, R = A/G, Y = C/T, and K = G/T. For amplification of the zFF1A and B transcripts, the upper strand primer was designed in the region shared by both zFF1A and B cDNAs, as I (5'-GGATCGCCGCCCTCCTTCCCT-3'); the lower strand primers were II (5'-GCACATCACGTAGTCCAGCAG-3') and III (5'-AGACGTGACTAGTCTGGCATC-3'), specific to zFF1A or -B, respectively. DNA amplified from the latter reaction was fractionated on 1.0% agarose gel and confirmed by Southern blotting with 32P-labeled zFF1A or -B cDNA. The exposure time to the x-ray film (Eastman Kodak, Rochester, NY) varied from 12 h (room temperature) to 48 h (-70 C).

Embryonic cDNA Library Screening and Identification of Zebrafish FTZ-F1 Homolog cDNAs
The embryonic cDNA library was constructed as previously described (49). A 273-bp fragment amplified from RT-PCR was excised from low melting point agarose gel (Sigma), subcloned into the pT7Blue T-vector (Novagen, Madison, WI), and sequenced. A search of the DNA database confirmed the identity of the fragment as a partial sequence of the zebrafish FTZ-F1 homolog.

The 273-bp fragment then served as the probe to screen 2 million phages, and a total 40 positive clones were obtained. After the first screening, all positive phages were eluted in sodium-magnesium buffer, and 2 µl of the elution were used to perform the PCR reaction by primers P and A. With this approach, 6 of 40 were identified as the real positives. The second screening was skipped by using massive in vivo excision and colony hybridization. Insertion of each selected bacterial colony was examined by PCR, and the plasmid DNA was restricted by a series of enzyme digestions. Two types of cDNA clone were identified based on their lengths and restriction patterns. Using a double stranded Nest deletion kit and a T7Sequencing kit (Pharmacia), both zFF1A and -B cDNAs were fully sequenced from their 3'-ends (T7 primer) and partially confirmed by SK and P primers. Further sequence confirmation was performed using internal primers deduced from the known sequences.

In Situ Hybridization
Zebrafish embryos were obtained from the natural cross, staged accordingly (50), fixed in 4% paraformaldehyde in PBS, and stored in 100% methanol. Plasmids used for the in vitro transcription labeling were constructed based on the pBluescript (Stratagene, La Jolla, CA). Briefly, a fragment specific to zFF1A DNA (nucleotides 1582–3105) was subcloned into the PstI site of pBluescript, while nucleotides 1144–2286 of zFF1B cDNA were inserted into PstI and XhoI sites of the vector. Antisense RNA probe generation and the whole mount in situ hybridization procedure were essentially similar to those previously described (51).

Plasmid Construction, Transfection, and CAT Assay
Both zFF1A and -B full-length cDNA fragments were released from their original plasmids, as was a PCR fragment containing only ORF-A, and were introduced into the eukaryotic expression vector pCDNA3 (Invitrogen, San Diego, CA) in the same fashion. The resulting constructs were named, in sequence, z.FF1.A, z.FF1.B, and ORF-A. ER, mSF-1, pCMV-lacZ, and (frgt pERE)-IIß-39 constructs were previously described (30, 37, 40). The reporter consGSE/CAT-39 was constructed as follows. Two oligo nucleotides (5'-AGCTGCTGACCTTGACACT-3' and 5'-AGCTAGTGTCAAGGTCAGC-3') were annealed and ligated with the HindIII-restricted -0.039-kb CAT. Only the plasmid containing the single fragment with the same orientation as that found in mammalian gonadotropin gene promoters was used in the transfection studies.

Cell culture, transfection, hormone treatment, and complete CAT assay were performed as previously described (30, 37, 40).

Gel Shift Assay
Whole cell extract was obtained from HeLa cells individually transfected with 5 µg z.FF1.A, z.FF1.B, pCMVmSF-1, and pCMVß-lacZ. Cells were washed twice in PBS and harvested in ice-cold PBS, swollen in cold 0.25 x PBS for 3 min, and resuspended in buffer I (30). After two freeze-thaw cycles, the supernatant was ready for the DNA binding assay. The consGSE fragment was labeled, and the binding reaction was set up as previously described (30). Similar amounts of proteins were used in each reaction. In addition to the cold consGSE, other competitors included were: sGSE2 (5'-AAGTAGAGGTCAGGA-3') (30), pERE (5'-ATTATGTCAATCTGACCCTAA-3'), and Homo (5'-TCTATGACAATTATGTCAATCT-3'). The putative GSE and palindromic sequences are italicized. The Homo palindrome sequence overlaps pERE in the sGTHIIß gene promoter and serves as a nonspecific competitor in the retardation assay.


