Expression of zebrafish Brn1.2 (Pou3f2) and two Brn-3a (Pou4f1) POU genes in brain and sensory structures

POU genes are characterized by a conserved POU DNA-binding domain, and are divided into six subclasses. Class III and IV POU genes are predominantly expressed in the developing nervous system. POU class III genes are critical for several neuronal cell differentiation and class IV POU genes serve important functions in the differentiation and survival of sensory neurons. In this study, we attempted to identify POU genes in the zebrafish and pufferfish genomes by using existing bioinformatics tools. We analysed the expression of zebrafish brn1.2 and brn3a genes (brn3a1 and brn3a2)) using whole-mount in situ hybridisation. Similarly to the mammalian orthologue, zebrafish brn1.2 was widely expressed in the forebrain, midbrain and hindbrain. During the late stages of embryogenesis, brn1.2 expressing cells were located in the preoptic area and in the auditory vesicles. Expression of both zebrafish brn3a genes was detected in trigeminal ganglia, cranial sensory ganglia, sensory neurons along the dorsal spinal cord, in the anterior and posterior lateral line placodes (ALL and PLL), retinal ganglion cell layer, optic tectum and in small cell clusters in the forebrain and hindbrain. Similar to mammalian Brn3a, zebrafish brn3a genes were detected in the retina and sensory structures. However, different domains of expression were also observed, namely in spinal sensory neurons, and lateral line system.


Introduction
POU genes encode a subclass of sequence-specific DNA-binding proteins within the family of homeodomain transcription factors. They are defined by the conserved POU DNA-binding domain, that was originally identified by sequence comparison of the mammalian Pit-1, Oct-1, Oct-2 homeodomain proteins and the Caenorhabditis elegans transcription factor unc-86 (Herr et al., 1988). Based on their sequence homologies, POU genes have been divided into six subclasses (I to VI) (Latchman, 1999;Ryan and Rosenfeld, 1997). Mammalian class III POU genes include four members, Brn-1,  and Oct-6/SCIP/Tst-1 that are predominantly expressed in the developing and adult nervous system (He et al., 1989;Monuki et al., 1989;Meijer et al., 1990;Hara et al., 1992;Mathis et al., 1992;Alvarez-Bolado et al., 1995;Zwart et al., 1996). POU class III genes are important for the neuronal cell differentiation. Knock out of the mouse Brn -2 gene results in the loss of neurons that produce oxytocin, vasopressin and corticotropin-releasing hormone (Nakai et al., 1995;Schonemann et al., 1995). Brn-4 mutants in mice show developmental defects in the inner ear causing deafness (Phippard et al., 1999). In humans, DFN3, an Xchromosome linked non-syndromic mixed deafness is caused by the naturally occurring mutations in Brn-4 gene (de Kok et al., 1995). Targeted deletion of Tst-1 has shown that Tst-1 is essential for the terminal differentiation of myelinating Schwann cells in the peripheral nervous system (Bermingham et al., 1996;Jaegle et al., 1996).
In zebrafish, five POU class III genes have been identified and characterized (Matsuzaki et al., 1992;Sampath and Stuart 1996;Spaniol et al., 1996;Hauptmann and Gerster 1996;Hauptmann and Gerster 2000). They include zp12, zp23, zp47, brn1.2 and zp50. The zebrafish POU class III genes show high sequence identity with their corresponding mammalian genes. Similar to their mammalian members, zebrafish class III POU genes lack introns within their POU-domain encoding sequences. zp50 is orthologous to mammalian Oct-6 (Levavasseur et al., 1998). zp12 and zp23 are the zebrafish orthologs of mammalian Brn-1 and are identical with each other (Spaniol et al., 1996). zp47 and brn1.2 are related to each other and are the zebrafish orthologs of mammalian Brn-2 gene (Spaniol et al., 1996;Sampath and Stuart 1996). The presence of additional genes in zebrafish could be attributed to the gene duplication in the genomic sequence of the teleost lineage. Previously only a partial sequence of brn1.2 was deduced and a detailed developmental expression analysis of this gene was not described (Sampath and Stuart et al., 1996). Therefore, we have attempted to describe the developmental expression pattern of zebrafish brn1.2 using whole-mount in situ hybridization.
Members of the class IV group of POU genes are characterized by an additional amino terminal consensus sequence, the POU-IV box (Gerrero et al., 1993;Xiang et al., 1995;Xiang et al., 1993) and are known to play important roles during development of the nervous system (Latchman, 1999;Ryan and Rosenfeld, 1997).
Drosophila I-POU (Acj6) and Caenorhabditis elegans unc-86 are the invertebrate homologues of mammalian class IV POU genes (Gruber et al., 1997). The three mammalian class IV POU genes, POU4F1 (also called Brn-3.0, Brn-3a, RDC1), , and POU4F3 (Brn-3.1, Brn-3c) display high sequence similarities and distinct but overlapping expression patterns in the developing CNS and PNS (Latchman, 1999). Murine Brn-3a is mainly expressed in dorsal root and trigeminal ganglia, medial habenula, red nucleus and inferior olivary nucleus (Xiang et al., 1996). Loss of Brn-3a function by targeted deletion in mice causes loss of neurons in the brain stem and trigeminal ganglion and leads to uncoordinated limb movement and impaired suckling (Xiang et al., 1996). Human Brn-3a is localised on chromosome 13 and was found expressed in subsets of peripheral nervous system tumours (Collum et al., 1992). Human Brn-3a has been reported to activate expression of p53 in human tumour cells (Budhram-Mahadeo et al., 2002). It has also been shown that human Brn3a can activate the NGF1-A promoter in primary neurons and neuronal cell lines (Smith et al., 1999).
Zebrafish brn3b and brn3c homologues have recently been characterized, while a brn3a gene of zebrafish has not been described. In zebrafish two brn3b cDNAs, a long and a short isoform, have been cloned and their expression reported in the retina, optic tectum, migrating posterior lateral line primordium and larval neuromast (DeCarvalho et al., 2004). Zebrafish brn-3c has also been cloned and was found to be expressed in the developing otic vesicle (DeCarvalho et al., 2004;Sampath and Stuart, 1996). In an effort to determine the complete set of POU genes in teleost fish by search through available genome sequence and EST databases, we identified two brn3a zebrafish orthologues named brn3a1 and brn3a2,. Similarly to brn3b, brn3a2 was found to be expressed as a long (brn3a2 (l)) and a short (brn3a2(s)) isoform. The developmental expression patterns of the two-zebrafish brn3a genes were analyzed by whole-mount in situ hybridisation (WISH).

