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Molecular Endocrinology, doi:10.1210/me.2005-0445
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Molecular Endocrinology 20 (11): 2630-2640
Copyright © 2006 by The Endocrine Society

Zebrafish dax1 Is Required for Development of the Interrenal Organ, the Adrenal Cortex Equivalent

Y. Zhao, Z. Yang, J. K. Phelan, D. A. Wheeler, S. Lin and E. R. B. McCabe

Department of Human Genetics (Y.Z., E.R.B.M) and Department of Pediatrics (J.K.P., E.R.B.M.), David Geffen School of Medicine at UCLA; Department of Molecular, Cell and Developmental Biology (Z.Y. S.L.), UCLA; Mattel Children’s Hospital at UCLA (E.R.B.M.); and UCLA Molecular Biology Institute, Los Angeles, California 90095; and Human Genome Sequencing Center (D.A.W.), Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Edward R. B. McCabe, Department of Pediatrics, David Geffen School of Medicine at UCLA, 10833 LeConte Avenue, Room 22-412 MDCC, Los Angeles, California 90095-1752, E-mail: EMcCabe{at}mednet.ucla.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the human nuclear receptor, DAX1, cause X-linked adrenal hypoplasia congenita (AHC). We report the isolation and characterization of a DAX1 homolog, dax1, in zebrafish. The dax1 cDNA encodes a protein of 264 amino acids, including the conserved carboxy-terminal ligand binding-like motif; but the amino-terminal region lacks the unusual repeats of the DNA binding-like domain in mammals. Genomic sequence analysis indicates that the dax1 gene structure is conserved also. Whole-mount in situ hybridization revealed the onset of dax1 expression in the developing hypothalamus at approximately 26 h post fertilization (hpf). Later, at about 28 hpf, a novel expression domain for dax1 appeared in the trunk. This bilateral dax1-expressing structure was located immediately above the yolk sac, between the otic vesicle and the pronephros. Interestingly, weak and transient expression of dax1 was observed in the interrenal glands (adrenal cortical equivalents) at approximately 31 hpf. This gene was also expressed in the liver after 3 dpf in the zebrafish larvae. Disruption of dax1 function by morpholino oligonucleotides (MO) down-regulated expression of steroidogenic genes, cyp11a and star, and led to severe phenotypes similar to ff1b (SF1) MO-injected embryos. Injection of dax1 MO did not affect ff1b expression, whereas ff1b MO abolished dax1 expression in the interrenal organ. Based on these results, we propose that dax1 is the mammalian DAX1 ortholog, functions downstream of ff1b in the regulatory cascades, and is required for normal development and function of the zebrafish interrenal organ.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DOSAGE-SENSITIVE SEX reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1 (DAX1), encoded by the gene NR0B1, is an unusual member of the orphan nuclear receptor family of transcription factors. DAX1 owes its name to its dual role in human pathology. Duplications of the 160-kb dosage-sensitive sex-reversal region in Xp21, containing the NR0B1 gene, result in male-to-female phenotypic sex reversal (1, 2). However, mutations in human NR0B1 gene cause X-linked AHC, a hereditary disorder of the adrenal cortex commonly manifested by early-onset adrenal insufficiency (3, 4, 5, 6). DAX1 is expressed in the hypothalamic-pituitary-adrenal-gonadal (HPAG) axis, i.e. ventromedial hypothalamic nucleus, pituitary gonadotropes, adrenal cortex, testis, and ovary (7, 8, 9, 10, 11). This pattern of expression reflects the phenotypic features of DAX1 deficiency, specifically AHC and hypogonadal hypogonadism.

DAX1 is conserved in gene structure and orthologs are present throughout vertebrates (3, 12, 13, 14, 15, 16, 17, 18). The DAX1 gene has a very simple genomic structure with two exons separated by a single intron, which is located in the same relative position across species (19). Structurally, the DAX1 protein is a member of the nuclear receptor superfamily (20). The C-terminal region of this orphan nuclear receptor contains the ligand binding-like (LBL) domain common to other members of the nuclear receptor superfamily. However, DAX1 lacks the conventional DNA-binding domain; instead, its N terminus is composed of 3.5 alanine/glycine rich repeats of a 65- to 70-amino acid motif that has no known homology to any other proteins except SHP (short heterodimer partner, NR0B2), the only other nuclear receptor NR0B family member present in mammals (19). The 3.5 N-terminal repeat structure with an unusual DNA binding-like (DBL) domain has been conserved among mammalian DAX1s. However, in nonmammalian vertebrates, including chick (16), alligator (15), frog (17), and tilapia (18), cloned DAX1 orthologs lack these repeats.