    ACKNOWLEDGMENTS
 
We thank Dr. Z. Gong for the preparation of the zebrafish embryonic cDNA library, and Dr. B. C. Chung for sharing the unpublished data. We are grateful to Drs. H. P. Elsholtz, C.-C. Hui, M. A. Akimenko, and C. Peng for their helpful discussion and technical advice. We thank L. Liao and G. Hatch for their technical assistance, and L. Mark for helping in the preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Choy L. Hew, Department of Clinical Biochemistry, University of Toronto, Room 351, 100 College Street, Toronto, Ontario, Canada, M5G 1L5.

This work was supported by the Medical Research Council of Canada (to C.L.H.).

1 Recipient of a Restracom Trainee Fellowships from the Hospital for Sick Children, Toronto. Back

Received for publication January 23, 1997. Revision received March 19, 1997. Accepted for publication March 20, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ueda H, Sonoda S, Brown JL, Scott MP, Wu C 1990 A sequence-specific DNA-binding protein that activates fushi tarazu segmentation gene expression. Genes Dev 4:624–635[Abstract]
  2. Lavorgna G, Ueda H, Clos J, Wu C 1991 FTZ-F1, a steroid hormone receptor-like protein implicated in the activation of fushi tarazu. Science 252:848–851[Medline]
  3. Sun G-C, Hirose S, Ueda H 1994 Intermittent expression of BmFTZ-F1, a member of the nuclear Hormone receptor superfamily during development of the silkworm Bombyx mori. Dev Biol 162:426–437[CrossRef][Medline]
  4. Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor 1, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor 1. Mol Endocinol 6:1249–1258[Abstract]
  5. Morohash K, Honda S, Inomata Y, Handa H, Omura T 1992 A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450 s. J Biol Chem 265:17913–17919
  6. Honda S, Morohash K, Nomura M, Takeya H, Kitajima M, Omura T 1993 Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily. J Biol Chem 266:7494–7502[Free Full Text]
  7. Ellinger-Ziegelbauer H, Hihi AK, Laudet V, Keller H, Wahli Q, Dreyer C 1994 FTZ-F1-related orphan receptors in Xenopus laevis: transcriptional regulators differentially expressed during early embryogenesis. Mol Cell Biol 14:2786–2797[Abstract]
  8. Tsukiyama T, Ueda H, Hirose S, Niwa O, 1992 Embryonal long terminal repeat-binding protein is a murine homolog of FTZ-F1, a member of the steroid receptor superfamily. Mol Cell Biol 12:1286–1291[Abstract]
  9. Mangeisdorf D, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[Medline]
  10. Wilson TE, Fahrner TJ, Milbrandt J 1993 The orphan receptor NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol Cell Biol 13:5794–5804[Abstract]
  11. Ueda H, Sun G-C, Murata T, Hirose, S 1992 A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein. Mol Cell Biol 12:5667–5672[Abstract]
  12. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz, G, Umesono K, Blimberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  13. Luo X, Ikeda Y, Parker KL 1994 A cell specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[Medline]
  14. Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleas. Mol Endocrinol 9:478–486[Abstract]
  15. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen W-H, Nachtigal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312[Abstract]
  16. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Morohashi K-I, Li E 1995 Developmental defects of the ventromedial hypothalamic nucleas and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dynam 204:22–29[Medline]
  17. Lynch JP, Lala DS, Peluso JJ, Luo W, Parker KL, White BA 1993 Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues. Mol Endocrinol 7:776–786[Abstract]
  18. Ikeda Y, Lala DS, Kim E, Moisan M-P, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract]
  19. Ikeda Y, Shen W-H, Ingraham HA, Parker KL 1994 Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654–662[Abstract]
  20. Hatano O, Takayama K, Imai T, Waterman MR, Takakusu A, Omura T, Morohashi K-I 1994 Sex-dependent expression of a transcription factor, Ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development. Development 120:2787–2797[Abstract/Free Full Text]
  21. Keeney DS, Ikeda Y, Waterman MR, Parker KL 1995 Cholestrol side-chain cleavage cyochrome P450 gene expression in the primitive gut of the mouse embryo does not require steroidogenic factor 1. Mol Endocrinol 9:1091–1098[Abstract]
  22. Zhang P, Mellon SH 1996 The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3', 5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17{alpha}-hydroxylase/c17–20 lysase). Mol Endocrinol 10:147–158[Abstract]
  23. Morohashi K-I, Iida H, Nomura M, Hatano O, Honda S-I, Tsukiyama T, Niwa O, Hara T, Takakusu A, Shibata Y, Omura T Functional difference between Ad4 BP, ELP, their distributions in steroidogenic tissues. Mol Endocrinol 8:643–653
