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Keywords:

  • pbx1;
  • swim bladder;
  • zebrafish

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

pbx1, a TALE (three–amino acid loop extension) homeodomain transcription factor, is involved in a diverse range of developmental processes. We examined the expression of pbx1 during zebrafish development by in situ hybridization. pbx1 transcripts could be detected in the central nervous system and pharyngeal arches from 24 hpf onwards. In the swim bladder anlage, pbx1 was detected as early as 28 hpf, making it the earliest known marker for this organ. Morpholino-mediated gene knockdown of pbx1 revealed that the swim bladder failed to inflate, with eventual lethality occurring by 8 dpf. The knockdown of pbx1 did not perturb the expression of prdc and foxA3, with both early swim bladder markers appearing normally at 36 and 48 hpf, respectively. However, the expression of anxa5 was completely abolished by pbx1 knockdown at 60 hpf suggesting that pbx1 may be required during the late stage of swim bladder development. Developmental Dynamics 239:865–874, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Pbx family belongs to the PBC (termed after the well-conserved domain found in PBX and ceh-20) group of the TALE (three amino acid loop extension) class of homeodomain proteins. PBX1 was originally identified in human pre-B acute lymphoblastic leukemia as a result of its disruption by t(1;19) chromosomal translocation (Kamps et al.,1990; Nourse et al.,1990). Subsequently, PBX2, PBX3, and PBX4 genes were identified based on their sequence conservation with PBX1 (Monica et al.,1991; Wagner et al.,2001). All mammalian Pbx isoforms share a highly similar sequence identity within and flanking their DNA-binding homeodomain. In addition, both Pbx1 and Pbx3 have longer (Pbx1a and Pbx3a) and shorter (Pbx1b and Pbx3b) isoforms derived from C-terminal differential splicing (Monica et al.,1991; Wagner et al.,2001). Pbx orthologs in Caenorhabditis elegans (ceh-20), Drosophila melanogaster (Exd), and Danio rerio (lazarus/pbx4) have also been characterized (Burglin and Ruvkun,1992; Rauskolb et al.,1993; Popperl et al.,2000; Vlachakis et al.,2000).

Pbx acts as an essential HOX cofactor through an interaction involving the TALE domain in the first helix of the homeodomain (Moens and Selleri,2006). A short C-terminal tail comprising 16 amino acid residues is also essential for maximal cooperative interactions with Hox partners, as well as for maximal monomeric binding of Pbx1 to DNA. Pbx also heterodimerizes with the Meis/Prep subfamily of TALE-class homeodomain proteins (Chang et al.,1997; Berthelsen et al.,1998; Kilstrup-Nielsen et al.,2003) to form trimeric complexes with Hox proteins on appropriate DNA sites (Berthelsen et al.,1998,1999; Jacobs et al.,1999). In addition, the binding of Pbx with other homeodomain-containing proteins, such as PDX1 and Engrailed, has been reported (Swift et al.,1998; Vlachakis et al.,2001; Erickson et al.,2007). The discovery of an increasing number of non-homeodomain transcription factors that can act as partners of Pbx suggests that it has a broader developmental role than previously assigned (Laurent et al.,2008).

The importance of Pbx1 in development is first observed in Pbx1 knockout mice, which showed multiple abnormalities and eventual embryonic lethality. Specifically, Pbx1 has been implicated in skeletal development and patterning (Selleri et al.,2001), maintenance of definitive hematopoiesis in the fetal liver (DiMartino et al.,2001; Selleri et al.,2001; Kim et al.,2002), pancreatic development and function (Kim et al.,2002), kidney morphogenesis, adrenal development, and urogenital differentiation (Schnabel et al.,2003b), and caudal pharyngeal pouch-derived organ formation and patterning (Manley et al.,2004). In contrast, despite its widespread expression, the loss of Pbx2 does not affect organogenesis, fertility, hematopoiesis, or immune function (Selleri et al.,2004). Mice deficient of Pbx3 develop to term but die within a few hours of birth due to respiratory failure as a result of abnormal activity of the inspiratory neurons in the medulla (Rhee et al.,2004). Finally, Pbx4 is found preponderantly in testes and is important for spermatogenesis (Wagner et al.,2001).