DAX1 is a transcriptional repressor of a number of other nuclear receptors expressed in the steroidogenic axis, particularly steroidogenic factor 1 (SF1) (21). DAX1 is a negative regulator of SF1-induced transactivation of many genes and inhibits the synergistic transcriptional activation of SF1 interacting with other SF1 heterodimeric partners like WT1 (21, 22). However, the exact mechanisms of DAX1 action at the different levels of the HPAG axis during normal development and adulthood are not fully understood (21).

One of the reasons for the lack of understanding of DAX1 function is the difficulty in studying mammalian embryogenesis. To better understand the roles of this gene in normal HPAG axis development, and adrenal development in particular, we investigated the zebrafish, Danio rerio, as a model organism, because zebrafish embryos are amenable to molecular manipulation and genetic dissection (23, 24). The adrenal cortex homolog in teleosts is called the interrenal gland, because together with chromaffin cells (counterpart of adrenal medulla), it is embedded inside the anterior part of the kidney, commonly referred to as the head kidney (25). The interrenal gland is the major site of steroid synthesis in most teleosts (26), as is the adrenal cortex in mammals (27, 28). Interrenal and adrenocortical cells both express genes encoding steroidogenic proteins, such as cyp11a, 3ßhsd, and star (28, 29, 30, 31).

In this study, we isolated and characterized the zebrafish dax1 by expressed sequence tag (EST) database searching and molecular cloning. Using whole-mount in situ hybridization (ISH), the spatial and temporal expression patterns of the dax1 gene throughout zebrafish embryogenesis were determined. Disruption of in vivo dax1 function by morpholino (MO) led to larval phenotypes that were suggestive of impaired interrenal function and very similar to ff1b (zebrafish SF1 ortholog) MO-injected embryos. Injection of dax1 MO also down-regulated the expression of steroidogenic genes, cyp11a and star. These lines of evidence strongly support the direct involvement of dax1 in zebrafish interrenal development. In addition, our gene knockdown experiments showed that dax1 MO did not affect the expression of ff1b, whereas MO knockdown of ff1b activity abolished the interrenal expression of dax1, suggesting the function of dax1 downstream of ff1b in the regulatory cascades underlying normal interrenal development in zebrafish.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A DAX1 Homolog, dax1, Is Identified in Zebrafish
The C-terminal consensus sequence of known DAX1 proteins was used as a probe to search zebrafish cDNA and EST databases for homologous sequences. A single EST clone (CK029520) was identified that encoded amino acids with significant homology to the DAX1 consensus sequence. Subsequent sequencing showed that this clone contained the polyadenylation signal and poly(A) tail (Fig. 1Go). This putative dax1 cDNA included the open reading frame encoding a predicted 264-amino acid protein, and a 69-bp 5'-untranslated region. The dax1 genomic sequence containing the complete coding sequence and a 1487-bp intron was also obtained by bacterial artificial chromosome (BAC) library screening and subsequent sequencing. The position of the intron was exactly the same as that of known DAX1 genes (19). The zebrafish dax1 gene was composed of two exons separated by a single intron between the first and second nucleotides of the codon 185 for the highly conserved amino acid aspartate (D185) in the LBL domain (Figs. 1Go and 2Go). We compared the amino acid sequences of the putative zebrafish dax1 with DAX1 orthologs in tilapia, frog, alligator, chick, mouse, rat, and human (Fig. 2Go). The predicted zebrafish dax1 protein contained the conserved 3'-LBL motif; but the 5'-region lacked the unusual repeat motif of the DBL domain in mammals. As a consequence of the truncated DBL, zebrafish dax1 contained only the third and fourth of four LXXLL motifs seen in mammals; this feature is typical of other nonmammalian DAX1 proteins (Fig. 2Go). Based on the alignment results, we constructed a phylogenetic tree of DAX1 proteins in vertebrates (Fig. 3Go). Not unexpectedly, the zebrafish dax1 is most closely related to tilapia DAX1 protein.


