The role of Zic transcription factors in regulating hindbrain retinoic acid signaling
© Drummond et al.; licensee BioMed Central Ltd. 2013
Received: 15 April 2013
Accepted: 5 August 2013
Published: 12 August 2013
The reiterated architecture of cranial motor neurons aligns with the segmented structure of the embryonic vertebrate hindbrain. Anterior-posterior identity of cranial motor neurons depends, in part, on retinoic acid signaling levels. The early vertebrate embryo maintains a balance between retinoic acid synthetic and degradative zones on the basis of reciprocal expression domains of the retinoic acid synthesis gene aldhehyde dehydrogenase 1a2 (aldh1a2) posteriorly and the oxidative gene cytochrome p450 type 26a1 (cyp26a1) in the forebrain, midbrain, and anterior hindbrain.
This manuscript investigates the role of zinc finger of the cerebellum (zic) transcription factors in regulating levels of retinoic acid and differentiation of cranial motor neurons. Depletion of zebrafish Zic2a and Zic2b results in a strong downregulation of aldh1a2 expression and a concomitant reduction in activity of a retinoid-dependent transgene. The vagal motor neuron phenotype caused by loss of Zic2a/2b mimics a depletion of Aldh1a2 and is rescued by exogenously supplied retinoic acid.
Zic transcription factors function in patterning hindbrain motor neurons through their regulation of embryonic retinoic acid signaling.
During development, the vertebrate hindbrain is transiently divided into a series of lineage-restricted segments, termed rhombomeres, through the expression of distinct transcription factors. Notably, anterior-posterior patterning and segmentation of the hindbrain is critical in appropriately specifying neuronal cell types [1–5]. The identity of each hindbrain segment is regulated by the Hox family of homeobox transcription factors, the anterior expression limits of which correlate precisely with rhombomere boundaries [6–18]. The correct complement of hox genes expressed within each hindbrain segment specifies the identity of cells within that segment by activating regional expression of cell migration and axon guidance molecules. Blocking the functions of Hox proteins or their Pbx (Pre-B cell leukemia) and Meis (Myeloid ecotropic virus integration site) cofactors within the hindbrain leads to changes in rhombomere identity and corresponding defects in cranial motor neuron migration and axon guidance [6, 17, 19–21].
The vitamin A-derived morphogen retinoic acid (RA) regulates anterior-posterior patterning of the neural tube, including defining regional identity of hindbrain segments [22–28]. For example, vitamin A-deficient quail embryos lack posterior rhombomeres r4-r8 [24, 25]. Maintaining the precise level of retinoic acid is critical, with increased levels known to result in teratogenic defects of the forebrain, heart, and eyes [24, 29]. In the hindbrain, segmentation defects associated with changes in retinoic acid are attributed to alterations in hox gene expression [9, 27, 30–33]. For example, an increase in retinoic acid levels causes expansion of the posterior hindbrain hox-4 expression [31, 32], while a deficiency in retinoic acid causes an embryonic loss of hox-1, hox-3, and hox-4 paralog expression domains [16, 17, 26, 28].
Regional specificity of retinoic acid signaling is achieved in part through restricted domains of Retinaldehyde dehydrogenase proteins (Raldh, encoded by the aldh1a gene family), the enzymes that catalyze the rate-limiting step in RA synthesis [34, 35]. Pharmacologic blockade of Raldh activity using diethylaminobenzaldehyde (DEAB) results in ablation of the posterior hindbrain, a phenotype that is highly analogous to the vitamin A-deficient quail [26, 36]. The heme-thiolate family of cytochrome p450 type 26 enzymes (cyp26a1/b1/c1) hydroxylate RA, a modification that targets it for degradation [37, 38]. The forebrain, midbrain and anterior hindbrain express cyp26 genes, thereby blocking RA signaling in these regions [39–43]. The combined activity of posteriorly expressed aldh1a with anterior-specific cyp26 genes creates a defined zone of RA signaling within the presumptive hindbrain. RA activity is mediated intracellularly by two nuclear receptor families, retinoid-X-receptor (RXR) and retinoic acid receptor (RAR) [44–46]. Ligand-bound heterodimeric RXR:RAR complexes activate transcription of genes containing retinoic acid response elements (RAREs). Analysis of conserved non-coding elements surrounding hox-1 and hox-4 paralogs has identified RAREs that are essential to rhombomere-specific expression of hox-1/hox-4 genes in the hindbrain [11, 31, 34, 47, 48]. In support, alterations in RA levels result in profound defects to hox-1 and hox-4 gene expression domains [11, 31, 34, 47, 48].