All four Pbx isoforms are encoded by the zebrafish genome (Popperl et al.,2000). The roles of pbx2 and pbx4 in hindbrain development have been reported (Popperl et al.,2000; Vlachakis et al.,2000,2001; Waskiewicz et al.,2002). More recently, pbx 2 and pbx 4 have been shown to interact directly with the basic helix-loop-helix transcription factors Myod and Hand2 in promoting skeletal and myocardial muscle differentiation, respectively (Maves et al.,2007,2009). In contrast to the well-characterized roles of mammalian Pbx1, the function of pbx1 in teleost has not been examined despite the availability of the zebrafish pbx1a and pbx1b cDNA sequences.

Given the expression of Pbx1 in the fetal lung mesenchyme layer and its involvement in mouse lung organogenesis (Schnabel et al.,2001), we speculate that zebrafish pbx1 might be similarly associated with the development of swim bladder. The teleost swim bladder shares a common evolutionary origin with the tetrapod lung and are both derived from the foregut during embryonic development (Warga and Nusslein-Volhard,1999; Shannon and Hyatt,2004). Like several digestive tract–associated organs such as liver and pancreas, swim bladder originates from the anterior endodermal precursors, where they initially form a multi-cellular rod before eventually developing into the alimentary canal and its associated organs (Warga and Nusslein-Volhard,1999; Ober et al.,2003; Finney et al.,2006).

Here we report the embryonic and adult expression pattern of pbx1 in zebrafish. In situ hybridization results revealed that pbx1 was expressed in central nervous system, pharyngeal arches, and swim bladder during early embryonic stages at 24 to 72 hours post-fertilization (hpf). The detection of pbx1 as early as 28 hpf in the swim bladder anlage makes it the earliest known gene marker for this organ. Concomitantly, the knockdown of pbx1 function by both translation-blocking and splice-blocking morpholinos (MOs) prevented the swim bladder from inflating, leading to a disruption of normal buoyancy and locomotory movements, and eventual lethality of pbx1 morphants.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The Zebrafish pbx1 Gene

pbx1 isoforms, pbx1a and pbx1b, are two differentially spliced forms with differing C-termini. pbx1a and pbx1b comprise 7 exons and 5 exons, respectively, with Pbx1a shorter by 37 amino acid residues (aa) due to alternative splicing at exon 5 (Fig. 1A). The N-terminus contains two conserved domains: the PBC domain, which is located at aa 38 to 231, and the homeodomain at aa 223 to 292. Zebrafish pbx1 is located in different contigs in the zebrafish genome database, with the first three exons localized to chromosome 7, while the remaining exons have been assigned to chromosome 2. Zebrafish pbx1a exhibits between 81 to 85% nucleotide sequence identities to Homo sapiens, Mus musculus, and Gallus gallus, respectively, and 92% identity in their amino acid sequences, whereas zebrafish pbx1b exhibited nucleotide sequence and amino acid identities of 83 and 92% to these 3 species, respectively.

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Figure 1. Pbx1 protein structure and reverse transcriptase-polymerase chain reaction (RT-PCR) expression pattern analysis. A: Protein domains of zebrafish Pbx1a and Pbx1b. Numbers refer to amino acid residues. The location of the alternatively spliced exon is marked by an arrow. Exons 1 to 5 are common to both Pbx1 isoforms. B: RT-PCR temporal expression pattern of zebrafish pbx1a and pbx1b during early embryonic development. Sizes of the amplified products were 641 bp for pbx1a, 530 bp for pbx1b, and 200 bp for β-actin. C: Expression of pbx1a and pbx1b in adult tissues obtained from 6-month-old zebrafish.

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Expression of pbx1a and pbx1b in Zebrafish Embryos and Adult Tissues

The tissue distribution and embryonic stage–specific expression of zebrafish pbx1a and pbx1b were analyzed using RT-PCR. Primers specific for pbx1a and pbx1b were designed to amplify these isoforms from developmental stages between 0 to 72 hpf, as well as the adult tissues. In embryos, only pbx1a was maternally expressed and by 24 hpf, both pbx1a and pbx1b mRNA expression was detected, with a gradual increase throughout the development of the embryo (Fig. 1B). In adult tissues, the expression of pbx1a was the strongest in brain followed by eyes, heart, muscle, ovary, spleen, swim bladder, and testis, and weak or no expression in gills, head kidney, intestine, and liver. In comparison, pbx1b was highly expressed in spleen and swim bladder, followed by testis (Fig. 1C).