Figure 1
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  		Fig. 1. Nucleotide and Amino Acid Sequences of Zebrafish dax1

The translated amino acid sequence is shown in standard one-letter code above the nucleotide sequence. The arrowhead indicates the position of the intron.

 

Figure 2
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  		Fig. 2. Alignment of the Amino Acid Sequences of Zebrafish dax1 with Those of Other DAX1s

GenBank accession numbers of DAX1 orthologs used for the analysis are: tilapia (AY135397), frog (AB079550), American alligator (AF180295), chick (AF202991), rat (NM_053317), mouse (U41568), pig (U82466), and human (NM_000475).

 

Figure 3
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  		Fig. 3. Phylogenetic Analysis of Zebrafish dax1 and Other Known DAX1s in Vertebrates

GenBank accession numbers are given in the legend of Fig. 2Go.

 
The similarities between the predicted zebrafish dax1 and other reported DAX1 proteins are listed in Table 1Go. The zebrafish dax1 shows the highest levels of identity and similarity to tilapia DAX1 (65.5% identity and 79.5% similarity) over the full-length protein sequence. It also shows more than 48% and 43% amino acid identity to other nonmammalian (frog, alligator, and chick) and mammalian (mouse, rat, human, and pig) DAX1s, respectively.


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  		Table 1. Amino Acid Sequence Identities and Similarities between Zebrafish dax1 and Other Vertebrate DAX1s

 
To determine whether a dax1 isoform analogous to human DAX1a (32, 33) exists in zebrafish, the 1487-bp intron sequence of the zebrafish dax1 gene was analyzed. The sequence comparison of the zebrafish dax1 and the human DAX1 introns did not reveal conserved regions. The zebrafish dax1 intron sequence was further searched for potential splice sites. Combined with polyA-addition signal analysis, we identified three potential splice sites, which had the potential to result in an exon 2a. RT-PCRs were then performed to amplify the original exon 2 and each potential exon 2a, respectively. RT-PCR detected the original dax1 gene, but not any of the potential dax1a isoforms (data not shown). In addition, we searched the zebrafish EST database with the exon 1 sequence. No EST indicating alternative splicing was found.

dax1 Is Expressed during Zebrafish Embryogenesis
In the central nervous system, expression of dax1 was first detected at around 26 h post fertilization (hpf) in two strips of cells extending from the midline of the rostral basal forebrain (Fig. 4Go, A and B). By 31 hpf, dax1 was strongly expressed in the rostral diencephalon between the eye fields (Fig. 4Go, C and D), immediately anterior to the pituitary, shown by double ISH with the pituitary marker pomc (34) (Fig. 4Go, E and F). Subsequent to 36 hpf, dax1 expression in the brain was down-regulated and by 4 days post fertilization (dpf), no specific dax1 expression could be observed in this region (Fig. 4Go, Q and R).


Figure 4
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  		Fig. 4. Expression Analysis of dax1

A–D, G, H, Q, and R, ISH for dax1 was performed with DIG-labeled dax1 riboprobe and visualized with purple. E, F, I–N, and P, Two-color ISH was performed with fluorescein-labeled dax1 riboprobe and DIG-labeled marker gene riboprobes and were visualized with purple and Fast Red staining, respectively. O, Two-color ISH was performed with fluorescein-labeled pax2.1 riboprobe (purple) and DIG-labeled dax1 riboprobe (Fast Red). Lateral views of dax1 expression at 26 hpf (A), 31 hpf (C), and 4 dpf (Q). Ventral views of dax1 expression at 26 hpf (B) and 31 hpf (D). Dorsal views of dax1 expression at 28 hpf (G), 32 hpf (H), and 4 dpf (R). Arrow in panel H indicates the expression of dax1 in the interrenal gland. Lateral (E) and lateral-ventral (F) views of 32 hpf embryos stained for dax1 and pituitary marker pomc. Lateral (I) and dorsal (J) views of 32 hpf embryos stained for dax1 and interrenal marker ff1b. Lateral (K) and lateral-dorsal (L) views of 32 hpf embryos stained for dax1 and ear marker otx1. Lateral (M) and dorsal (N) views of 32 hpf embryos stained for dax1 and pectoral fin bud marker tbx5. Dorsal view (O) of 32 hpf embryo stained for dax1 and pronephric tubule marker pax2.1. Arrowhead indicates stained pronephric tubules. Dorsal view (P) of 38 hpf embryo stained for dax1 and tooth germ marker pitx2a.