Although the role and requirement of retinoic acid metabolism genes during embryogenesis has been extensively studied, the factors acting to initiate and maintain expression of RA metabolism genes remain largely unknown. Within vertebrates, transcription factors from the Zic (Zinc Finger of the Cerebellum) family of transcription factors are dynamically expressed in partially overlapping regions of the neural tube, indicative of a role in neural development. Recent evidence suggests a connection between Zic transcription factors and the retinoic acid signaling pathway: Maurus et al. demonstrated that loss of zebrafish Zic1 causes a decrease in presumptive forebrain expression of cyp26a1 and an increase in RA signaling as detected by RARE:eGFP transgenics . Further, mutations in human ZIC2 result in holoprosencephaly (HPE), a forebrain defect where the cerebral hemispheres fail to separate during development [50, 51] and HPE phenotypes have been connected to aberrant RA signaling, thus providing a plausible link between Zic2 and retinoic acid metabolism [29, 36, 52, 53]. Based on these observations, we tested the hypothesis that Zic transcription factors play a key role in the initiation and maintenance of RA metabolism gene expression during zebrafish embryogenesis. The data presented here demonstrate that zebrafish zic2 genes act upstream of retinoic acid metabolism and suggest a novel regulatory interaction between Zic2a and Zic2b transcription factors and the RA-synthesizing gene aldh1a2. Further, we show that Zic2 signaling is necessary for proper hindbrain patterning.
Results and discussion
Zic transcription factors are expressed during the initiation of RA metabolism genes
Zics act upstream of early retinoic acid metabolism genes, cyp26a1 and aldh1a2
Zic depletion causes mild alterations to retinoic acid-responsive genes and hindbrain patterning
We took advantage of manipulating RA levels to further examine the regulatory loops present in the early zebrafish embryo. Given that zic2a2b morpholinos cause a loss in cyp26a1 expression, it is plausible that this causes an indirect effect on the aldh1a2 domain. To test this, we examined expression of aldh1a2 in mutants lacking cyp26a1. Notably, aldh1a2 levels are unaffected in cyp26a1(giraffe) mutants, arguing against a scenario whereby the aldh1a2 reduction in zic2a2b morphants occurs because of reduced hydroxylation of RA by Cyp26a1 (Figure 5M, N).
Zic2a2b depletion reduces retinoic acid signaling
Vagal neurons are sensitive to alterations in retinoic acid levels
Zic2a2b knockdown causes loss of vagal neurons
We have identified a novel regulatory mechanism between Zic2a2b transcription factors and early retinoic acid signaling levels. We propose a model whereby Zic2a and Zic2b act upstream of both retinoic acid synthesis (aldh1a2) and degradation (cyp26a1) genes in an early embryo. We see a persistent reduction in retinoic acid signaling in Zic2a2b morphants at 26 hpf that is similar to that observed in embryos depleted of the main retinoic acid synthesis enzyme, Aldh1a2. Consistent with reduced retinoic acid levels, Zic2a2b knockdown results in reduced vagal motor neuron formation within the posterior hindbrain and spinal cord. This phenotype is nearly identical to Aldh1a2-depleted embryos and is rescued by exogenous retinoic acid treatment.
While the role and requirement for retinoic acid metabolism genes has been well studied, less is known about the upstream regulators of these factors. During mouse development, Hoxa1-Pbx1 (Pre-B-cell leukemia homeobox 1) complexes directly regulate Raldh2 transcription within mesodermal tissue . Additionally, deficiencies in zebrafish Tgif and Hmx4 cause defects associated with reduced retinoic acid levels and reduced aldh1a2 transcription [36, 67]. Interestingly, Tgif-depletion also results in a concomitant reduction in cyp26a1 mRNA levels, similar to defects observed in Zic-depleted embryos. As expected, the Cyp26a1 murine mutant has defects associated with increased retinoic acid levels . More notably, rescue of these defects is observed with heterozygous disruption of Aldh1a2 in a Cyp26a1 murine mutant, suggesting co-dependency between these genes, where less retinoic acid degradation is necessary when retinoic acid synthesis is reduced . Thus, the fact that the transcription factors Zic2a and Zic2b regulate expression of both retinoic acid and synthesis genes strongly suggests that they play an important role in regulating retinoic acid levels during early embryogenesis.