The Zebrafish pbx1 Is Expressed During Ontogenic Development of Central Nervous System, Pharyngeal Arches, and Swim Bladder

To determine the spatio-temporal expression of pbx1, we performed whole-mount in situ hybridization on zebrafish embryos from 24 to 72 hpf. An in situ hybridization probe that spans the region of differential spliced exon, which detected both pbx1a and pbx1b, was used (Fig. 2). In situ hybridization using probes that are specific to pbx1a or pbx1b revealed identical expression patterns (data not shown). Results showed that pbx1 was expressed in the central nervous system, including the forebrain (diencephalon and telencephalon), midbrain, and hindbrain (cerebellum and rhombomere) throughout embryonic development until 72 hpf (Fig. 2). A second domain of expression could be seen at the posterior pharyngeal arches (3rd–7th arches) as early as 28 hpf (Fig. 3A), based on its colocalization with dlx2a, which is a specific marker for pharyngeal arches (data not shown; Sperber et al.,2008). They were located bilaterally and situated anterior to the first somite. This expression domain remained as bilateral clusters when observed at 32, 48, and 72 hpf, respectively (Fig. 3B–D).

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Figure 2. Expression of pbx1 in the head region. A–D: Lateral view at 24 (A), 36 (B), 60 (C), and 72 hpf (D). E–H: Dorsal view at 24 (E), 36 (F), 60 (G), and 72 hpf (H) of pbx1 expression in the forebrain (FB), midbrain (MB), and hindbrain (HB).

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Figure 3. Spatio-temporal expression of pbx1 at the trunk region. A–D: Posterior pharyngeal arches (5th–7th arches) seen as early as 28 hpf located bilaterally anterior to the first somite. A: Expression of pbx1 in the swim bladder anlage at 28 hpf. B: Swim bladder anlage at 32 hpf. C: Swim bladder bud at 48 hpf. D: Elongated swim bladder sac at 72 hpf. E: Inflated swim bladder at 6 dpf. F: prdc expression in the swim bladder overlapped with pbx1 at 32 hpf. G: foxA3 expression at the swim bladder bud and other gut-derived organs at 48 hpf. H: Colocalization of pbx1 (blue) and anxa5 (red) in the swim bladder at 60 hpf. I, intestine; L, liver; PP, posterior pharynx; P, pancreas; black arrow, swim bladder; red arrow, posterior pharyngeal arches.

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Another domain of pbx1 expression was detected in the trunk from 28 hpf onwards. The expression was initially faint but could be seen clearly at the midline, just above the yolk sac (Fig. 3A). Progressively, the cluster became more prominent by 32 hpf and was localized to a region between the first to second somite (Fig. 3B). By 48 hpf, pbx1 expression could be localized clearly at the swim bladder bud (Fig. 3C). Consequently, at 72 hpf the entire sac-like swim bladder was observed to express pbx1 and persisted in the inflated swim bladder at 6 days post-fertilization (dpf) (Fig. 3D,E). Comparison with prdc, which was previously the earliest reported marker of swim bladder primordium at 32 hpf (Muller et al.,2006), and foxA3, a marker for gut-derived endodermal organs including swim bladder at 48 hpf (Ober et al.,2003), further confirmed the expression of pbx1 in the swim bladder (Fig. 3F,G). Finally, pbx1 expression overlapped with anxa5, a late-stage marker for swim bladder at 60 hpf (Winata et al.,2009) (Fig. 3H).

Morpholino Knockdown of pbx1 Function Disrupts Swim Bladder Development

A morpholino-mediated gene knockdown strategy was used to study the functional role of pbx1. An antisense morpholino, Pbx1MO, was designed such that it was complimentary to the AUG start codon and 23 adjacent bases of the pbx1 mRNA. A standard control antisense MO (StdMO) was used as the negative control. Prior to knockdown experiments, the efficacy of Pbx1MO in targeting and blocking protein translation from pbx1 transcripts was evaluated using in vitro transcription and translation. Pbx1MO successfully reduced the amount of Pbx1a to 37% and Pbx1b to 5%, respectively, indicating the effectiveness of Pbx1MO in blocking translation of both pbx1 transcripts (Fig. 4A). In contrast, StdMO had no significant effects on the translation of pbx1. In addition, a splice-blocking morpholino (Pbx1-Ex2MO) was also designed and its efficacy was confirmed by RT-PCR, showing greatly reduced pbx1 transcripts as compared to its missense MO control, Pbx1-Ex2MM (Fig. 4B).