Phylogenetic analysis of the zebrafish Pou gene class.
Studies on vertebrate genome evolution provide increasing evidence of a whole genome duplication in the teleost lineage. In order to determine the effect of the proposed teleost genome duplication on the number of POU genes, we searched the zebrafish and pufferfish genome databases (Zebrafish version 3 and 4 (Zv3, Zv4) and FUGU 2.0) (www.ensemble.org). Our database search revealed 18 POU genes in zebrafish and 17 POU genes in pufferfish. To classify the identified POU genes into different subclasses, multiple sequence alignment was performed using the identified POU domain sequences and a phylogenetic tree was constructed ( Fig 1A). The phylogenetic tree clustered the different zebrafish and pufferfish genes into the known six subclasses. Zebrafish and pufferfish genes corresponding to each of the six subclasses were identified. When compared to the mammalian set of 15 POU genes, it became eveident that several POU genes were present in two copies, while others were missing in the two teleost genomes. Surprisingly, genes corresponding to POU5F2 (Sprm-1) and POU3F4 (brn-4) could not be identified through the genome search in zebrafish and pufferfish. Perhaps, zebrafish and pufferfish sprm-1 and brn-4 may be secondarily lost during evolution ( Fig 1A).

Identification of zebrafish brncDNA
We identified the zebrafish brn1.2, fugu brn1.2 and zp47 gene by TBLASTN search of the genomic zebrafish and fugu sequence at wwww.ensemble.org using zebrafish zp47 (acc.no. P79746). The constructed sequence was used to screen the zebrafish EST database for zebrafish brn1.2 ESTs. Two ESTS for zebrafish brn1.2 were identified and sequenced. We found that only one of them contained the complete open reading frame, therefore we used this clone for further analysis. This clone contained only a partial fragment of the 5ÚTR (20nt), 1639nt 3ÚTR and 1014nt ORF.
Multiple sequence comparison between the zebrafish POU class III proteins Zp47 and Brn1.2 with the corresponding proteins of other species revealed high conservation in the POU domain region (Fig 1B). The POU domain sequence of zp47 and brn1.2 are highly similar to human, mouse and pufferfish. zp47 showed an overall sequence identity of 97.4% to human, mouse and pufferfish while brn1.2 was 95.5% identical to human and mouse and 96.8% identical to pufferfish.

