The function of DrPax 1 b gene in the embryonic development of zebrafish

Vertebrate Pax1 gene is a member of Pax gene family and encodes a transcription factor associated with crucial roles in the development of pharyngeal pouch, scletrotome and limb bud. In zebrafish, the genome contains two Pax1 paralogs, DrPax1a and DrPax1b, which share high sequence similarity with other Pax1 genes. To elucidate the function of zebrafish DrPax1b gene, we first examined the gene expression pattern and found that it was mainly expressed in the endodermal pharyngeal pouch, caudal somites, notochord, and fin bud. Then, we performed knockdown experiments using antisense morpholino oligonucleotides, which lead to the defects in the vertebral column, tail, pharyngeal skeleton, and pectoral fin. Additionally, we also found that the mouse MmPax1 mRNA, but not the amphioxus AmphiPax1/9 mRNA, could rescue the MO-induced defects. Furthermore, sequence alignment revealed that the N-terminal region of vertebrate Pax1 and amphioxus Pax1/9 were highly conserved, whereas their C-terminal regions were relatively divergent. However, the chimeric Am(N)Dr(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 mRNA could partially rescue the defects, while the Dr(N)Am(C)Pax1 mRNA could not. In conclusion, our data demonstrate a conserved function of DrPax1b in the development of the vertebral column, pectoral fin and pharyngeal skeleton formation in zebrafish and also provide critical insight into the functional evolution of Pax1 gene by changing its C-terminal sequence.


INTRODUCTION
Pax proteins are a group of transcription factors that play key roles in cell proliferation, cell differentiation, organogenesis and embryonic pattern formation.All Pax proteins contain a highly conserved paired domain of 128 amino acids, and many of them also have a homeodomain and an octapeptide (Mansouri et al., 1999).To date, total of nine Pax genes have been determined in mammalian and divided into four subfamilies: Pax1/9, Pax2/5/8, Pax3/7 and Pax4/6, based on the sequence homology and structural similarity (Noll et al., 1993).Among those genes, Pax1 is a member that plays important roles in embryonic development and its homologous gene is widely distributed through animal phyla (Dahl et al., 1997).
In protostome drosophila, the Pax1 gene (Pox meso) was expressed in the somatic mesoderm during myogenesis, indicating a crucial role in the development of larval body muscles (Bopp et al., 1989;Duan et al., 2007).In deuterostome amphioxus, the gene (AmphiPax1/9) was strongly expressed in the endoderm of the developing pharynx and weakly in the somitic mesoderm at the posterior end of larvae (Holland et al., 1995;Kozmik et al., 2007).In mouse, Pax1 gene was mainly expressed in the ventromedial part of the sclerotomes, and also in the pharyngeal region, limb bud.Its mutation led to the morphological abnormalities of vertebral body, intervertebral disks and pectoral girdle (Capellini et al., 2008;Peters et al., 1999;Timmons et al., 1994;Wallin et al., 1994).Similarly, the expression of Pax1 appeared in the pharyngeal region, somite, sclerotomes and wing bud of developing chick embryos, and the knockdown of Pax1 caused somite dysmorphogenesis of the embryos (Barnes et al., 1996;Leclair et al., 1999;Senthinathan et al., 2012;Smith and Tuan, 1995).Moreover, expression of Pax1 was also observed in pharyngeal pouches as well as caudal somites of Xenopus embryos (Handrigan and Wassersug, 2007).In teleost medaka, the similar expression of Pax1 was X. LIU et al. observed in the pharyngeal pouch and ventromedial part of the sclerotomes, and once it was knocked down, the development of vertebral column was affected (Mise et al., 2008).
However, two Pax1 genes DrPax1a and DrPax1b were found in the genome of another teleost zebrafish (Wang et al., 2010), and the expression of DrPax1a was previously used as a sclerotomal marker gene (Clements et al., 2011).Since the gene function of DrPax1b has not been detected yet, this report presents the gene spatial-temporal expression pattern and elucidates the gene function by morpholino-mediated knockdown experiment.Furthermore, we perform rescue experiments with amphioxus Pax1/9, mammalian mouse Pax1, and chimeric Pax1 mRNAs to analyse the conservation and diversification of Pax1 gene function during the evolution.