Holoprosencephaly (HPE) is the most common forebrain birth defect, occurring when the cerebral hemispheres fail to separate. Mutations in human ZIC2 and TGIF are known to cause HPE. Our results from zebrafish studies provide evidence that both of these genes function to regulate RA metabolism . Although both Zic and Tgif genes are implicated in regulating multiple signaling pathways, it is now plausible that altered levels of RA predispose embryos to HPE.
Danio rerio were maintained in accordance with published protocols . Zebrafish adults and embryos were maintained at 28.5°C. Embryos were raised in embryo medium (EM) with 10 ml/l Penicillin/Streptomycin (Sigma) added to prevent bacterial growth, and with phenyl-thiourea (0.003%) used to prevent pigmentation after 24 hpf. The stage of developing embryos was assessed using reported morphological guidelines (Kimmel et al., 1995). Wild type AB, Tg(isl1:eGFP), and Tg(12xRARE-ef1:eGFP) strains were used as described [61–63]. Animal protocols were approved by the University of Alberta’s Animal Care and Use Committee-Biosciences with protocol #427.
Splice-blocking Zic2aMO [ 2 ng/ml; 5’-CTCACCTGAGAAGGAAAACATCATA-3’; ], splice-blocking Zic2bMO [2 ng/ml; 5’-CACGAATTGAAATAATTACCAGTGT-3’], splice-blocking Zic3MO [3 ng/ml; 5’-GGAATTTAATTTCCTTACCTGTGTG-3’], translation-blocking Aldh1a2MO [2 ng/ml; 5’-GCAGTTCAACTTCACTGGAGGTCAT-3’; [54, 70]], translation-blocking Cyp26a1MO [2 ng/ml; 5’-CGCGCAACTGATCGCCAAAACGAAA-3’; ], and p53MO morpholinos [1 ng/ml] were designed to produce a non-functional protein . Embryos were injected at the one-cell stage and allowed to develop until the desired time-point for phenotypic characterization.
Examination of retinoic acid signaling
To manipulate retinoic acid levels in developing zebrafish embryos, we used pharmacological treatment (1 or 5 nM of all-trans retinoic acid (Sigma); 1 or 5 μM diethylaminobenzaldehyde (DEAB, Sigma)) or morpholinos (Cyp26a1 or Aldh1a2) that target enzymes known to regulate degradation and synthesis of endogenous RA. Embryos were treated at approximately 50% epiboly with DEAB or RA in Embryo Medium, with DMSO and ethanol used as vehicle controls. Embryos were manually dechorionated at 26 hpf and, maintaining treatment concentration, media was changed once per day. All treatments were carried out in 60 mm petri dishes each containing approximately 40 embryos grown at 28.5°C. Tg(12xRARE- ef1a:eGFP) transgenic zebrafish allow visualization of RA-signaling levels by assaying fluorescence (or by in situ hybridization for eGFP mRNA).
Whole mount in situhybridization
mRNA in situ hybridization procedure is based on previously published methods . Probes were synthesized via a PCR-based approach whereby primers are designed to amplify the 3’ untranslated region (primer sequences available on request) . Embryos of desired stage were fixed in 4% paraformaldehyde (PFA), permeabilized in Proteinase K (10 μg/ml), re-fixed in 4% PFA and pre-hybridized for 2 hours at 65°C. 100 μg of probe was added and hybridization allowed to proceed overnight. Unbound probe was removed with three 20-minute high-stringency washes (0.2× SSC + 0.1% Tween-20; 0.1× SSC + 0.1% Tween-20; 0.1× SSC + 0.1% Tween-20). Embryos were first incubated in Blocking Solution (2% Sheep Serum + 2 mg/ml Bovine Serum Albumin in PBST) for one hour at room temperature, or 4°C overnight, before transfer into primary antibody (Blocking Solution + 1/5000 dilution of sheep anti-DIG-AP-FAB fragments antibody (Roche)) for two hours at room temperature, or 4°C overnight. Embryos are washed out of antibody using five-fifteen minute PBST washes. The coloration reaction is performed with either nitro-blue tetrazolium (NBT)/bromo-4-chloro-3-indolyl phosphate (BCIP) stock (Roche) in Coloration Buffer or, for embryos before bud stage, with BM purple (Roche). Coloration was stopped via 100% methanol/0.1% Tween-20 washes. Deyolked embryos were dehydrated in 50% glycerol, then 70% glycerol before mounting. Images are taken on Zeiss Axio Imager.Z1 using Axovision SE64 Rel.4.8 software. Embryos in early development (6-14 hpf) or with yolk attached are photographed on Olympus SZX12 stereoscope using QImaging micropublisher camera.