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Figure 4. Efficacy of pbx morpholinos. A: Efficiency of translation-blocking morpholinos (Pbx1MO, Pbx2MO, Pbx4MO) in blocking translation as shown by in vitro translation of corresponding mRNA alone, with standard control antisense morpholino (StdMO) or with morpholinos. B: Efficacy of splice-blocking morpholinos. RT-PCR shows decreased mRNA expressions level for embryos injected with splice-blocking morpholino (Pbx1-Ex2MO, Pbx2-Ex4MO, Pbx4-Ex3MO) as compared to their respective missense control (Pbx1-Ex2MM, Pbx2-Ex4MM, Pbx4-Ex3MM).

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Pbx1MO-injected embryos developed relatively normally until 3 dpf, after which the morphants developed a curvature in body shape and whirlpool swimming motion. About the swim bladder, 70% of the Pbx1MO microinjected larvae failed to inflate and as a result were unable to remain buoyant, leading eventually to lethality by 8 dpf. None of these defects was observed in embryos injected with the StdMO. A non-inflating swim bladder phenotype was also obtained with the splice-blocking Pbx1-Ex2MO. In addition, Pbx1-MO-mediated gene knockdown of 6-dpf embryo from the gutGFP transgenic line (Field et al.,2003) further demonstrated the failure of normal swim bladder development and inflation, as a result of the loss of pbx1 function (Fig. 5A,B).

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Figure 5. Morpholino-mediated knockdown of pbx1 disrupts swim bladder, pancreas, and pharyngeal arches development. A,B: Swim bladder development in 6-dpf gut GFP transgenic embryo injected with StdMO (A) and Pbx1MO (B) showing non-inflated swim bladder in pbx1 morphants. C–H: In situ hybridization of swim bladder markers prdc, foxA3, and anxa5 using embryos injected with StdMO and Pbx1MO at 36 (C, D), 48 (E,F), and 60 hpf (G,H). I,J: In situ hybridization for anxa5 in 60-hpf embryos injected with Pbx1-Ex2MM (I) and Pbx1-Ex2MO (J). K,L: Expression of pbx1 in mutants deficient in hedgehog signaling. Reduced pbx1 expression in the syu mutant at 72 hpf (K) and absence of expression of pbx1 in the swim bladder of smu mutant at 72 hpf (L). M–P: Splice-blocking morpholino-mediated knockdown of pbx2 or pbx4 disrupts swim bladder development. In situ hybridization for anxa5 in 60-hpf embryos injected with Pbx2-Ex4MM (M) and Pbx2-Ex4MO (N), Pbx4-Ex3MM (O), and Pbx4-Ex3MO (P). Q,R: In situ hybridization for pancreas marker, pdx-1, in 36-hpf embryos injected with StdMO (Q) and Pbx1MO (R). S,T: In situ hybridization for dlx2a, a pharyngeal arches marker in 36-hpf embryos injected with StdMO (S) and Pbx1MO (T). Red arrow, swim bladder; black arrow, pancreas; blue arrow, posterior pharyngeal arches.

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The role of pbx1 in swim bladder development was further addressed by subjecting pbx1 morphants to in situ hybridization with relevant swim bladder markers at various developmental stages. There were no significant differences in prdc and foxA3 expression at 36 hpf and 48 hpf, respectively, for embryos injected with StdMO or Pbx1MO (Fig. 5C–F). The swim bladder bud in pbx1 morphants failed to develop into an elongated sac-like structure as compared to embryos injected with StdMO when examined at 60 hpf. The relatively smaller pouch in pbx1 morphants failed to inflate even when examined at later stages. Also, the expression of anxa5 was completely lost with the knockdown of pbx1 using both Pbx1MO and Pbx1-Ex2MO morpholinos (Fig. 5G–J). In addition, we have chosen two hedgehog signaling pathway mutants, syu and smu, to determine the involvement of pbx1 in swim bladder development. Our results showed that pbx1 expression was reduced in the swim bladder of syu mutants and was completely absent in smu mutants (Fig. 5K,L).