MATERIALS AND METHODS
Animals AB strain zebrafish (Danio rerio) was maintained at 28.5°C on a 14-hour light/10-hour dark cycle and the embryos were collected by natural mating in the laboratory.C57BL/6 strain mouse embryo was supplied by the Laboratory Animal Center of Xiamen University and amphioxus (Branchiostoma belcheri) was cultured according the previous report in the laboratory (Zhang et al., 2007).(AmphiPax1/9) (DQ991501), Drosophila melanogaster Pox meso (NM_001043222), and aligned them using MEGA 5.0.Then a phylogenetic tree was constructed using the neighbor-joining method.Further, we designed primer pairs to amplify Pax1 genes (Table 1).For cDNA cloning, we extracted total RNAs respectively from the embryos of mouse and zebrafish as well as adult amphioxus using Trizol reagent (Tiangen Co.), synthesized cDNAs using Primescript TM RT reagent kit (Takara Co.), and amplified the full-length coding sequences of DrPax1a, DrPax1b, MmPax1 and AmphiPax1/9.Then we respectively subcloned them into pXT7 vector in the sense orientation downstream the promoter.

Real-time quantitative PCR
To reveal the temporal expression pattern of DrPax1b gene during the embryonic development, we separately extracted RNA templates from zebrafish embryos at 9 different developmental stages (Kimmel et al., 1995) and reverse-transcribed them into cDNAs.For real-time quantitative PCR (RTqPCR), we designed a pair of specific primers according to the coding sequence of DrPax1b gene (Table 1) and carried out the reactions on Rotor-Gene 6000 Real-Time System (Corbett Robotics Co.) using SYBR Green (Promega Co.) for fluorescence detection with an inner-control of zebrafish β-actin gene.All samples were predenatured for 2 min at 94°C and subjected to 40 cycles of 10 s at 94°C, were scaled at the same standard and related to copy numbers using the 2 −ΔΔCT method (Livak and Schmittgen, 2001).Three independent reactions were performed for each detected gene.

Whole-mount in situ hybridization
In order to detect the expression pattern of DrPax1b by whole-mount in situ hybridization, we subcloned the full coding sequence of DrPax1b into pBluescript KS vector (pBKS), synthesized digoxigenin-labeled antisense RNA probe using Riboprobe ® in vitro transcription system (Promega Co.), and purified it with RNeasy mini kit (Qiagen Co.).Then the wholemount in situ hybridization (WISH) for zebrafish embryos was carried out according to the previous description (Westerfield, 1995).The embryos were observed and photographed under a stereo microscope.Additionally, in order to test whether the DrPax1b played a role in the development of pectoral fin bud in zebrafish, we also conducted WISH to reveal the pectoral fin development in DrPax1b-MO-injected embryos using antisense RNA probe of the pectoral fin marker gene Axin2 (Thisse et al., 2004).).These chimeras were also subcloned into pXT7 in the sense orientation downstream of the T7 promoter.

Construction of chimeric molecules
mRNA synthesis For mRNA synthesis, the correctly recombined DrPax1b-pXT7, MmPax1-pXT7, AmphiPax1/ 9-pXT7 and chimeric Pax1-pXT7 vectors were applied to transcribe mRNA in vitro.First, we linearized those recombinant plasmids with XbaI and synthesized capped mRNAs using T7 Cap-Scribe kit according to the manufacturer's instruction (Roche Co.).Then, we removed the DNA templates by treating the products with RNase-free DNaseI (Takara Co.), purified the mRNA using RNeasy mini kit (Qiagen Co.), and dissolved them in nuclease-free water.The products were stored at -80°C for use.
For rescue experiments, we respectively injected DrPax1b, MmPax1, AmphiPax1/9 and chimeric mRNAs (400 pg of each) into the embryos to rescue the MOinduced defects immediately after the microinjection of *The letters indicate as: F, forward primer; R, reversed primer; Am, AmphiPax1/ 9; Dr, DrPax1b; Mm, MmPax1; N, N-terminal region; C, C-terminal region.
X. LIU et al.
MO. Then the injected embryos were incubated and observed at 48 hpf stage for data collection.