Identification of zebrafish brn3a cDNAs.
Through the zebrafish genome search ENSDARP00000007850 and ENSDARP00000013808 were identified. Similarity search with Blast program followed by multiple sequence alignment of POU domain and phylogenetic tree construction was performed to identify the class of POU genes these hits belonged.
The similarity analyses revealed that ENSDARP00000007850 and ENSDARP00000013808 were paralogues to each other and orthologues to mammalian Brn3a.
For the murine Brn3a gene a short and a long cDNA isoform has been characterized (acc. Nos: AAO60105, AAO60106) (Thomas et al., 2004). In order to explore the possible existence of zebrafish brn3a1 and brn3a2 isoforms, we attempted to theoretically construct a long and a short isoform transcript form the available genome sequence using in silico methods. It was possible to construct a long and a short isoform for zebrafish brn3a2 (brn3a2(l) and brn3a2(s)) but not for zebrafish brn3a1.
The coding region of zebrafish brn3a2(l) was derived from two exons (E1,E2) separated by an intron (I1). Incontrast, brn3a2(s) was made up from the 3' region of intron (I1) and exon E2 ( Fig 1D). The genome sequence analyses of the members of the brn3 family in zebrafish and fugu demonstrated that the mRNA was mainly derived from two exons and some members of the family also formed shorter isoforms ( Fig 1D).
The amino acid sequences from the assembled zebrafish brn3a1, brn3a2(l) and brn3a2(s) were taken to mine the zebrafish expressed sequence tag database available at NCBI (www.ncbi.nlm.nih.gov). One EST for zebrafish brn3a1 was identified and Mouse Brn3a is 93.7% identical to zebrafish brn3a1 and 96.2% identical to zebrafish brn3a2). Even higher sequence identity was found between zebrafish and pufferfish class IV POU domain sequences (pufferfish brn3a1 is 97.5% identical to zebrafish brn3a1 and pufferfish brn3a2 is 97.5% identical to zebrafish brn3a2).

Multiple sequence alignment of Pou class IV sequences.
Multiple sequence alignment of class IV amino acid sequences revealed a high conservation of the POU domain region (POU+Linker+Homeodomain) between different species, but significant conservation was also observed outside of the POU domain ( Fig 1C). A poly-glycine stretch at the N-terminus of mammalian Brn-3b and in mammalian Brn-3a was seen; this region was absent in the zebrafish and pufferfish sequences. Distinct region specific for brn3b sequences, mammalian brn3a, brn3b sequences were observed ( Fig 1C). A specific region of 11 amino acid was identified to be present in fish (zebrafish and pufferfish) brn3a2 and mammalian brn3a. This region was absent from the fish brn3a1 ( Fig 1C). Analysis of the genomic sequence of Pou class IV genes from human, mouse, zebrafish, and pufferfish showed that the coding region is derived from two exons. The position of the intron was conserved among all the species compared. The length of the exons of different brn3 genes in zebrafish and fugu were very similar and zebrafish brn3a2 and brn3b also formed shorter isoforms ( Fig 1D).