Comparison of pbx1, pbx2, and pbx4 Knockdown on the Swim Bladder

Diffused pbx2 and pbx4 expression pattern could be detected among the endodermal organs by in situ hybridization at 2 dpf as reported previously (diIorio et al.,2007). Therefore, we examined whether pbx1, pbx2, and pbx4 share a common role in swim bladder development by knockdown using both translation-blocking and splice-blocking morpholinos for pbx2 and pbx4 (Fig. 4A,B). We confirmed that there was no cross-reaction among the different pbx morpholinos by coupled transcription/translation assay or RT-PCR (data not shown). The effects of their knockdown on the swim bladder were examined by in situ hybridization at 60 hpf using anxa5 (Fig. 5M–P). For pbx4, no anxa5 expression could be detected in all Pbx4MO-injected embryos, while 87% of splice-blocking Pbx4Ex3MO-injected embryos showed loss of anxa5 expression (Table 1, Fig. 5P). In contrast, anxa5 could still be detected in the majority of the pbx2 morphants injected with either the translation-blocking or splice-blocking MOs. As expected, all the 3 missense splice-blocking MOs did not perturb anxa5 expression (Table 1). Finally, the simultaneous knockdown of pbx1, pbx2, and pbx4, using the translation-blocking MOs, did not reveal further perturbations of prdc and foxA3 as compared to pbx1 morphants.

Table 1. Effect of pbx1, pbx2, and pbx4 Knockdown by Translation-Blocking and Splice-Blocking Morpholinos (8.64 μM) on anxa5 Expression as Observed by In Situ Hybridization at 60 hpf
Morpholinoanxa5 expression (%)Total number of injected embryos
PresenceAbsence
StdMO98.51.5132
Pbx1MO0.0100.0147
Pbx2MO83.017.0141
Pbx4MO0.0100.0141
Pbx1-Ex2MO30.469.646
Pbx2-Ex4MO84.215.838
Pbx4-Ex3MO13.386.945
Pbx1-Ex2MM98.02.049
Pbx2-Ex4MM98.02.050
Pbx4-Ex3MM95.84.248

Effect of pbx1 Knockdown on Other Abdominal Organs

In situ hybridization experiments were also performed on pbx1 knockdown embryos to study their effects on the development of the pancreas, pharyngeal arches, liver, interrenal, kidney, and hindbrain (Table 2). The knockdown of pbx1 resulted in 80% of the embryos showing decreased pdx-1 expression in the pancreas at 36 hpf (Fig. 5Q,R) and 35.4% of embryos showing lesser dlx2a expression in pharyngeal arches at 36 hpf (Fig. 5S,T). Liver, interrenal, kidney, and hindbrain were not much affected by Pbx1MO as the percentage of reduction was consistently 20% or below.

Table 2. Effects of pbx1 Knockdown on Selected Organs at 36 hpf as Determined by In Situ Hybridization Using Specific Markersa
MarkerOrganExpression level in Pbx1MO (8.64 μM) morphants (%)Total number of injected embryos
++/−
  • a

    −, lack of expression; +, presence of expression; +/−, weak expression.

pdx-1Pancreas0.080.020.0135
dlx2aPharyngeal arches64.635.40.0144
ff1bInterrenal87.10.012.9186
prox1Liver69.010.021.0126
Hindbrain54.823.821.4126
wt1Kidney100.00.00.0144

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The strong pbx1a expression detected in the adult brain tissues by RT-PCR is consistent with the results from mouse, where Pbx1a expression is detected primarily in the neural tissues (Schnabel et al.,2001). Also pbx1b has a more ubiquitous expression pattern in adult tissues compared to pbx1a. In addition, both isoforms were expressed in the spleen, which correlates well with the finding of mammalian Pbx1 (Schnabel et al.,2001; Brendolan et al.,2005). In vertebrates, the spleen is a lymphoid organ that serves important roles in hematopoiesis and generation of primary immune responses. Pbx1 has been shown to be a central hierarchical coregulator in spleen genesis in mice (Brendolan et al.,2005). This indicates that the function of pbx1 in these organs may have been preserved.