Western blot analysis
To detect the efficiency of the DrPax1b-MO, we compared the protein expression levels of Pax1b in the WT embryos with that in the MO-injected embryos.Both of WT and MO-injected embryos were separately lysed with lysis buffer, eluted with SDS sample buffer, and then analyzed by Western blotting with rabbit polyclonal antibody to Pax1 or β-Actin (Abcam Co.).After incubation with HRP-conjugated secondary antibody, proteins were detected using chemiluminescence.
Cartilage staining To detect whether the DrPax1b gene was required for the development of the pharyngeal cartilage, we selected WT and MO-injected embryos at 5 dpf stage and fixed them in 4% paraformaldehyde con- fected with PBS for 2 h.After 1 h washing in 70% ethanol, the samples were transferred into a 0.1% Alcian blue solution dissolved in 80% ethanol and 20% glacial acetic acid for at least 4-6 h or overnight.Then, the larvae were gradually rehydrated in 70% ethanol, 50% ethanol and PBST for 1 h each, washed in solution (3% H 2 O 2 and 1% KOH in PBST) for 1 h and finally imaged them in 50% glycerol under the stereo microscope.

RESULTS
Two Pax1 genes in zebrafish genome From the zebrafish genome (http://asia.ensembl.org/Danio_rerio/Info/Index), we retrieved two Pax1 genes DrPax1a and DrPax1b.These genes respectively located on chromosome 17 and 20 and presented different exon-intron structures in contrast to that of DrPax9 (Fig. 2A).Besides, we constructed a phylogenetic tree with drosophila Pax1 (Pox meso) as an out-group (Fig. 2B), and the tree again confirmed that two Pax1 genes existed in the zebrafish genome.Moreover, we respectively cloned partial cDNA segments of the two genes from zebrafish embryos.These results not only demonstrated the existence of two Pax1 genes but also indicated the activation of them during the embryonic development.

The expression pattern of DrPax1b in zebrafish embryos
To analysis the function of DrPax1b gene, we examined the temporal and spatial expression pattern in the developing zebrafish embryos.First, we detected the temporal expression pattern of DrPax1b gene at 9 different embryo developmental stages using RTqPCR with a primer pair specific for DrPax1b (Fig. 3A).The results displayed a high expression level of DrPax1b in unfertilized eggs indicating a maternal expression gene.After fertilization, the copy number of mRNA decreased gradually with the early cell division of the embryos.Till 75%epiboly stage, the expression was hard to be detected, suggesting the exhaustion of maternal mRNA of DrPax1b.After that, the gene expression strikingly rose again at 18-somite and later development stages, exhibiting a zygotic expression profile.
Further, we detected the spatial expression pattern of DrPax1b during embryonic development from 2-cell stage to 48 hpf stage using WISH.And the antisense RNA probe was specific for DrPax1b gene, as its sequence displayed 29.6% diversity with DrPax1a at nucleoside sequence level.The results revealed a strong hybridizing signal of DrPax1b throughout the 2-cell stage embryos (Fig. 3B), which well matched with the result from RTqPCR assay.At 30% and 75%-epiboly stages, the signal gradually became weaker and weaker (Fig. 3, C and  D).But at 8-somite stage, zygotic expression of DrPax1b started obviously in the pharyngeal region (Fig. 3E).
Whereafter the expression continued in the pharyngeal endoderm and more restrictedly in the pharyngeal pouch at 18-somite stage (Fig. 3, F and G).At 24 hpf stage, DrPax1b was apparently expressed in the pharyngeal pouch and moderate signals was found in the caudal part of somites (Fig. 3, H and I).At 48 hpf stage, DrPax1b was continually expressed in the pharyngeal pouches, fin bud, notochord and weakly expressed in the dorsal aorta and axial vein (Fig. 3, J-L).