 
From 28 hpf, a second dax1-expressing domain appeared in the trunk, emerging as two clusters of cells symmetric to the embryo body midline (Fig. 4GGo). Expression of dax1 mRNA in this bilateral structure peaked at around 32 hpf (Fig. 4HGo) and disappeared by 48 hpf. To characterize further this novel dax1-expressing domain, we performed double ISH of dax1 with the interrenal marker ff1b (35, 36, 37) (Fig. 4Go, I and J), otic vesicle marker otx1 (38, 39) (Fig. 4Go, K and L), pectoral fin bud marker tbx5 (40) (Fig. 4Go, M and N), pronephric tubule marker pax 2.1 (41) (Fig. 4OGo), and tooth germ marker pitx2a (42) (Fig. 4PGo). The bilateral dax1-expressing structure was clearly distinct from all five organs/tissues investigated. It was located immediately above the yolk sac, ventral to both the otic vesicle and the pectoral fin buds, caudal to the otic vesicle, cephalic to the pectoral fin buds and head kidney, and laterally adjacent to the tooth germ (Fig. 4PGo).

Weak and transient expression of dax1 was observed in the interrenal organ at around 31–32 hpf (Fig. 4HGo, arrow), determined by its colocalization with the interrenal marker ff1b (Fig. 4JGo). Another intriguing result for dax1 ISH is that this gene was expressed in the liver after 3 dpf in the zebrafish larvae (Fig. 4Go, Q and R).

MO-Mediated Knockdown of dax1 Function Down-Regulates Expression of the Steroidogenic Genes cyp11a and star
To study the potential role of dax1 in interrenal development, MO was used to block its translation during zebrafish embryogenesis. We injected either dax1 MO or dax1 mismatch MO (mMO), the mismatch control, into one- to two-cell embryos and subsequently examined the effects of MO injection on the expression of the steroidogenic genes, cyp11a and star, both of which were shown to be expressed in the developing zebrafish interrenal organ (29, 31), by ISH. Embryos injected with 9 ng of dax1 MO and dax1 mMO were collected at 36 hpf for ISH with cyp11a. Injection of dax1 MO significantly down-regulated cyp11a expression in 77.1% (81/105) of embryos, whereas at the same dosage, only 5.4% (5/93) of dax1 mMO-injected embryos showed the same degree of down-regulation of cyp11a (Fig. 5Go, A and B). Similarly, knockdown of dax1 function by MO significantly reduced the number of embryos showing star expression in the interrenal organ (Fig. 5Go, C and D). Of dax1 MO-injected embryos 80.2% (77/96) were star negative at 40 hpf. However, 96.6% (85/88) of embryos injected with dax1 mMO showed positive star staining, therefore establishing the specificity of dax1 MO effects.


Figure 5
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  		Fig. 5. Effects of dax1 MO Injection on the Expression of cyp11a and star

A and B, ISH for cyp11a on 36 hpf embryos injected with 9 ng per embryo of dax1 MO (panel A) and dax1 mMO control (panel B). C and D, ISH for star in dax1 MO (panel C) and dax1 mMO control (panel D)-injected embryos (40 hpf). Embryos were oriented with anterior to the left.

 
Morpholino Knockdown of dax1 Function Leads to Impaired Osmoregulation
Embryos injected with 9 ng of dax1 MO and dax1 mMO were allowed to develop to larval stages. Injection of dax1 MO led to severe phenotypes similar to ff1b (SF1) MO-injected embryos (37). These phenotypes could be grouped into three different classes as previously described by Chai et al. (37). The percentages of larvae exhibiting different classes of phenotypes are presented in Table 2Go. Morphological changes had begun to appear by 4 dpf. Some dax1 MO-injected larvae showed signs of fluid accumulation in their body cavities, predominantly in the abdomen. By 7 dpf, varying degrees of sc edema were observed, consistent with ff1b morphants. Edema of the optic sacs led to protrusion of the optic cup. Edema of the pericardial sac interfered with cardiac pumping, leading to slowing or even cessation of heartbeat. As a result, blood circulation slowed or ceased, and blood islands formed in the ventral tail. About 8.7% of control embryos injected with dax1 mMO displayed phenotypes ranging from classes A to C.