In situ hybridization results of zebrafish pbx1 showed discrete expression patterns in the central nervous system, whereas pbx2 and pbx4 were strongly expressed throughout the central and peripheral nervous system. In mouse, Pbx1 and Pbx3 levels were marked in the forebrain (especially intense in the ventral thalamus), midbrain, and hindbrain. In zebrafish, the knockdown of pbx1 resulted in decreased prox1 expression in the hindbrain. Thus, pbx function in the mammalian and teleost central nervous system is likely to be conserved. The expression of all four zebrafish pbx members in the central nervous system permits functional redundancy and this is reflected by the ability of pbx1 and pbx3 to rescue lzr phenotype when ectopically expressed by mRNA injection (Popperl et al.,2000). Specific combinations of pbx4 and its coregulators, meis3b and hoxb1b, lead to differing development fates of the zebrafish hindbrain development (Vlachakis et al.,2001), indicating that coregulators may play an important role in the determination of Pbx function in the central nervous system.

pbx1 expression was observed at the five posterior arches (3rd–7th arches), which will eventually develop into the gills (3rd–7th arches) and teeth (only the 7th arch). Zebrafish lack teeth on the oral jaws, but have well-developed pharyngeal teeth that form a bilaterally symmetric pattern, posterior and medial to the seventh pharyngeal arches (Yelick and Schilling,2002). Elsewhere, pbx4 has been implicated to play a role in zebrafish pharyngeal endodermal segmentation (Popperl et al.,2000; Waskiewicz et al.,2002; diIorio et al.,2007). In mouse, Pbx1 knockdown caused abnormal caudal pharyngeal pouch development, while Pbx2 and Pbx3 are speculated to have redundant roles in pharyngeal region development (Manley et al.,2004; Selleri et al.,2004; Di Giacomo et al.,2006).

The loss of Pbx1 markedly reduces urogenital ridge outgrowth, impairs differentiation of the mesonephros and kidneys, and obliterates the Mullerian ducts (Schnabel et al.,2003a). Pbx1 is important in mouse adrenal development primarily by regulating expression of SF-1 in the fetal zone (Zubair et al.,2006). Furthermore, Pbx1 plays a role in mouse pancreatic islet physiology by serving as a co-factor for Cdx-2 in the regulation of proglucagon expression (Liu et al.,2006). Unlike the broad expression pattern we observed for mouse Pbx1, we failed to detect pbx1 in the liver, interrenal, pancreas, and kidney. One possibility is that zebrafish pbx1 expression level in these organs is below the in situ hybridization detection limit, or that pbx1 is only expressed in these organs at a much later stage. Nevertheless, the lack of perturbation in the morpholino knockdown experiment suggests that embryonic development of these organs in zebrafish do not seem to require pbx1.

The earliest known swim bladder marker previously is prdc, which marks the budding site of swim bladder at 32 hpf (Muller et al.,2006). In the present study, we have shown that pbx1 expression predated prdc and could clearly demarcate the swim bladder anlage from the foregut by 28 hpf. Despite the early appearance of pbx1, loss of pbx1 function does not seem to affect the swim bladder budding process as shown by the unperturbed expression patterns of prdc and foxA3. Instead it appears that pbx1 may be required for subsequent stages of swim bladder development, which involves differentiation of three tissue layers and the attendant inflation process (Winata et al.,2009). At the same time, we could not rule out the possibility of compensation for the loss of pbx1 functions by other pbx members during the budding stage but this needs to be further clarified by the knockdown of all 4 Pbx members. In mouse, the loss of Pbx2 does not perturb organogenesis despite its widespread embryonic expression and this has been attributed to the functional compensation by other Pbx family members (Selleri et al.,2004; Di Giacomo et al.,2006). Besides pbx1, our results also showed that the knockdown of pbx4 function also resulted in the complete loss of anxa5 expression in swim bladder at 60 hpf. The elimination of prep1.1, a Meinox protein known to form complexes with Hox and Pbx, by morpholino knockout also resulted in morphants lacking a proper swim bladder (Deflorian et al.,2004). Taken together, it seems that the regulation of zebrafish swim bladder development may be shared by pbx1 and pbx4 and could involve coregulators.

Swim bladder is primarily a buoyancy organ and shares similar features with the mammalian lung in being an air-filled organ rich in elastin and surfactants (Perrin et al.,1999; Prem et al.,2000). Pulmonary surfactant is a complex of lipids and proteins that line the interior surfaces of the lung and prevents alveolar collapse by reducing the surface tension across the air/liquid interface of the alveoli (Clements,1977). In non-mammalian vertebrates, the pulmonary surfactant is generally less disaturated and hence its surface tension activity tends to be relatively low (Daniels et al.,1994; Smits et al.,1994). These considerations have led to the notion that in non-mammalian vertebrates pulmonary surfactant has other functions, such as acting as an anti-adherent for an inflating/deflating organ with an air/liquid interface, for instance in the swim bladder (Veldhuizen et al.,1998). The failure of swim bladder inflation in pbx1 morphants indicates that pbx1 may play a role in regulating genes related to surfactant production. In this regard, excessive thyroid hormone has been reported to exert a late effect on swim bladder morphogenesis, by suppressing surfactant production crucial for the inflation of swim bladder (Liu and Chan,2002).