DrPax1b knockdown in the embryos of zebrafish
To further assay the function of DrPax1b gene in zebrafish, we designed an antisense morpholino oligonucleotides against the DrPax1b mRNA to block its translation.In the early developmental stages (75%epiboly and 8-somite stages), the MO-injected embryos developed normally (Fig. 1, A-D).At 18-somite stage, the MO-injected embryos showed shorter tail compared to the WT embryos (Fig. 1, E and F).At 24 h and 48 h stage, most of the MO-injected embryos obviously showed curving vertebral column in contrast with the normally developing WT embryos (Fig. 1, G-L).The moderate morphant showed mildly curving vertebral column with almost normal tail (Fig. 1, H and K), and the severe morphant showed relatively serious curving vertebral column and particularly depauperate tail (Fig. 1, I and L).
To clearly show the development of pharyngeal skeleton, we stained both MO-injected and WT embryos at 5 dpf stage using Alcian blue.Compared to the WT controls (Fig. 1M), the moderate morphant embryos showed hypoplasia at pharyngeal cartilage (Fig. 1N), and the severe morphant ones showed relatively serious loss of the pharyngeal cartilage (Fig. 1O).The WISH with Axin2 probe revealed that the fin bud could be found apparently at 48 hpf normal larvae (Fig. 1P) but it was stunted in the moderate morphant (Fig. 1Q) or hard to be observed in the severe morphant (Fig. 1R).

Western blot and rescue experiments
The western blot assay revealed that the protein expression level of Pax1b in the MO-injected embryos was dramatically decreased in contrast to that in the WT embryos (Fig. 1S).The results indicated that the DrPax1b-MO effectively blocked DrPax1b protein translation.
The rescue experiments further validated the specificity of the MO.Statistically, when injecting DrPax1b-MO only, 25.3% of the embryos showed moderate abnormal phenotype and 71.7% of the embryos showed severe abnormal phenotype.But 52.6% of embryos displayed moderate abnormal phenotype and 43.3% of embryos dis-played severe abnormal phenotype by co-injection of DrPax1b-MO and DrPax1b mRNA in the rescue experiments.The percentage of severe phenotype embryos significantly decreased by 28.4%, which suggested that the DrPax1b mRNA could rescue the knockdown phenotype and again confirmed the specificity of the DrPax1b-MO Considering that the paired domain of Pax gene is highly conservative across chordate lineages, we performed additional rescue experiments using mRNAs of MmPax1 and AmphiPax1/9 to evaluate the functional evolvement of Pax1.The results showed that 44.8% of embryos displayed moderate phenotype and 49.7% of embryos displayed severe phenotype in contrast to 22.6% moderate phenotype and 73.6% severe phenotype embryos in positive control (Fig. 1U).The percentage of severe phenotype embryos significantly decreased by 23.9%, suggested that MmPax1 mRNA could partially rescue the DrPax1b knockdown defect in zebrafish embryos.However, 21.8% and 75.2% of zebrafish embryos respectively displayed moderate and severe phenotypes after rescue experiment using AmphiPax1/9 mRNA, and the positive control with DrPax1b-MO injection presented 24.5% moderate abnormality and 72.1% severe abnormality.No distinct difference was observed between the rescue experiment and the positive control (Fig. 1U).We also tested different injection quantities (100 pg, 200 pg, 400 pg, 800 pg) of AmphiPax1/9 mRNA but still no clearly improved phenotype appeared in the developing embryos (data not shown).The results suggested that AmphiPax1/9 mRNA could not rescue the DrPax1b knockdown defects of zebrafish embryos.Compared to the DrPax1b-MO injected embryos, the percentage of embryos displayed severe phenotype decreased by 18.09% in the rescue injection of Am(N)Dr(C)Pax1 mRNA, indicating that phenotype of the DrPax1b knockdown was partially rescued (Fig. 1V).However, the percentage of severe phenotype was not decreased obviously by the injection of Dr(N)Am(C)Pax1 mRNA, hinting the fail of the rescue experiments with Dr(N)Am(C)Pax1 (Fig. 1V).Furthermore, the percentage of embryos displayed severe phenotype reduced by 22.40% in the co-injection with Mm(N)Dr(C)Pax1 mRNA, and those decreased by 25.05% in the co-injection with Dr(N)Mm(C)Pax1 mRNA (Fig. 1V).The observations also indicated that both the Mm(N)Dr(C)Pax1 mRNA and the Dr(N)Mm(C)Pax1 mRNA could partially rescue the MO-defects in zebrafish.

