- Research article
- Open Access
Pbx homeodomain proteins pattern both the zebrafish retina and tectum
© French et al; licensee BioMed Central Ltd. 2007
- Received: 03 February 2007
- Accepted: 16 July 2007
- Published: 16 July 2007
Pbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined.
Analysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth.
These data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.
- Retinal Ganglion Cell
- Optic Tectum
- Retinal Pigment Epithelium
- Wild Type Embryo
- Retinal Pigment Epithelium Cell
Vertebrate embryos use a combination of transcription factors to specify position along the anterior-posterior (A-P) axis. Of particular importance is the Pbx (pre-B cell leukemia homeobox)-family of TALE (Three Amino acid Loop Extension)-class transcription factors, which are required globally to pattern the A-P axis of the developing vertebrate embryo. Using mouse knockout and zebrafish knockdown models, researchers have shown that Pbx proteins are required to specify cell fate in the midbrain, hindbrain, somites, pancreas, and blood [1–4]. In the hindbrain, trimeric DNA-binding complexes of Pbx, Hox and Meinox (Meis/Pknox) proteins specify rhombomere identity. In the midbrain, Pbx cooperates biochemically with Engrailed (Eng) proteins to maintain both midbrain-hindbrain and the diencephalic-mesencephalic boundaries [2, 5].
Pbx clearly also plays a role in patterning regions outside of the midbrain and hindbrain. For example, mouse knockouts have demonstrated a critical role for Pbx during pancreatic development, in which interactions between Pbx and Insulin promoter factor 1 (Ipf1) are required for transcriptional activity, and subsequent expansion of pancreatic cell lineages . Pbx also plays a role in the development of blood, as Pbx – Prep1 (also known as Pknox1) complexes are required for the production of normal populations of CD4 and CD8 T-lymphocytes . Furthermore, Pbx – Meis complexes have been implicated in megakaryocyte differentiation in rats, through the ability to initiate transcription from the platelet factor 4 (PF4) promoter . Recently, it has been shown during the development of skeletal muscle that Pbx is constitutively bound to the Myogenin promoter, can bind directly to the bHLH transcription factor MyoD, and is thus required for the development of muscle cell fates . A zebrafish mutant, lazarus (lzr), that contains a null mutation in the pbx4 gene , displays global defects in embryonic patterning including hindbrain, muscle, blood, and midbrain tissues.
The Meinox (Meis/Pknox/Prep) family of TALE-class transcription factors are well characterized as DNA-binding cofactors for both Pbx and Hox proteins . Zebrafish contain at least six meis genes that are expressed in dynamic and tissue specific manners [1, 10–12]. In addition, zebrafish possess at least two pknox/prep genes that are both widely expressed . meis genes are prominently expressed in the developing retina [1, 10–12] suggesting a role for meis in regulating eye formation or patterning. In zebrafish, overexpression of hoxb2 throughout the embryo causes ectopic expression of hindbrain markers exclusively within the retina, demonstrating the existence of retinal specific Hoxb2 competency factors . Pbx and Meinox proteins function as two of these retinal Hoxb2-competency factors, demonstrating that both Pbx and Meis can function in the retina [1, 8, 15, 16]. The idea that Meinox proteins play a role in eye formation is supported by studies in Drosophila, where the pbx homologue extradenticle (exd) and the meis homologue homothorax (hth) inhibit eye formation , and in mice where the Pknox1 hypomorphic and Meis1 knockout phenotypes include defects in retinal development. [16, 18]
Zebrafish make an excellent model for the study of retinal development. The optic primordium is distinct from surrounding tissues as early as 12 hours post fertilization (hpf), and by 24 hpf, eyecups have developed to include lens tissue and the surrounding neural retina . By 30 hpf, the first post-mitotic neurons have differentiated  and by 50 hpf, lamination is evident across the retina. Two partially redundant pbx genes, pbx2 and pbx4, are expressed during early development when the optic primordium and optic cup are developing. The other zebrafish pbx genes, pbx1 and pbx3.1, are expressed more strongly starting at 24 hpf. Thus, inhibition of Pbx2 and Pbx4 in a homozygotic lazarus (lzr) (pbx4 mutant) embryo allows for analyses of development in the absence of Pbx function, prior to 24 hpf (see methods). The retina of lazarus (pbx4-/-) zebrafish embryos is noticeably smaller than wild type embryos, but no analysis of patterning has yet been undertaken. This manuscript provides an analysis of Pbx and Meis function in the developing zebrafish retina, as well as a role for Pbx in patterning the tectum, and includes a microarray screen for pbx dependent transcripts in zebrafish to identify gene regulatory networks downstream of this critical homeodomain protein. We propose a model in which Pbx functions to specify regional identity in the developing zebrafish retina via activation of the dorsal/temporal specific growth factor, gdf6a.