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  		Table 2. Classification of dax1 Morphant Phenotypes at 7 dpf

 
We also investigated the effects of dax1 MO at the histological level. Either dax1 MO or mismatch control MO-injected embryos were collected at 36 hpf and 48 hpf, respectively. These embryos were stained by ff1b ISH and randomly sampled for cross-sectioning. Examination of the sections revealed that dax1 MO injection did not cause any obvious structural difference in the interrenal tissues, labeled by ff1b staining (data not shown). In addition, the apoptosis profiles of dax1 morphants and controls were examined using acridine orange staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay. dax1 morphants presented similar numbers of apoptotic cells in the interrenal region compared with those in control embryos (data not shown).

dax1 May Function Downstream of ff1b during Zebrafish Interrenal Development
As previously described, the disruption of dax1 activity by dax1 MO led to severe phenotypes that are similar to those reported in ff1b morphants; MO knockdown of either protein was sufficient to interrupt the expression of steroidogenic enzymes. To explore the relationship of these two genes in the regulatory network involved in the interrenal development and function, we first examined the effects of dax1 MO injection on the expression of ff1b in the interrenal organ. Embryos injected with 9 ng of dax1 MO and dax1 mMO were collected at 36 hpf for ISH with ff1b. Similar proportions, 91.3% (73/80) of embryos injected with dax1 MO and 93.1% (67/72) embryos injected with dax1 mMO, showed positive ff1b staining in the interrenal organ (Fig. 6Go, A and B). These results indicated that the disruption of dax1 function did not affect the normal expression of ff1b.


Figure 6
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  		Fig. 6. Effects of ff1b MO Injection on the Expression of dax1

A and B, ISH for ff1b on 36 hpf embryos injected with dax1 MO (panel A) and dax1 mMO control (panel B). C and D, ISH for dax1 on approximately 31–32 hpf embryos injected with ff1b MO (panel C) and ff1b mMO control (panel D). Arrow indicates the expression of dax1 in the interrenal organ.

 
Furthermore, we investigated the interrenal expression of dax1 in ff1b morphants. An antisense morpholino and a mutated antisense morpholino were synthesized and injected as previously described by Chai et al. (37). Because dax1 expression in the interrenal organ is weak and transient at approximately 31–32 hpf, we collected both ff1b MO and ff1b mMO-injected embryos at 30–33 hpf for ISH with dax1. We observed that 16.4% (29/177) of ff1b mMO (control)-injected embryos displayed weak expression of dax1 in the interrenal organ. In contrast, only 4.8% (9/186) of embryos injected with ff1b MO retained comparable dax1 staining (Fig. 6Go, C and D). Thus, the injection of ff1b MO significantly down-regulated the interrenal expression of dax1 (P < 0.001).

To characterize further the relationship of dax1 and ff1b during zebrafish early development, we determined whether there is the classical SF1 response element sequence in the dax1 promoter region. We sequenced the 5'-flanking region of the dax1 gene and aligned the putative promoter sequences of human, mouse, rat, and zebrafish. No consensus SF1 response element was identified in the zebrafish dax1 gene, unlike the human, mouse, and rat DAX1s (Fig. 7Go). We did find, however, conserved blocks of sequence, all relatively short, in the promoter regions for these four species, e.g. the region shown in Fig. 7Go. This conservation suggests the possibility that regulatory strategies may have been maintained from fish to human.


Figure 7
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  		Fig. 7. Alignment of the Promoter Sequences of Zebrafish dax1 with Mammalian DAX1s

A small portion of the alignment, including the SF1 response element (SF1-RE), is shown. The nucleotides are numbered based on their positions relative to the start codon ATG in the dax1 sequences of human (top) and zebrafish (bottom). The mammalian SF1 consensus response element does not appear to be present in this teleost. Blocks of sequence are conserved, however, in the 5'-untranslated regions of these four species, as, for example the 5'-CATGG-3' at position –157 to –161, suggesting conservation of certain aspects of dax1 expression throughout vertebrates.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we cloned and characterized a DAX1 homolog, dax1, in zebrafish embryos and larvae. We provide several lines of evidence to support a role for dax1 in the development of the interrenal organ in the zebrafish. The dax1 gene encodes a protein of 264 amino acid residues, including the conserved 3'-LBL motif, and the 5'-region resembles other nonmammalian DAX1 proteins and lacks the unusual repeat motif of the DBL domain in mammals. The genomic structure of the dax1 gene is highly conserved when compared with those of known DAX1s. Whole-mount ISH revealed that dax1 shares a similar expression pattern with other DAX1 orthologs as well. Knockdown of dax1 activity by MO led to severe phenotypes that are consistent with impaired osmoregulation and down-regulated the expression of steroidogenic genes. The fact that the injection of ff1b MO abolished the interrenal expression of dax1 whereas knockdown of dax1 did not significantly affect ff1b expression suggests that dax1 is downstream of ff1b in transcriptional regulation during zebrafish interrenal development.