DISCUSSION
The conserved function of Pax1 in vertebrates Usually, single Pax1 gene was found in the most of vertebrate genomes.The gene expression pattern and functional analysis revealed that Pax1 was mainly involved in the development of pharyngeal region, pectoral girdle, axial skeleton and tail of vertebrates (Chi and Epstein, 2002;Wallin et al., 1994).However, two paralogues DrPax1a and DrPax1b were previously identified in the zebrafish genome (Wang et al., 2010) and one of them DrPax1a was already related to the development of centrums (Clements et al., 2011).In this study, we cloned the DrPax1b gene from zebrafish embryos.WISH demonstrated that the DrPax1b was mainly expressed in the endodermal pharyngeal region, as well as in the caudal somites, notochord and fin bud indicating the function involved in those organic development.The DrPax1b knockdown experiments lead to the defects in the vertebral column, tail, pharyngeal skeleton and pectoral fin.And the rescue experiments with DrPax1b or MmPax1 mRNA could relieve the defects of knockdown zebrafish embryos.All those above results allowed us safely infer that the function of DrPax1b was conserved during evolutionary of vertebrates, comparing with its homologs in vertebrates.
Neofunctionalization of vertebrate Pax1 gene during evolution Amphioxus AmphiPax1/9 is an ancestral gene of vertebrate Pax1 and Pax9 genes and also shares a conserved paired domain with zebrafish DrPax1b.However, AmphiPax1/9 mRNA could not relieve the defects of DrPax1b knockdown zebrafish embryos.WISH revealed that AmphiPax1/9 was weakly expressed in the somitic mesoderm at the posterior end of the larva (Kozmik et al., 2007).This mesodermal function of AmphiPax1/9 is comparable to the myogenic role of Pax1 homologs in drosophila.However, vertebrate Pax1 derived from amphioxus AmphiPax1/9 by gene duplication, which occurred after the divergence between cephalochordates and vertebrates, and mainly involved in the development of axial skeleton.Presumably, vertebrate Pax1 in the somitic mesoderm are most obviously involved in the development of the somites into elements of the axial skeleton, deriving from the original myogenic function in cephalochordate.
Actually, the bone of vertebrates, including skeleton of pectoral girdle/fin, axial skeleton and the pharyngeal skeleton, was preformed in cartilage anlages in the early embryonic development.Based on above observations, we might infer that all morphants of DrPax1b knockdown were results of abnormal development of cartilage anlage.This inference also hinted that DrPax1b might be fundamentally involved in the development of cartilage anlage.Therefore, we speculated that vertebrate Pax1 developed new function in vertebrates for the cartilage anlage, which first appeared in vertebrates but not in their ancestor cephalochordates.

Fig. 1 .
Fig. 1.DrPax1b knockdown in zebrafish embryos.(A-L) DrPax1b knockdown causes developmental defects in zebrafish embryos.The WT and DrPax1b-MO (5 ng each) injected embryos are photographed at 75%-epiboly (A and B), 8-somite (C and D), 18-somite (E and F), 24-h (G-I), 48-h (J-L).At 24 and 48 hpf stage, the embryos are scored morphologically for different degree of defects into three categories: normal (G and J), moderate morphant (H and K), severe morphant (I and L).(M-O) Staining of pharyngeal skeleton in the WT and MO-injected embryos: WT (M), moderate morphant (N), severe morphant (O).(P-R) Axin2 gene WISH in the WT and DrPax1b MO-injected embryos: WT (P), moderate morphant, (Q), severe morphant (R).Black arrows indicate the location of fin bud.Scale bars in panel (A-L) are 500 μm and those in other panels are 100 μm.(S) Western blot analysis with antibody to Pax1b and β-Actin.The injection of DrPax1b-MO effectively blocked DrPax1b protein translation in lane 2 comparing with the DrPax1b protein in lane 1. (T) Schematic of primer localities and chimeric genes.The white, grey and black rectangles respectively denote the AmphiPax1/9, DrPax1b and MmPax1.Arrows indicate the localities and directions of primers used to amplify the N-terminal or Cterminal regions.Am(N)Dr(C)Pax1 composes AmphiPax1/9 N-terminal (white) and DrPax1b C-terminal (grey) regions.Dr(N)Am(C)Pax1 contains DrPax1b N-terminal (grey) and AmphiPax1/9 C-terminal (white) regions.Mm(N)Dr(C)Pax1 composes MmPax1 N-terminal (black) and DrPax1b C-terminal (grey) regions.Dr(N)Mm(C)Pax1 contains DrPax1b N-terminal (grey) and MmPax1 C-terminal (black) regions.(U and V) A histogram displayed the results of the rescue experiments with DrPax1b, MmPax1, AmphiPax1/9 and chimeric mRNAs.The MO injections and co-injections with MO plus mRNAs were respectively performed in parallel on the same batch of embryos.Zebrafish embryos were respectively co-injected with DrPax1b-MO (5 ng each) and DrPax1b mRNA, MmPax1 mRNA, AmphiPax1/9 mRNA and chimeric mRNA (400 pg each).The phenotypes are scored at 48 hpf stage and the percentages of normal (white bar), moderate (grey bar) and severe (black bar) are indicated.Rescue experiments were performed in triplicate for each analyzed mRNA.The letter n denotes the total number of embryos.The P values less than 0.05 are considered as statistically significant and those more than 0.05 are not shown.

Fig. 2 .
Fig. 2. Exon-intron structure, phylogenetic analysis and sequence alignment.(A) Exon-intron structures of DrPax1a, DrPax1b and DrPax9 genes.The exons are represented by boxes and the introns by short lines, with the length in nucleotides written above.The coding regions are shown in black and the 5′ and 3′ untranslated regions in white.(B) Phylogenetic tree of Pax1 and Pax9 genes.(C) Alignment of MmPax1, DrPax1a, DrPax1b and AmphiPax1/9 full coding sequence.The paired domain is boxed with solid lines.The N-terminal domain is underlined with a real line, and the C-terminal domain is underlined with a broken line.

Fig. 3 .
Fig. 3. Temporal and spatial expression pattern of DrPax1b gene in the developing zebrafish embryos.(A) Temporal expression analysis of DrPax1b gene.Relative expression levels of DrPax1b gene at 9 different embryo developmental stages were evaluated by RTqPCR.(B-L) Spatial expression pattern of DrPax1b gene in zebrafish embryos detected by WISH.(B) 2-cell stage.(C) 30%epiboly stage.(D) 75%-epiboly stage.(E) 8-somite stage.(F-G) 18-somite stage.(H-I) 24 hpf stage.(J) 48 hpf stage.(K) Cross section through level K in photo J. (L) Cross section through level L in photo J.All the panels are lateral view except G, I, K and L which are dorsal view or cross sections.Abbreviations: pp, pharyngeal pouch; so, somite; fb, fin bud; nc, notochord; da, dorsal aorta; av, axial vein.Scale bars in panel I, K, and L indicate 50 μm and those in other panels indicate 500 μm.
Although highly conserved paired domain at the N-terminal region was shared by Pax1 genes, it was MmPax1 mRNA rather than AmphiPax1/9 mRNA possessed the ability to rescue the DrPax1b morphant.The sequence alignment showed that the C-terminal of Pax1 existed less homology.In order to determine which part of Pax1 performed an essential function in the embryonic development, we constructed four chimeric Pax1s: Am(N)Dr(C)Pax1, Dr(N)Am(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 (See Fig. 1T for meaning of the abbreviations), and then respectively injected these chimeric mRNAs into the DrPax1b knockdown embryos to assay their rescue efficiency.
The C-terminal region of Pax1 gene plays important role in gene function evolution Further rescue experiments revealed that all chimeric molecules except Dr(N)Am(C)Pax1 mRNA were able to partially relieve the defects.The results proved the two parts of Pax1 gene were, to some extent, functionally independent.The chimeric Dr(N)Am(C)Pax1 failed to rescue the defect phenotype though its counter-molecule Am(N)Dr(C)Pax1 could improve developing status.This The function of DrPax1b gene in zebrafish result demonstrated that the less conserved C-terminal region of DrPax1b also performed important roles in zebrafish embryonic development.The divergence between the C-terminal region of AmphiPax1/9 and DrPax1b possibly endowed DrPax1b with a new function contributing in the cartilage anlages development.

Table 2 .
Primer sequences used for amplification of N-terminal region or C-terminal region of Pax1 genes