Retinal laminar structure is normal but retinal ganglion cell axon outgrowth is aberrant in pbx2/4null embryos
Whole embryo microarray analysis of Pbx mutants identifies known hindbrain patterning genes
Microarray identified Pbx-dependent transcripts expressed in the hindbrain, eye, and tectum
Average fold change 18hpf
rhombomere 3, 5
central nervous system, neural crest
posterior retina, somites
heart, pectoral fin, posterior retina
lens, dorsal retina
eye, tectum, midbrain, hindbrain
tectum, midbrain, mid/hindbrain boundary
eye, tectum, midbrain, somites
Microarray and whole mount in situanalysis demonstrates that Pbx proteins regulate gene expression in the developing retina
The role of Meis proteins in regulating retinal gene expression
Pbx is required for both early and late eye patterning genes
It is likely that pbx2/4 function is also needed later in eye development as processes such as retinal ganglion cell outgrowth are also affected when Pbx function is inhibited. We therefore analyzed the expression of atonal homolog 7 (atoh7), which is expressed in a wave-like fashion (Figure 5K–M) in the inner retina prior to retinal ganglion cell differentiation . There is a marked reduction in the expression of atoh7 in the dorsal retina at both 36 hpf (18/27 embryos) and 41 hpf (22/32 embryos) (Figure 5O and 5P), while expression in the rest of the inner retinal tissue remains unaffected. This indicates that a lack of atoh7 in the dorsal retina may affect retinal ganglion cell differentiation in this domain, which may contribute to the observed defects in retinal ganglion cell outgrowth.
Pbx proteins regulate polarized gene expression in the optic tectum
As development proceeds, the tectum becomes highly organized and is characterized by the polarization of gene expression. At 48 hpf, the expression of otx2 and ephrinA5a (efnA5a) is strongest at the periphery of the tectum, with the highest expression observed distal to the eye while decreasing proximally (Figure 6G and 6I). These gradients are disrupted in pbx2/4 null embryos, whereby expression of otx2 is greatest proximal to the eye (Figure 6H), and expression of efnnA5a is confined to the posterior tectum (Figure 6J). By 3 dpf, the optic tectum is smaller and disorganized in lzr-/-, pbx2/4 morphant embryos (Figure 6L), as shown by visualization of acetylated tubulin-positive axons. These data clearly indicate a requirement for Pbx proteins in patterning the optic tectum.
Pbx is critically required for expression of gdf6a, itself a regulator of dorsal retina patterning
In this paper, we present evidence that pbx2/4 gene function is essential for proper retinal ganglion cell axon pathfinding. This phenotype likely results from aberrant Pbx mediated gene transcription in both the eye and tectum of pbx2/4 null embryos. Although the eye laminar structure is relatively normal after 5 days of development, connections between the RGC axons and the optic tectum are not made. Through microarray and in situ analysis, we have identified numerous candidate genes that may contribute to RGC axon guidance phenotype seen in embryos with reduced Pbx function. Abnormalities in eye patterning begin during early somitogenesis, and persist throughout development. Defects in the differentiation of retinal ganglion cells may also contribute to this phenotype, as the expression of atoh7 is eliminated in the dorsal region of the neural retina in pbx2/4 null embryos. The data presented here builds on current knowledge of Pbx proteins in embryonic patterning, and provides a novel role for Pbx in patterning the eye and tectum, and subsequently, the regulation of RGC axon outgrowth and pathfinding.
An absence of Pbx function results in reduced expression of genes involved in eye patterning. In pbx2/4 null embryos, defects are seen in the expression of rx2 during early somitogenesis, and at 10 somites, rx3 expression has not been downregulated as seen in wild type embryos . This could lead to defects in retinal ganglion cell and photoreceptor development, as rx2 and rx3 are also expressed in these subsets of cells later in development. We have identified aldh1a2, hmx4, and tbx5 as Pbx-dependent transcripts at 18 hpf. Each of these genes has the potential to play a strong role in regulating retinal patterning. For example, aldh1a2, which is involved in the synthesis of retinoic acid, is likely to affect development of the visual system in zebrafish as retinoic acid is required for development of photoreceptor cells and pigmented epithelium in the developing zebrafish eye . By 3 dpf, the photoreceptor layer is highly disorganized in fish with reduced pbx gene function, indicating a possible effect of aldh1a2 misexpression. Moreover, we also observed a downregulation of tbx5 via both microarray and in situ analysis, a downstream effector of retinoic acid signaling that is essential for establishment of the dorsoventral boundary of the eye [30, 31].