  24. Schafer AJ 1995 Sex determination and its pathology in man. Adv Genet 33:275–329[Medline]
  25. Shen W-H, Moore CCD, Ikeda Y, Parker KL, Ingraham HA 1994 Nuclear receptor steroidogenic factor 1 regulates the müllerian inhibiting substance gene: a link to the sex determination cascade. Cell 77:651–661[Medline]
  26. Luo X, Ikeda Y, Schlosser DA, Parker KL 1995 Steroidogenic factor 1 is the essential transcript of the mouse Ftz-F1 gene. Mol Endocrinol 9:1233–1239[Abstract]
  27. Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of luteinizing hormone ß gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. J Biol Chem 271:6645–6650[Abstract/Free Full Text]
  28. Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site is required for activity of the luteinizing hormone ß subunit promoter in gonadotropes of transgenic mice. J Biol Chem 271:10782–10785[Abstract/Free Full Text]
  29. Asa SL, Bamberger A-M, Cao B, Wong M, Parker KL, Ezzat S 1996 The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 81:2165–2170[Abstract]
  30. LeDrean Y, Liu D, Wong AOL, Xiong F, Hew CL 1996 Steroidogenic factor 1 and estradiol receptor act in synergism to regulate the expression of the salmon gonadotropin IIß subunit gene. Mol Endocrinol 10:217–229[Abstract]
  31. Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina G, Milbrandt J 1996 Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273:1219–1221[Abstract]
  32. Galarneau L, Pare J-F, Allard D, Hamel D, Levesque L, Tugwood JD, Green S, Belanger L 1996 The {alpha}-1 fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family. Mol Cell Biol 16:3853–3865[Abstract]
  33. Moore MJ, Query CC, Sharp PA 1993 Splicing of precursors to mRNAs by the spliceosome. In: Gesteland RF, Atkins JF (eds) The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 303–357
  34. Kendall SK, Samuelson LC, Saunders TL, Wood RI, Camper SA 1995 Targeted disruption of the pituitary glycoprotein hormone {alpha}-subunit produces hypogonadal and hypothyroid mice. Genes Dev 9:2007–2019[Abstract]
  35. Tanaka M, Tanangonan JB, Tagawa M, de Jesus EG, Nishida H, Isaka M, Kimura R, Hirano T 1995 Development of the pituitary, thyroid and internal glands and applications of endocrinology to the improved rearing of marine fish larvae. Aquaculture 135:111–126[CrossRef]
  36. Puschel AW, Gruss P, Westerfield M 1992 Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. Development 114:643–651[Abstract]
  37. Xiong F, Liu D, Elsholtz HP, Hew CL 1994 The chinook salmon gonadotropin IIß subunit gene contains a strong minimal promoter with a proximal negative element. Mol Endocrinol 8:771–781[Abstract]
  38. Ellinger-Ziegelbauer H, Gläser B, Dreyer C 1995 A naturally occurring short variant of the FTZ-F1-related nuclear orphan receptor xFF1rA and interactions between domains of xFF1rA. Mol Endocrinol 9:872–886[Abstract]
  39. Tsukiyama T, Niwa O, Yokoro K 1989 Mechanism of suppression of the long terminal repeat of moloney leukemia virus in mouse embryonal carcinoma cells. Mol Cell Biol 9:4670–4676[Medline]
  40. Liu D, Xiong F, Hew CL 1995 Functional analysis of estrogen-responsive elements in chinook salmon (Oncorhynchus tschawytscha) gonadotropin IIß subunit gene. Endocrinology 136:3486–3493[Abstract]
  41. Smith GWJ, Patton JG, Nadal-Ginard B 1989 Alternative splicing in the control of gene expression. Annu Rev Genet 23:527–577[CrossRef][Medline]
  42. Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487[Medline]
  43. vom Baur E, Zechel C, Heery D, Heine MJ, Garnier JM, Vivat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124[Abstract]
  44. Renaud J-P, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-{gamma} ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  45. Perlmann T, Umesono K, Rangarajan PN, Forman BM, Evans RM 1996 Two distinct dimerization interfaces differentially modulate target gene specificity of nuclear hormone receptors. Mol Endocrinol 10:958–966[Abstract]
  46. Ohno CK, Ueda H, Petkovich M 1994 The Drosophila nuclear receptors FTZ-F1{alpha} and FTZ-F1ß compete as monomers for binding to a site in the fushi tarazu gene. Mol Cell Biol 14:3166–3175[Abstract]
  47. Lavorgna G, Karin FD, Thummel CS, Wu C 1993 Potential role for a FTZ-F1 steroid receptor superfamily member in the control of Drosophila metamorphosis. Proc Natl Acad Sci USA 90:3004–3008[Abstract]
  48. Gong Z, Fletcher GL, Hew CL 1992 Tissue distribution of fish antifreeze protein mRNAs. Can J Zool 70:810–814
  49. Gong Z, Yan T, Liao J, Lee SE, He, J, Hew CL, Rapid identification and isolation of zebrafish genes. Gene, in press
  50. Westerfield M 1994 The Zebrafish Book, ed 2.1. University of Oregon Press, Eugene
  51. Akimenko MA, Ekker M, Wegner J, Lin W, Westerfield M 1994 Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. J Neurosci 14:3475–3486[Abstract]