dax1 Is Required for the Development of the Steroidogenic Component of the Zebrafish Interrenal Organ
Microinjection of MOs into one- to four-cell zebrafish embryos has proven to be an effective and specific means to inhibit the translation of a target gene. The inhibitory effects can persist in all cells throughout the first 72 h of development, allowing many early developmental pathways to be analyzed (43).

A role for zebrafish dax1 in interrenal organ development and function is demonstrated by MO-mediated knockdown of dax1 activity in vivo. Injection of the dax1 MO clearly abolishes the expression of the steroidogenic protein genes cyp11a and star in the interrenal organ. Detailed histological analysis revealed that dax1 MO injection did not cause any obvious structural difference in the interrenal tissues. In addition, no abnormality in the pattern of apoptosis was observed in those dax1 morphants. These results suggest that the zebrafish dax1 is required not for the structural or the organizational development of the interrenal organ, but for some other functional characteristics of this organ, e.g. perhaps the acquisition of the steroidogenic identity for the interrenal cells. This hypothesis is also consistent with the expression of dax1 after ff1b and after the coalescence of the paired interrenal structures in the midline.

The phenotypes of larvae caused by the knockdown of dax1 expression are consistent with impaired osmoregulation. In teleosts, the interrenal organ plays a critical role in osmoregulation by producing corticosteroids, principally cortisol, which is essential for hydromineral control in both seawater and fresh-water fish (44, 45, 46). Fresh-water fish produce highly diluted urine, which is hypotonic, to excrete excess water. Therefore, the edema phenotype observed in dax1 morphants is likely due to reduced ability to produce hypotonic urine, as the result of the disruption of interrenal cortisol production.

dax1 Functions Downstream of ff1b in Regulation of Interrenal Organ Development
SF1 (NR5A1), an Ftz-F1 member of the nuclear receptor superfamily, is a key transcriptional factor critical for adrenal development in mammals (47). SF1 shares a similar tissue expression profile with DAX1 and could be involved in a common developmental pathway with DAX1 (21).

ff1b, an SF1 homolog, was identified in zebrafish embryos and was specifically expressed in the hypothalamus and the interrenal organ (35). Knockdown of ff1b gene function led to the loss of structural component of the interrenal organ (37). To date, ff1b is the earliest known molecular marker for teleost interrenal development (35, 36, 37). In zebrafish, the primordial interrenal cells first appear as bilateral clusters expressing ff1b ventral to the third somite by 22 hpf (Zhao, Y., and E.R.B. McCabe, unpublished results). These cells then migrate toward the axial midline, coalesce at around 30 hpf, and subsequently begin to acquire a steroidogenic identity (36, 37). However, dax1 expression in the interrenal region is first observed at around 31 hpf, right after the primordial interrenal paired cell clusters fuse together and before these cells develop their steroidogenic identity. These data suggest that dax1 acts downstream of ff1b in interrenal gland development.

In this report, we present MO knockdown data for both dax1 and ff1b that suggest the relative hierarchical roles of these two factors in regulation of normal interrenal development and function. Whereas dax1 MO did not alter the ff1b gene expression in the interrenal organ, knockdown of ff1b activity abolished the interrenal expression of dax1. These observations are consistent with the temporal expression patterns, strongly suggesting that ff1b acts upstream of dax1 in regulation of interrenal gland development.