It has been documented that the homeodomain transcription factor soho1 represses the expression of epha3 in chicks, and is necessary for eye development. The zebrafish Pbx dependent gene hmx4 is likely the functional ortholog of chick soho1, since it is expressed in the retina and shares extensive sequence similarity. misexpression of soho1 in chicks affects epha3 expression , and loss of epha3 leads to premature arborization by RGC axons that originate in the nasal retina, while those that originate in the posterior retina appear unaffected . In pbx2/4 null embryos, hmx4 is down regulated during early eye patterning, and thus may also play a role in phenotypes observed in eyes with reduced pbx gene function, such as a lack of RGC axon outgrowth.
Given the profound changes in gene expression, it is surprising that laminar structure and positioning of the optic nerve is relatively normal without Pbx2/4 function. As such, there is no evidence for overt dorsal/ventral patterning defects or improper cell differentiation. We believe a partial answer to this paradox lies in the data from the 28 hpf microarray, in which each of these Pbx-dependent transcripts has more normal levels of expression (data not shown). We confirmed these results with in situ analysis, showing that changes in aldh1a2 and tbx5 expression are more subtle, while the expression of hmx4 is near wild-type levels at 24 hpf, indicating that Pbx2/4 are essential for proper initiation of these transcripts, but that other factors are capable of regulating their transcription in the absence of Pbx2/4. At 24 hpf, other pbx genes, including pbx1 and pbx3.1, are expressed in the zebrafish embryo. As such, these Pbx proteins or other transcription factors might also regulate eye patterning genes at 24 hpf.
Although not identified in our microarray screen for Pbx dependent transcripts, we analyzed the expression of otx2, which in wild type embryos, is expressed throughout the optic vessicle early in development , but is restricted to the RPE between 22–24 hpf. otx2 is also strongly expressed in the optic tectum, and could therefore be responsible for axon guidance through either eye or tectal patterning. We noticed an expansion of expression to include the entire eye in pbx2/4 null embryos at 24 hpf, indicating that these embryos are unable to restrict the expression of otx2 as development proceeds. A loss of retinoid signaling in mice leads to reduced expression of tbx5 and expanded expression otx2 throughout the retina, placing both genes downstream of aldh1a2 . This provides a possible signaling mechanism for the expansion of otx2 in pbx2/4 null zebrafish whereby Pbx is required for expression of aldh1a2, which, in turn, is required for the restriction of otx2 to the RPE. As restriction of otx2 expression is required for specification of the RPE layer, it is logical that pbx2/4 null embryos would have defects in the specification of RPE cells. In both lzr and pbx2/4 null embryos, the RPE is thickened in a subtle, but reproducible manner. Thus it is likely that the ectopic expression of otx2 in pbx2/4 null embryos leads to the thickening of the RPE, but aberrant expression of other retinal transcripts may also be involved.
Many Pbx functions, such as hindbrain patterning and blood development, are known to involve Meinox binding partners. Meinox proteins can stabilize Pbx proteins, and are often essential for Pbx function through cooperative DNA binding. [1, 9]. Meis1 knockout mice and Prep/Pknox mutants have morphological defects in retinal formation including an inappropriately positioned optic nerve . The position of the optic nerve implies that Meis1 is required for proper dorsal-ventral patterning of the retina, although marker analysis has yet been performed. To create zebrafish with reduced Meis1.1 function, we injected embryos with meis1.1 translation blocking morpholinos. As with pbx2/4 null embryos, knockdown of Meis1.1 function reduced or eliminated aldh1a2 expression. Thus, it is likely that Pbx2/4 and Meis1.1 act cooperatively to regulate at least some aspects of eye patterning. This is in concert with previously published reports that indicate that both proteins are needed to pattern tissues such as the hindbrain [1, 9].
Using our microarray approach, we have also identified reduced tectal expression of fabp7a, nat10, and efna2 in pbx2/4 null embryos. Interestingly, fabp7a and nat10 also show decreased expression in the eye in pbx2/4 null embryos, and thus may be important in patterning both structures in a Pbx dependent fashion. FABP7 has been identified as a downstream target of Pbx/Meinox signaling in humans, whereby increased expression of PKNOX1 (human prep1 homologue) leads to overexpression of FABP7 in fetal trisomy leading to Down's Syndrome .