Developmental expression pattern of zebrafish brn1.2
We performed whole-mount in situ hybridization (WISH) (Hauptmann andGerster, 1994, 2000) to characterize the spatial expression of brn1.2 in the developing zebrafish brain. Expression of brn1.2 was first detected at the tail bud stage in an area that corresponds to the midbrain primordium (Fig 2A). At the 3-, 5-and 10-somite stages the expression became quite strong in the midbrain region. From the 5-somite stage on brn1.2 was also detected in the hindbrain and spinal cord (Fig 2B,C,D).
A weak expression of brn1.2 transcripts was found in the ventral diencephalic region at the 10-somite stage ( Fig 2D). We compared the expression of brn1.2 with pax2.1 (Krauss et al., 1991) andkrx-20 (Oxtoby andJowett, 1993) in order to locate the position of brn1.2 transcripts in the midbrain and hindbrain. Two color in situ hybridisation with pax2.1 showed that brn1.2 was located anterior to pax2.1 expression domain indicating that brn1.2 expression domain was located in the anterior midbrain ( Fig 2E). Co-labelling studies with krx-20 revealed that brn1.2 expression in the hindbrain is confined to r3 and r5 (Fig 2G,H).
From 24hpf on, brn1.2 showed a widespread expression in the CNS. In the forebrain, brn1.2 was detected in the diencephalon, epiphysis, ventral thalamus, dorsal thalamus and pretectum while the telencephalon was devoid of brn1.2 expression. At 1dpf, brn1.2 was expressed throughout the hindbrain with strong expression levels in r3 and r5 (Fig 2I). At 30hpf a lamda shaped expression domain with two arms were observed in the diencephalon (Fig 2J). To define the position of the two arms more precisely we performed whole-mount in situ hybridisation to visualize brn1.2 followed by immunohistochemistry to detect acetylated α-tubulin (Piperno and Fueller, 1985). One arm of the lamba domain was found to be lying in-between the optic recess and the tract of the postoptic commissure. The other arm extending from the ventral thalamus was broader in shape and was widespread across the tract of the commissure of the posterior tuberculum (Fig 2J).
At 36hpf and 2dpf the expression of brn1.2 became more complex in the brain (  , 1996). Zebrafish brn3a1 was found to be expressed in the ventronasal region of the retina at 25 hpf ( Fig 5D).
Retinal brn3a1 expression spread dorsally and temporally and throughout the RGC layer by 36hpf (Fig. 5J). The three retinal layers (ganglion cell layer, inner nuclear layer and outer nuclear layer) are clearly visible at 48hpf, and intense expression of brn3a1 was seen throughout the RGC layer (Fig 5M-N). The early expression profile of zebrafish brn3a1 indicated that brn3a1 may precede the expression of brn3b and brn3c. The sequence of expression of Brn3a, Brn3b and Brn3c is different in mouse where the expression of Brn3b precedes the expression of Brn3a and Brn3c (Xiang, 1998). Axonal projections from the RGC's exit the retina by 34hpf and innervate optic tectum by 72hpf (Burrill and Easter, 1995). Expression of brn3a1 was also found in the optic tectum. Tectal expression of brn3a1 was detected at 30 hpf ( Fig 5F) and fully established by 48 hpf (Fig 5L-M), Zebrafish brn3a2 was very similarly expressed as brn3a1.
Expression of brn3a2(l) in the trigeminal placode and presumed Rohon Beard sensory neurons started around the 7 somite stage (Fig. 6A,B) and became stronger by 10 somite stage (Fig 6C,D). Expression in the trigeminal ganglion ceased at 24 hpf.
Expression of brn3a2(l) was detected in the anterior lateral line (ALL) and posterior lateral line (PLL) by 15 somites and continued to be expressed there until 48hpf ( Fig   6E-J, 7A-M). Clusters of cells expressing brn3a2(l) were also seen in the forebrain, hindbrain (Fig 7A,D,G,I) and along the dorsal cells of the spinal cord (Fig 7C,F). The expression in the spinal cord goes down by 48hpf (Fig 7M).In the midrain tectum and RGC layer, brn3a2(l) expression was established by 48hpf (Fig 7I,J,L,M). In summary, apart from expression of both brn3a genes in the RGC layer and sensory ganglia, expression of these genes was also detected in sensory neurons along the dorsal spinal cord and in small cell clusters within the forebrain and hindbrain.
Similar to zebrafish brn3b (DeCarvalho et al., 2004), brn3a1 and brn3a2(l) were also detected in the lateral line system.

Zebrafish maintenance
Wild type Zebrafish were maintained at 28.5 °C and under standard conditions of feeding, care and egg collection. Embryos were collected by natural mating . The collected embryos were staged according to Kimmel et al. (1995). Embryos were staged in hours post fertilization (hpf) and days post fertilization (dpf), and embryo stages older than 24hpf were subjected to 0.03% phenylthiourea treatment. The collected embryos were fixed at different stages in 4% paraformaldehyde overnight and then washed with phosphate buffered saline containing 0.1% Tween-20 (PBSTw) and stored in 100% methanol until usage for in situ hybridization.

Sequence analysis
Zebrafish and Pufferfish genome sequence databases were mined to identify and construct POU class genes. Mammalian POU genes were used as query. A database using Filemaker Pro software was developed to collect and maintain the assembled POU sequences from zebrafish and pufferfish as well as published sequences from other species. The assembled sequences were translated and POU, Linker region and Homeodomain were extracted and multiple sequence alignments were performed with the Clustal X program (Thompson et al., 1997). A neighbour joining tree with bootstrap value of 1000 was constructed based on the multiple sequence alignment obtained in the Clustal X program. The neighbour joining tree helped to cluster the newly found zebrafish and pufferfish sequences to their mammalian counterparts. The tree was viewed with NJ plot software (Perriere and Gouy, 1996).
Zebrafish brn1.2, brn3a1 and brn3a2 genomic sequences (ENSDARG00000023662, ENSDARP00000007850 and ENSDARP00000013808) were identified using zebrafish zp47 for brn1.2 and murine Brn3a protein sequences (acc No P79746 and acc. Nos. S69350) as query against the ongoing zebrafish genome project (Zv3) using TBLASTN search. The long and the short isoforms of brn3a2 were assembled manually using conceptual translation. Amino acid sequences of brn3a1, brn3a2(l), brn3a2(s) were obtained by conceptual translation.

Immunohistochemistry
Some embryos processed for WISH to visualize brn1.2 were further processed for immunohistochemical detection of the position of the primary axons using a monoclonal antibody against acetylated alpha-tubulin (Piperno and Fuller, 1985). The experiments were performed as described previously (Hauptmann and Gerster, 1996).
Axon tracts were visualized in red using Fast red as alkaline phosphatase substrate.