Previous investigations have shown that mammalian SF1 directly regulates transcription of the DAX1 gene (21). In the present study we did not find any evidence of the consensus SF1 response element in the zebrafish dax1 gene, unlike the mammalian DAX1s. To determine the mechanistic relationship of dax1 and ff1b, a series of transfection analyses in appropriate zebrafish cell lines will be necessary in future investigations.

dax1 May Have Novel Functions outside the HPAG Axis
In this report, we describe the zebrafish dax1 gene expression not only in the hypothalamus and adrenal, where other known DAX1s are specifically expressed, but also in novel bilateral structures and in the liver at a later larval stage, suggesting novel functions for dax1 outside of the HPAG axis.

The later expression of dax1 in the liver is particularly intriguing, because liver is one of the major expression sites for another orphan nuclear receptor SHP (48). SHP and DAX1 belong to the same nuclear receptor family. SHP functions as a transcriptional repressor, as does DAX1 (49, 50, 51). The mammalian SHPs possess the equivalent of one of the DAX1 repeats in the DBL domain and show high homology to nonmammalian DAX1s (18, 48). Moreover, phylogenetic analysis of DAX1s, SHPs, and homologous EST fragments indicates a paralogous relationship between DAX1 and SHP with origin from duplication of a common ancestral gene (18).

Our search for an SHP ortholog in zebrafish yielded an EST that shares significant sequence similarity to tilapia and mammalian SHPs. Subsequently we obtained the potential zebrafish shp full-length cDNA and studied its expression pattern by ISH. Surprisingly, this potential shp gene is expressed neither in the liver, where mammalian SHPs are expressed, nor in any internal organs. Instead, it is expressed diffusely in the superficial cell layers of the yolk sac, the yolk sac extension, and part of the trunk (data not shown). We are now in the process of further characterizing the function of this zebrafish shp gene and the fine structure of those tissues that express this gene.

The lack of shp expression and the atypical expression of dax1 in the liver raise the possibility that zebrafish dax1 may carry out certain functions that are usually executed by SHPs in mammals. To evaluate the potential function of the relatively late expression of dax1 in the liver, we examined the expression of liver markers in both the dax1 morphants and the control-injected embryos. We did not observe any significant difference between the two panels, possibly due to the limitations of the morpholino technique beyond 72 h of development (data not shown). Future analysis of the relationship between zebrafish dax1 and the classical target genes of SHP may help characterize the potential involvement of dax1 in zebrafish liver development, and eventually offer new insights into the relationship of these two unusual nuclear receptors.

Summary
We propose that zebrafish is an appropriate vertebrate model in which to explore the roles of DAX1 and other regulatory factors in the development and normal function of the HPAG axis, and, in particular, the adrenal cortex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Zebrafish Stock and Embryo Collection
Wild-type zebrafish were maintained at 28.5 C. Embryos were obtained by natural spawning and cultured in embryo medium following standard procedures. Staging of embryos was carried out according to Kimmel et al. (52). Embryos used for expression analysis were cultured in 0.03% phenylthiourea (Sigma Chemical Co., St. Louis, MO) solution from 10 h post fertilization (hpf) to inhibit pigment formation.

Cloning of dax1 cDNA and Phylogenetic Analysis
One DAX1-like EST clone was identified from the zebrafish genome databases (http://www.ensembl.org/Danio_rerio/blastview and http://www.ncbi.nlm.nih.gov/blast/tracemb.shtml) by BLAST with the consensus sequence of the tilapia DAX1 and other known DAX1 proteins, and was purchased from Open Biosystems (Huntsville, AL). After sequencing and multiple alignments with known DAX1 homologs, the complete coding sequence of the putative zebrafish DAX1 gene was determined from this EST. Homology analyses of nucleotide sequences and deduced protein alignments and the subsequent phylogenetic analysis were performed with Vector NT1 software (Infomax, North Bethesda, MD). Sequences used for alignments other than those reported here were extracted from the NCBI UniGene and Nucleotide databases. GenBank accession numbers of DAX1 orthologs used for the analysis are listed below: tilapia (AY135397), frog (AB079550), American alligator (AF180295), chick (AF202991), rat (NM_053317), mouse (U41568), pig (U82466), and human (NM_000475).

Cloning of dax1 Genomic Sequence
The zebrafish genome database from Sanger (http://www.sanger.ac.uk/Projects/D_rerio/) was screened by BLAST search with the putative zebrafish DAX1 cDNA and a single BAC clone from the zebrafish CHORI-211 BAC library was identified that contained the putative exon 1 sequence. Subsequent sequencing of this BAC clone confirmed that it actually contained the complete coding sequence and the intronic sequence of the putative zebrafish DAX1 gene.