Further in development, the optic tectum becomes highly polarized in wild type embryos, which is required for proper retinal ganglion cell axon outgrowth and mapping. By 48 hpf, tectal gradients of otx2 and ephrinA5a expression have been disrupted in embryos with reduced Pbx function, indicating its vital role for patterning this tissue. Given these results, it is possible that RGC axon pathfinding defects may result in part from pbx dependent patterning defects in the tectum in concert with pbx patterning defects in the retina. An alternative hypothesis would indicate that forebrain patterning, specifically via sonic hedgehog (shh) signaling could lead to the observed axon outgrowth defect, as shh has been shown to negatively regulate growth cone movement . This is not the case in pbx2/4 null embryos as no changes in shh expression, nor two of its downstream target genes titf1a and titf1b, are observed (data not shown).
It has recently been reported that a knockdown of gdf6a, a growth factor of the BMP family, results in smaller eyes in both zebrafish and frogs [28, 37]. Furthermore, the expression of marker genes in the dorsal/temporal domain of the retina is dependent on Gdf6a function. We have expanded on the marker analysis in gdf6a morphants to include aldh1a2 and tbx5, which are both eliminated in gdf6a morphants. Moreover, gdf6a expression is strongly reduced in pbx2/4 null embryos, indicating that gdf6a acts genetically downstream of both pbx, and is essential for patterning the dorsal/temporal retina. As similar phenotypes are seen with ectopic expression of FGF3/8 in the temporal retina , it is likely that FGFs and GDFs represent opposing signals to pattern the nasal and temporal domains of the retina, respectively. Given that our data suggest that Pbx proteins are genetically upstream of gdf6a expression in the retina, it appears that Pbx may play an important role in regulating the Fgf-Gdf antagonism that leads to correct retinal patterning.
The results presented support a model where aberrant eye and tectal patterning in pbx2/4 null embryos leads to RGC axon outgrowth errors. Eye patterning in the dorsal-temporal domain of the retina involve the function of meis1.1 and a downstream growth factor, gdf6a.
Zebrafish strains, morpholinos, and in situhybridization
The mutant pbx4 allele (b557), also known as lazarus, (lzr), was identified by altered expression patterns of egr2b (krox20) in the developing hindbrain, and has been described previously . For the microarray experiments, pbx2/4 null embryos were created by injecting pbx2 (CCGTTGCCTGTGATGGGCTGCTGCG) translation blocking morpholinos into one-cell maternally and zygotically mutant lzr embryos (mz lzr). Germ line transplantation was used to create mz lzr embryos and has been described previously . For all other experiments, pbx2/4 null embryos were created by injecting pbx2 and pbx4 (AATACTTTTGAGCCGAATCTCTCCG) translation blocking morpholinos into one-cell zygotically mutant lzr embryos. These embryos are phenotypically indistinguishable from mz lzr embryos injected with pbx2 morpholinos. Approximately 3 nl of pbx2/4 morpholino was injected at a concentration of 1 mg/ml. To create a similarly mispatterned embryo for microarray studies, we injected lzr embryos with morpholinos directed against hoxb1a (GGAACTGTCCATACGCAATTAA) and hoxb1b (AATTCATTGTTGACTGACCAAGCAA and ACCAAGCAAAATTGATTAAGCAGGG).
For gdf6a knockdown, a splice blocking morpholino (GCAATACAAACCTTTTCCCTTGTCC) was utilized. This morpholino results in the production of gdf6a messenger RNA containing its lone intron. This allows for normal maternal gdf6a function, as gdf6a is required for patterning the early embryo, but inhibits zygotic gdf6a function prior to eye development. As high levels of necrosis are observed in gdf6a morphants, gdf6a morpholinos were co-injected with a p53 translation blocking morpholino (GCGCCATTGCTTTGCAAGAATTG) . To block meis1.1 function, we injected a translation-blocking morpholinos (GTATATCTTCGTACCTCTGCGCCAT) into 1-cell zebrafish embryos. To rescue the meis1.1MO phenotype, we injected approximately 200 pg of 6X-myc tagged meis1.1 mRNA synthesized in vitro using the Ambion SP6 mMessage machine kit.