After in situ hybridization, embryos were post-fixed for 2 hours at room temperature in 4% paraformaldehyde. Embryos were then rinsed in PBS, dehydrated through a series of ethanol washes, and incubated over one hour in two rinses of JB-4 infiltration solution (made as per manufacturer’s instructions, Polysciences Inc.). After transferring the embryos to molds, the infiltration solution was replaced with JB-4 embedding media and the blocks were left to harden at room temperature overnight. Seven μM sections were cut on a Leica RM2235 microtome and images were captured on a Zeiss Axio Imager.Z1 using Axovision SE64 Rel.4.8 software.
Quantitative real-time PCR
Quantitative Real-Time PCR was used to quantify in vivo mRNA levels to ascertain aldh1a2 gene expression in morpholino-injected embryos. Primers were designed using Roche Universal Probe Library for Zebrafish [Forward 5’-AACCACTGAACACGGACCTC-3’ and Reverse 5’-ATGAGCTCCAGCACACGTC-3’]. After RNA isolation (Ambion RNAqueous), DNA was removed by DNAseI digestion (19 μl DEPC-treated H2O, 10 μl 10× DNAseI buffer, 1 μl DNAseI (Ambion)) for 30 minutes at 37°C. RNA was further purified using Qiagen RNeasy columns and cDNA synthesized using AffinityScript qPCR cDNA Synthesis kit (Ambion) by the recommended protocol. Quantitative RT-PCR and primer validation was carried out as previously described [74, 75]. For primer validation, control (AB) cDNA was isolated and used to create a dilution series: 1/8, 1/16, 1/32, 1/64, 1/128, 1/256. Ambion Brilliant II qPCR kit with thermocycler conditions 40× denaturation at 95°C for 30 seconds; annealing at 55°C for 1 minute; and extension at 72°C for 30 seconds. Finally, relative gene expression level was determined using the comparative Ct method (2−∆∆Ct method) and unpaired t-test calculations for significance [74, 75]. Reaction mixtures and thermocycler program were identical to primer validation protocol; a 1/16 dilution of experimental and control cDNA was used with each primer pair. Reactions were completed with at least 7 technical replicates from RNA isolated from 50 embryos, comparing to ef1a as endogenous control.
Imaging of cranial motor neurons
The Tg(isl1:eGFP) transgenic line allows visualization of a subset of motor neurons including those that are located within midbrain and hindbrain: oculomotor, trochlear, trigeminal, facial, glossopharyngeal, and vagal neurons . For fluorescent analysis, embryos were fixed with 4% PFA for 4 hours at room temperature. Yolks were removed manually and embryos dehydrated in 50% and 70% glycerol before mounting and imaging using a Zeiss LSM 510 confocal microscope and Zeiss Zen software. Image preparation and analysis (quantification of vagal neuron fluorescent area and length of vagal domain) was done with ImageJ software. To calculate vagal area, Z-stacked images were converted to 8-bit color and scale was set to correspond with that of image (pixel length 1024 or 2048, length of field of view 319.93 μm). Image threshold was set capturing the greatest number of neurons while keeping noise low. Regions of fluorescence were selected and the area calculated with numerical information transferred to Microsoft excel, where totals and averages were calculated. Statistical significance was determined using ANOVA followed by Tukey’s HSD post hoc test, or by an unpaired t-test.
This research was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) award to AJW. Additional funding was obtained from Research, Scholarly Activity and Creative Achievement Fund (internal award from MacEwan University) awarded to LBP. DLD was the recipient of a Banting and Best scholarship from CIHR. LGS is the recipient of a Canadian Graduate Scholarship-Master’s Award (NSERC). JCH is the recipient of an Alberta Innovates-Health Solutions postdoctoral fellowship. AJW is the recipient of a Canada Research Chair Award.
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