Analysis of the dax1 Promoter Region
The sequence of the dax1 promoter region was obtained by sequencing the BAC clone that contained this gene. The mRNA sequences of the human, mouse, and rat DAX1s were used to search the NCBI genome databases to identify the contigs that contained the complete gene sequences. Subsequently the promoter regions were determined by comparing the genomic sequences and the corresponding mRNAs. Multiple alignments were performed with Vector NT1 software.

Whole-Mount ISH
Digoxigenin (DIG)- or fluorescein-labeled dax1 riboprobes were synthesized from AvrII linearized dax1 cDNA using T7 RNA polymerase. DIG-labeled riboprobes were synthesized from plasmids containing zebrafish cDNAs for ff1b, pomc, tbx5, otx1, pax2.1, cyp11a, and StAR, respectively. Plasmids for pomc, tbx5, otx1, pitx2a, and cyp11a were digested with NotI and transcribed with T7 RNA polymerase. ff1b and star plasmids were linearized with NcoI and BamHI, respectively, and transcribed with Sp6 polymerase. pax2.1 plasmid was linearized with BamHI and transcribed with T7 polymerase. Single-color ISH for dax1 was performed on zebrafish embryos at 10 different developmental stages with DIG-labeled dax1 riboprobe as previously described (53). For two-color ISH, embryos at around 32 hpf were hybridized with both fluorescein-labeled dax1 and DIG-labeled pomc, ff1b, tbx5, otx1, or pax2.1, respectively, as previously described (54); and double ISH of dax1 and pitx2a was performed on embryos at around 38 hpf. DIG-labeled probes were detected with alkaline phosphatase (AP)-conjugated anti-DIG antibody and visualized with BM Purple AP substrate (Roche Applied Sciences, Indianapolis, IN), whereas fluorescein-labeled probes were detected with AP-conjugated antifluorescein antibody and stained with Fast Red (Roche). Fully stained embryos were washed three times for 10 min in PBS/Tween and postfixed in 4% paraformaldehyde, followed by tissue clarification in gradient concentrations of glycerol (in PBS/Tween). Specimens were then mounted on glass slides and photographed under Normaski optics on a Zeiss Axio microscope system (Carl Zeiss, Thornwood, NY).

Morpholino Injection
MOs were synthesized at Gene Tools (Corvallis, OR). A stock solution of 10 µg/µl was prepared by dissolving the lyophilized powder in doubly distilled water. The stock solution was diluted to a working concentration of 2 µg/µl in 1x Danieau solution (58 mM NaCl; 0.7 mM KCl; 0.4 mM MgSO4; 0.6 mM Ca(NO3)2; 5 mM HEPES, pH 7.6).

Freshly laid embryos were collected and placed into the embryo medium (55) and transferred onto an agrose platform. MO solution (1–6 nl) was injected into the yolk of one- to two-cell embryos from the vegetal pole. Microinjection was performed using a Nanojet injector.

The morpholino sequences were as follows

dax1 MO: 5'-CAGAGCTGCTTATGTTTCGTTGCTG-3'

dax1 mismatch control, dax1 mMO: 5'-CAcAGgTGCTTATcTTTCcTTGgTG-3'

ff1b MO: 5'-AATCCTCATCTGCTCTGAAGTC-3'

ff1b control, ff1b mMO: 5'-AATC-TCATC-GCTC-GAAGTCat-3'

Both the ff1b MO and the ff1b control were synthesized with the sequence previously described by Chai et al. (37).


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant RO1 HD39322.

Disclosure statement: the authors have nothing to disclose.

First Published Online July 13, 2006

Abbreviations: AHC, Adrenal hypoplasia congenital; BAC, bacterial artificial chromosome; DAX, dosage-sensitive sex reversal, AHC critical region on the X chromosome; DBL, DNA binding-like; DIG, digoxigenin; dpf, days post fertilization; EST, expressed sequence tag; HPAG, hypothalamic-pituitary-adrenal-gonadal; hpf, hours post fertilization; ISH, in situ hybridization; LBL, ligand binding-like; mMO, mismatch MO; MO, morpholino oligonucleotides; SF1, steroidogenic factor 1; SHP, short heterodimeric partner.

Received for publication November 7, 2005. Accepted for publication July 3, 2006.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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