To date, studies have reported that fibroblast growth factors, sonic hedgehog (shh), transforming growth factors and retinoids are the main molecular regulators of mammalian lung development (Cardoso,2001). So far, only hedgehog signaling has been reported to play pivotal roles in zebrafish swim bladder development (Bellusci et al.,1997; Winata et al.,2009). In zebrafish, three hedgehog genes, sonic hedgehog (shh), indian hedgehog (ihh), and desert hedgehog (dhh), have been identified (Currie and Ingham,1996; Schauerte et al.,1998; Avaron et al.,2006). Two of the zebrafish shh mutants identified are sonic-you (syu) mutant, which is defective of shh function (van Eeden et al.,1996; Schauerte et al.,1998), and the slow muscle omitted (smu) mutant, which has a mutation that disrupts smoothened, resulting in an inability to respond to any hedgehog signals (Schauerte et al.,1998; Chen et al.,2001). Detailed characterization of swim bladder formation in syu mutant revealed severe defects in the epithelium, mesenchyme, and mesothelium of the swim bladder (Winata et al.,2009). The disruption of the swim bladder is more serious in smu mutant, with both the epithelium and mesenchymal layers being absent and disorganized mesothelial layers (Winata et al.,2009). Our result showed reduced expression and complete loss of pbx1 expression in the syu and smu mutants, respectively, and is in concordance with the degree of severity in swim bladder disruption. Recent studies have shown that Pbx cooperates with Hox to regulate Shh in mammalian limb-bud development (Capellini et al.,2006). The transcriptional activation of fast muscle–related gene expression by shh in zebrafish embryos also requires Pbx proteins (Maves et al.,2007). In swim bladder, early hedgehog signals are crucial for both epithelial and mesenchymal specification (Winata et al.,2009). Further studies should detail the actual role of pbx1 in relation to the hedgehog signaling in swim bladder development.

In conclusion, we have shown that the development of a fully inflated swim bladder is dependent on the ontogenic expression of pbx1 in the swim bladder anlage, with eventual onset of lethality taking place by 8 dpf. Our study has demonstrated that zebrafish could be an alternative model for the functional analysis of pbx1, particularly as it has also overcome the problem of embryonic lethality presented by the Pbx1 knockout mice. Clarification of its role in swim bladder development may lead to a better understanding of its homologous functions in mammalian lung development. Furthermore, it would also be of interest to explore pbx1 regulation during swim bladder development by generating a transgenic fluorescent protein lineage marker of pbx1.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Embryos

Zebrafish were maintained in the fish facilities at the Department of Biological Sciences, National University of Singapore, according to established protocols. Embryos were collected by natural spawning and staged according to Kimmel et al. (1995).

RT-PCR

pbx1a and pbx1b cDNA were PCR amplified and cloned using the GenBank sequences AJ245962 and AJ245963. DNA sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer, Waltham, MA) and analyzed using the automated sequencer ABI 377 (Perkin Elmer). Total RNAs were isolated from embryos and various adult tissues harvested from 6-month-old fish with TRIzol reagent and RT-PCR reaction was performed. The forward primer used for RT-PCR reaction was 5′-TAGAGAAGTATGAGCAGGC GTGTA-3′ for both pbx1a and pbx1b, while the reverse primer for pbx1a and pbx1b was 5′-TCCCCGGAGTTCATGTTAAA-3′ and 5′-AGAGTATCCACC-GGCCGAAT-3′, respectively. β-actin was amplified using the primers: 5′-CCGTGACATCAAGGAGAAGCT-3′ and 5′-TCGTGGATACCGCAAGATTCC-3′.