Whole mount in situ hybridization analysis was performed essentially as previously described , with the following modifications: probes were not hydrolyzed; Proteinase K treatment (10 ug/ml) was performed for 30 seconds (tailbud- 10 somite embryos), 3 minutes (18–24 hpf embryos), and 20 minutes (embryos between 24–48 hpf). Embryos were manually deyolked, and photographed using a Zeiss AxioImager Z1 compound microscope and an Axiocam HR digital camera or on an Olympus steroscope with a Qimaging micropublisher camera. Embryos were raised at 25–31°C and staged according to published staging hallmarks .
cDNA microarray analysis
For each microarray hybridization, target cDNA was synthesized from either mz lzr, or lzr embryos injected with either pbx2 or hox-1 morpholinos, respectively. cDNA was generated from RNA templates isolated at both 18 hpf and 28 hpf. cDNA was labeled with either Cy3 or Cy5, and hybridized to microarrays containing 8448 cDNA probes, including 768 controls spots (e.g. negative, positive, and normalization controls). An Axon scanner (Molecular Devices) and Genepix software (Molecular Devices) were used to determine the intensities of each spot and normalize the data. All experiments were conducted in biological duplicate while simultaneously switching fluorescent labeling compounds.
Identification of hmx4
A previously uncharacterized EST clone was identified by microarray analysis as having reduced expression in pbx2/4 null embryos. in situ analysis identified expression in the lens and dorsal retina. The clone was sequenced and genomic location identified in zebrafish, and compared to sequences in other organisms. The EST (DY55561.1), is highly similar to that of hmx3 in zebrafish (55% amino acid identity), and is also highly similar to chick soho1 (59% amino acid identity). Based on the homology of the homeodomain and genomic organization, we have named this gene hmx4.
Eye cross sections
Wild type and pbx2/4 morpholino injected embryos were grown to 5 dpf, then fixed in 4% PFA. Embryos were embedded in Spurr's Resin for 1 μm ultramicrotome sectioning with a diamond blade, and stained with Richardson's blue for visualization of retinal layers. Sections were visualized using Zeiss Axioimager Z1 coumpound microscope and photographed with Zeiss Axiocam HR. Eye retinal pigmented epithelium and total eye area were quantified using ImageJ software.
DiI and DiO RGC labeling
Two lipophylic dyes, DiI and DiO (Molecular Probes), were used to label retinal ganglion cell axons. DiI was dissolved in dimethylformamide (DMF), at a concentration of 12.5 mg/ml. DiO was dissolved in chloroform at a concentration of 12.5 mg/ml. Embryos were raised in 0.0015% phenylthiourea (PTU) to inhibit pigment formation and fixed in 4% paraformaldhyde at 3.5 and 5.5 days. Embryos were mounted dorsal side up in 1% in low melting point agrose for injections. DiI was injected into the nasal domain of the right eye, while DiO was injected into the temporal domain of the right eye. Embryos were removed form the agrose and incubated in PBS 0.01% tween20 overnight at 28°C. Embryos were flat mounted in 70% glycerol, dorsal side up. Imaging was accomplished with a SP2 confocal microscope (Leica) using 20X objective.
The acetylated tubulin antibody (Sigma-Aldrich) staining protocol has been published previously . Briefly, zebrafish embryos (3 dpf) were fixed in Dent's fix (80% methanol/20%DMSO), and primary antibody incubation was completed overnight at 4°C (1:500) after blocking in PBS-DBT (1% DMSO/1% BSA/0.5% tween-20, 10% goat serum). After 5 washes in PBS 0.5% tween, a secondary antibody (anti-mouse Alexaflour 546, Molecular Probes, 1:1000), was added and incubated overnight at 4°C.
For zpr-1 (1:200) staining, embryos were fixed in 4% PFA overnight at 4°C. Embryos were blocked in PBS 2% goat serum, 2 mg/ml BSA, 0.1% triton X-100. After washing in PBSDTT, a secondary antibody (anti-mouse Alexaflour 488, Molecular Probes) was diluted 1:1000 and incubated overnight at 4°C. Embryos were mounted in 70% glycerol for imaging on a SP2 confocal microscope (Leica) using both 20X and 40X (oil) objective.
We would like to thank Chi-Bin Chien who kindly provided his protocol for retinal axon labeling. We would also like to thank Stephen Ekker for sharing his unpublished data regarding the use of p53 morpholinos to reduce morpholinos induced cell death. This work was supported by grants from Alberta Ingenuity (TE, AJW), NSERC (CF, TE, and AJW), NIH (JN and CBM), HHMI (CBM), and CIHR (AJW). Andrew Waskiewicz is a Canada Research Chair and Alberta Ingenuity New Faculty.
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