Whole Mount In Situ Hybridization

Whole mount in situ hybridization of zebrafish embryos was performed using digoxigenin-labeled antisense RNA probes as described previously (Chai and Chan,2000). The embryos were fixed in 4% PFA overnight and underwent serial dehydration through graded methanol solutions. A 694-bp pbx1 fragment including the 3′ UTR was generated by RT-PCR with the following primer pair: (5′-CTAACGCAACCAGCGTCTC-3′ and 5′-GCTGAGAG-GTAGAATGAAGCA-3′), and cloned into the pGEMT-Easy vector. Additional antisense RNA probes include: prdc (NM_001017704, nucleotides (nt) 5–767), foxA3 (NM_131299, nt 280–1,191), anxa5 (BC164112, nt 37–839), pdx1 (NM_131443, nt 54–835), and dlx2a (NM_131311, nt 6–800). The vector was linearized with NdeI and the antisense probe was generated by T7 polymerase. DIG-labeled riboprobes were detected with alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche, Nutley, NJ) and stained with NBT/BCIP (Promega, Madison, WI) to produce purple precipitate. For two-colour in situ hybridization, inactivation of the first antibody was performed by incubating the stained embryos in 100 mM glycine, pH 2.2, for 10 min followed by four washes with PBST for 10 min each, before proceeding with the blocking step prior to incubation with the second antibody. The second staining was performed with AP-conjugated anti-Fluorescein antibody (Roche) using Fast Red (Roche) as substrate. Stained embryos were post-fixed in 4% PFA and washed two times for 15 min in PBST. This was followed by tissue clarification in 50% glycerol in PBS. Digital pictures were obtained with a Zeiss (Thornwood, NY) Axioscope and processed with Adobe Photoshop.

Antisense Morpholino Knockdown

Antisense translation-blocking MOs (Gene Tools LLC) were designed to target the AUG codon of pbx1, 5′-GG CTGGTCATCCATCCTCGCCGCTG-3′ (Pbx1MO); and 5′ UTR of pbx2, 5′-CTAGATGAGTGTGTGACTAACTGCG-3′ (Pbx2MO), and pbx4, 5′-TAATACT- TTTGAGCCGAATCTCTCC-3′ (Pbx4-MO). The MO was resuspended in 1 × Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6) and microinjected into the yolk of single-cell embryos at a concentration of 8.64 μM per embryo. The specificity of the Pbx1MO was tested using an in vitro transcription and translation assay (Promega). pbx1 mRNA (0.5 μg) containing a T7 promoter was added to the TNT® T7 PCR Quick master mix and incubated for 90 min at 30°C for the synthesis of biotin-labeled proteins using an in vitro rabbit reticulocyte lysate translation kit (Ambion, Austin, TX) under conditions described by the manufacturer. The translation of mRNA was tested in the presence of 4 μg of Pbx1MO. StdMO (Gene Tools, Philomath, OR), which has no sequence homology to any known zebrafish sequence, was used as a control. The entire reaction mixture (20 μl) of each in vitro translation assay was separated by SDS-PAGE and transferred to PVDF membrane by Western blot. Streptavidin-HRP-conjugated antibody against the biotinylated protein was added and the detection was made using chemiluminescence substrate (SuperSignal West Pico, Pierce, Rockford, IL).

Splice-blocking MOs targeting the splice donor site of exon 2, 4, and 3 for pbx1, pbx2, and pbx4, respectively, were used to corroborate translation-blocking MOs. The sequences are Pbx1-Ex2MO: 5′-ATATGTGCCCTCTCACCTGCTCATA-3′, Pbx2-Ex4MO: 5′-CATTAGCATGCAGACGTCACCTGGC-3′, and Pbx4-Ex3MO: 5′-AGAGGAAACACTGCTCTGACCTGTT-3′. Additional morpholinos were designed as missense control for each of the pbx morpholinos. The sequences are Pbx1-Ex2MM: 5′-ATtTcTGCCgTCTCAC gTcCTCATA-3′, Pbx2-Ex4MM: 5′-CATTAcCATcCAcACGTgACCTcGC-3′, and Pbx4-Ex3MM: 5′-AGAcGAAAgAC TcCTCTcACCTcTT-3′. Even at concentrations as high as 17.28 μM, all three missense control MOs did not perturb the wild-type expression of pbx1, pbx2, and pbx4, respectively.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Svetlana Korzh and Cecilia Winata (DBS, NUS) for the syu and smu mutant zebrafish and Didier Stainier (University of California, San Francisco) for the gutGFP transgenic zebrafish. Funding from the Academy of Sciences Malaysia (SAGA 304/PBIOLOGI/653006.A118) and A* STAR, BMRC (CWK – BMRC 07/1/21/19/527) is gratefully acknowledged.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES