miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2

MicroRNAs (miRNAs) are 19-25 nucleotide, single stranded RNAs that regulate gene expression by binding target mRNAs, modulating their translation and turnover.^1-3  However, the biologic role of most individual miRNAs and the targets they modulate remain to be determined.

miRNA expression profiling has been a useful starting point for selecting miRNAs that may influence tissue- or cell-specific processes such as hematopoiesis. High or increasing levels of an miRNA in a specific cell lineage may promote differentiation by repressing transcripts whose persistence or presence would impede cellular commitment (eg, transcripts that promote an alternative cell lineage). On the other hand, high miRNA levels may act to preserve the cellular status quo, as has been proposed in a model of miRNA action in CD34⁺ hematopoietic progenitor cells (HPC).⁴  Conversely, miRNAs of low or falling abundance may be acting to release repression of a set of transcripts necessary to drive changes of cellular state. In view of these various possible mechanisms of action, functional studies are required to understand how individual miRNAs actually influence biologic processes and to determine the molecular mechanisms by which each one acts.

Erythropoiesis is a process of progression from stem cells, characterized by pluripotency and self-renewal, through intermediate states retaining proliferative capacity but of increasingly restricted potential, through to lineage-specific cells that undergo a morphologically recognizable pattern of terminal differentiation. This process requires cells to possess mechanisms for progressive modification of their transcriptomes to acquire new capabilities and to discard residual now-redundant or deleterious transcripts from a former cellular state. miRNAs provide a potential mechanism for achieving this.

Several miRNAs are down-regulated as erythropoiesis proceeds, removing constraints that act to preserve a more immature stem or progenitor cellular phenotype. In erythropoietic-driven cultures of human CD34⁺ HPCs, miR-221 and miR-222 are down-regulated.⁵  This contributes to erythroid expansion by derepression of at least one of their targets, KIT, through a mechanism verified by HPC transduction with miRNA duplexes or lentiviral overexpression vectors.⁵  Likewise, miR-24 has higher expression in HPCs and is down-regulated during erythroid differentiation.^4,6  miR-24 represses HPC erythroid differentiation in part through repression of its target, the type I activin receptor (ALK4), thereby gate-keeping the pro-erythropoietic action of activin (in conjunction with erythropoietin) mediated by this receptor.⁶ 

In contrast, several other miRNAs are strongly up-regulated as erythrocytes mature. Broad expression profiling surveys have recurrently identified miR-144 and miR-451 for their high erythropoietic-restricted expression in several species, including zebrafish,^7,8  mice,^9-11  and humans.^11-15  In mice, they arise from a single transcript.⁹  Overexpression or loss of expression of miR-451 in murine erythroleukemia (MEL) cells promoted or impaired erythrocyte differentiation, respectively, assessed by β-globin transcript abundance and hemoglobinization,¹¹  consistent with miR-451 being a positive regulator of erythroid maturation. A recent study has shown the murine miR-144/451 locus is transcriptionally regulated by Gata1.⁹  Loss of miR-144/451 expression in the zebrafish gata1-mutant vlad tepes was interpreted as further evidence for Gata1-dependent activation of miR-144/451 locus.⁹  This study also examined loss and gain of miR-451 function in zebrafish embryos, concluding that miR-451 was required for either the maintenance/survival or late-stage maturation of committed erythrocytes.

We independently examined the function of the miR-144/451 in zebrafish hematopoiesis by an alternate experimental approach. We exploited a new zebrafish mutant, meunier (mnr), a nonanemic mutant expressing gata1 normally but found to have miR-144/451–deficient erythrocytes. mnr freed miR-144/451 from their otherwise epistatic relationship with gata1 and provided a unique, genetically stable, miR-144/451–deficient but erythrocyte-replete background for experimentation. Using rigorously validated reagents, we demonstrate that miR-451 (but not miR-144) functions to accelerate the kinetics of erythrocyte maturation in zebrafish. Furthermore, we validated gata2 as one bona fide target of miR-451 in zebrafish, and demonstrate that miR-451–mediated clearance of gata2 is a crucial influence on the rate of zebrafish erythrocyte maturation.

Zebrafish strains used were: AB*, cloche (clo^m39),¹⁶  Tg(fli1:GFP)^y1,¹⁷ meunier (mnr^gl9, a novel mutant isolated in our ethylnitrosourea mutagenesis screen¹⁸  for its lack of myeloperoxidase [mpx] expression) and Tg(mpx:EGFP)ⁱ¹¹⁴.¹⁹  Fish were housed in the Ludwig Institute for Cancer Research Aquarium using standard husbandry practices. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee. Zebrafish gene, protein, and mutant naming follows the recommended conventions.²⁰ 

Fertilized 1- to 2-cell embryos were microinjected with 1 to 2 nL synthetic mRNA (50 μg/μL in H₂O), miRNA morpholino oligonucleotides (MOs; 100-500 μmol/L in H₂O; Gene Tools, Philomath, OR), control or gata2 MOs²¹  (130 μmol/L in H₂O; Gene_Tools) and synthetic miRNA duplexes (2-20 μmol/L in H₂O; Sigma-Proligo, St Louis, MO), traced where appropriate by mixing 1:1 with 5% rhodamine dextran (in 0.2 mol/L KCl). We customarily delivered different nucleic acid reagents by separate microinjections to avoid ex vivo mixing. As summarized schematically (Figures 3,Figure 4,Figure 5–6 and Figure S8, available on the Blood website; see the Supplemental Materials link at the top of the online article), we selected reagent combinations according to experimental purpose. Some assays required optimization and the specific reagent concentrations used were (1) miRNA duplex/mRNA 3′untranslated region (UTR) validation (Figure S8B,D,E): 50 μg/μL RNA, 2 μmol/L synthetic miRNA duplex; (2) MO validation (Supplementary Figure 8C,F,G): 50 μg/μL RNA, 2 μmol/L synthetic miRNA duplex, 500 μmol/L MO; and (3) miRNA rescue experiments (Figure 3) and gata2 target validation tests (Figures 4,6), 20 μmol/L synthetic miRNA duplex.

Table S1 lists oligonucleotide sequences. The green fluorescent protein (GFP) reporter/sensor constructs were derived from pCS2+GFP-F by standard cloning techniques²²  and linearized with NotI for in vitro transcription of capped mRNA using the mMESSAGE mMACHINE kit (Ambion, Austin, TX). Triplicate miRNA binding sites were constructed by annealing complementary oligonucleotides. The gata2 miR-451 binding sites were mutated by sequential inverse polymerase chain reaction (PCR) and ligation via an acquired restriction enzyme site. All constructs were validated by sequencing.

Whole mount in situ hybridization (WISH) was performed by using standard techniques and in vitro transcribed digoxigenin- or fluorescein-labeled antisense gata1, gata2, hbbe3, lcp1, and mpx riboprobes^23-26  and 4-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate or fast red for detection. miRNA WISH with locked nucleic acid (LNA) probes (Exiqon, Vedbaek, Denmark) was performed as described previously⁸  and with reference to the manufacturer's instructions, using hybridization at 35°C for miR-144, 50°C for miR-451, and 53°C for miR-206 LNA probes. We used standard techniques for double WISH (Figure 2B), probing first with the hbbe3 riboprobe at 68°C followed by stringent washing and reprobing with the miR-144 LNA probe at 35°C. For adult tissues, dissected organs were diced (1 mm fragments) and processed for WISH as for embryos, with proteinase K (20 μg/mL) treatment for 30 minutes. Organ fragments were aligned in 1% agarose, sectioned, and counterstained with nuclear fast red.

The miR144/451 locus RT-PCR used a Superscript III One-Step reverse transcription (RT)-PCR kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, primers as in Table S1, 30 amplification cycles at 94°C, 60°C, and 68°C for 15, 30, and 30 seconds, respectively, and displaying PCR products by 2% agarose gel electrophoresis.

Low-power images were collected using a Nikon SMZ1500 or Nikon 90i fluorescence microscope equipped with a DXM1200c camera and Nis-Elements AR software (Nikon, Tokyo, Japan), and high-power images with a Nikon Optiphot-2 microscope with a Zeiss AxioCam MRc5 digital camera and AxioVision AC (Release 4.5) software (Zeiss, Welwyn Garden City, United Kingdom). Images were imported into Adobe Photoshop CS2 9.0.2 or Illustrator CS2 12.0.1 (Adobe Systems, Mountain View, CA) for orientation and figure preparation. To ensure valid documentation of comparative assessments of fluorescence intensity, arrays of multiple embryos were photographed together in a single image.

Embryonic zebrafish erythrocytes were collected by transecting tails of 6 to 8 embryos in 600 μL of balanced salt solution with 5 mmol/L ethylenediaminetetraacetic acid (EDTA) and 4% fetal calf serum. Cytospin slides were prepared, stained with May-Grünwald/Giemsa stain, and examined at high power. Nuclear and cytoplasmic areas of 20 to 30 randomly selected erythrocytes were measured using either the outline tool of AxioVision AC software or NIS Elements software, and the nuclear/cytoplasmic (N:C) area ratio was calculated. O-dianisidine staining for hemoglobin was as described previously.²³ 

clo embryos were recognized as a Mendelian proportion (Figure 1E). mnr embryos were recognized at 48 hours postfertilization (hpf) by their characteristic syndrome of small eyes, cerebral degeneration, and thinner yolk extension. Younger mnr embryos were PCR-genotyped at the closely linked (3 centimorgans) simple sequence length polymorphism (SSLP) marker z3984 (oligonucleotides, Table S1; 10 μL reactions; Taq polymerase [New England Biolabs, Ipswich, MA] with supplied buffer; 40 cycles at 95°C, 60°C, and 72°C for 20, 20, and 30 seconds, respectively; PCR products were separated by 2% agarose gel electrophoresis).

Single images are representative of at least 30 embryos or representative of x of y embryos as stated. Quantitative data derives from at least 3 independent experiments; descriptive statistics are either medians or mean plus or minus SD of data for n individuals, or mean plus or minus SE of n independent experiments. Prism 4.0c (GraphPad Software, San Diego, CA) was used for analytical statistics, using unpaired, 2-tailed t tests for normally distributed continuous variables. P less than .05 indicated statistically significant difference.

miR-144 and miR-451 form a clustered pair in the genome of zebrafish, separated by only 65 nucleotides (Figure S1). We demonstrated, using RT-PCR, that miR-144 and miR-451 were present on a single transcript (Figure 1A). To determine the anatomical and temporal constraints on their direct function, we examined their expression pattern by whole-mount in situ hybridization. miR-144/451 expression in zebrafish hematopoietic tissues has been previously noted.^7-9  The locus was first expressed at 18 hpf in the cytoplasm of individual erythrocytes in the intermediate cell mass (ICM), the axial hematopoietic tissue of the early zebrafish embryo (Figure 1B,C). As expected, embryonic miR-144 expression overlapped with miR-451 expression (Figure 1A,E; data not shown). Early miR-144/451 expression tracked exactly with other genes expressed in erythroid-lineage cells (eg, the erythroid-determining transcription factor gata1 and globin [hbbe3]^23,26  [Figure 1D,E]) and required the presence of erythrocytes. For example, cloche (clo) is an early hematopoietic/vascular-failure mutant with absent/reduced expression of all known hematopoietic genes.^16,27,28  miR-144/451 expression was absent in clo (Figure 1E), confirming their restricted expression in hemovascular derivatives. The restricted expression of this locus is maintained into adulthood in clusters of cells in the hematopoietic organs (spleen and kidney; Figure 1Fi,ii) and the cytoplasm of circulating erythrocytes (Figure 1Fiii).

The expression of the miR-144/451 locus and other erythroid genes (gata1, hbbe3) was surveyed in a panel of other hematopoietic-failure mutants. In 8 of 9 mutants from the Melbourne Myeloid screen,¹⁸  there was concordance between miR-144/451 locus and other erythroid gene expression. The single exception was meunier (mnr), which showed greatly reduced (but not absent) miR-144/451 expression by WISH and semiquantitative RT-PCR despite normal gata1 and hbbe3 expression (Figure 2A,B; Figure S2) and normal O-dianisidine hemoglobin staining (Figure 2C; Figure S3A).

mnr is an ethylnitrosourea-induced recessive mutant initially identified for its reduced number of myeloperoxidase (mpx)–expressing cells (Figure 2D). Expression of myeloid markers that are less lineage-specific (lcp1, lyz) is reduced, but early hematopoietic transcription factors tal1 and spi1 are normal (Figure S4).²⁵ mnr develops later nonhematopoietic defects, most significantly cerebral degeneration leading to death at 4 to 5 days postfertilization (dpf). However, until approximately 38 hpf, mnr embryos are morphologically indistinguishable from wild-type (WT) siblings (confirmed by longitudinal observation), after which they can be reliably identified by reduced head and eye size (Figure 2C).

The loss of miR-144/451 expression in mnr did not merely reflect globally reduced miRNA expression/processing, because expression of the somite-specific miR-206 was normal (Figure 2A),^8,29  and there was no accumulation of the primary miRNA transcript detected by RT-PCR (Figure S2). Therefore, mnr uniquely dissociates miR-144/451 locus expression from its otherwise tight epistatic relationship to gata1⁹ without being a global miRNA biogenesis mutant and provides an erythrocyte-replete, genetically stable, miR-144/451–deficient background for further experimentation.

Two observations suggest that the miR-144/451–deficiency and mpx-deficiency phenotypes of mnr are not caused by 2 independent mutations. First, offspring from 3 of 3 independent mnr heterozgyote pairs genotyped on the basis of their mpx deficiency phenotype all showed reduced miR-144/451 expression. Second, embryos sorted by either phenotype showed tight linkage to the chromosome 14 SSLP marker z3984 (Figure S5). This tight linkage also means that mnr is not a mutation of the miR-144/451 locus itself, because the miR-144/451 locus is on chromosome 5 (Zebrafish Genome Assembly Zv7³⁰ ).

We hypothesized that mnr erythrocytes may exhibit an abnormal phenotype that would point to the nature of their requirement for miR-144/451, and we therefore carefully scrutinized erythroid development in mnr mutant embryos. During zebrafish development, there is a characteristic change in erythrocyte morphology associated with developmental age.^31,32  Proerythroblasts, characterized by deeply basophilic cytoplasm and large finely granular nucleus, mature through stages displaying an increasingly acidic cytoplasm, progressively denser nuclear chromatin, and a decreasing N:C area ratio despite an overall concomitant reduction in erythrocyte diameter (Figure 2E).

Comparing erythrocyte morphology between mnr and its WT siblings revealed delayed erythroid maturation in mnr. Although evident morphologically (mnr erythrocytes were larger, had more basophilic cytoplasm, and more open nuclear chromatin), the N:C ratio (48 hpf) provided a robust objective numeric variable reflecting this delay that was statistically different between mnr and WT erythrocytes (Figure 2E,F; Figure S6).

The immaturity of mnr erythrocytes was not due to a population skewing effect from loss of mature erythrocytes by apoptosis or from an erythropoietic drive resulting from accelerated erythrocyte destruction, because acridine orange staining failed to demonstrate excess death of ICM hematopoietic cells or later erythroid cells (Figure S7). Overall, mnr shows that miR-144/451 are not essential for erythroid specification, early development including hemoglobinization, and survival but suggests their involvement in promoting erythroid maturation.

To determine whether miR-144 and/or miR-451 absence alone was sufficient to cause erythrocyte immaturity observed in mnr, we used antisense MO knockdown of miR-144 and miR-451.^9,29  The capability and efficacy of each MO to intercept repressive miRNA/3′UTR interactions was demonstrated by their activity in an in vivo reporter assay based on microinjection of combinations of MO, synthetic miRNA duplexes, and mRNA encoding enhanced green fluorescent protein (EGFP) with a 3′UTR containing canonical miRNA target-sites (Figure S8). MO-451 (but not MO-144) resulted in a statistically significant dose-dependent increase in N:C ratio reflecting morphologic erythrocyte immaturity (Figure 3A; Figure S9). However, despite maximal MO doses, this phenotype was not as marked as that observed in mnr. MO-451 specifically affected erythrocyte development; MO knockdown of miR144/451 did not affect neutrophil number (Figure 3B), nor did it recapitulate the nonhematologic mnr phenotypes. A second MO, MO-451B (of the “multiblocking” type, also interfering with miRNA biogenesis), proved toxic at a dose achieving only modest inhibition in the reporter assay (data not shown). MO-451 affected the rate of erythrocyte maturation but did not abort it, because O-dianisidine staining demonstrated wild-type hemoglobin levels at 48 hpf (Figures 3A, S3B).

The selective miR-144/451 deficiency of mnr provided a scenario for testing whether miRNA replenishment was sufficient to rescue erythrocyte maturation in an endogenously miRNA-deficient environment. Overexpression was achieved by microinjecting a synthetic RNA duplex that mimics the endogenous dicer processed product. The activity of synthetic duplexes was confirmed by an in vivo reporter assay (Figure S8). Overexpression of miR-451 (but not miR-144) partially rescued the mnr erythrocyte maturation defect (Figure 3C-E), reflected morphologically and by a statistically significant reduction in the N:C ratio. This indicates that the mnr maturation defect was at least partially due to miR-451 deficiency. In a WT background, overexpression of miR-144/451 did not affect erythrocyte maturation or gross embryo morphology (Figure 3E; Figure S10; data not shown). Furthermore, several nonhematopoietic mnr phenotypes were not altered by miR-144/451 overexpression (data not shown), suggesting that they resulted from a role of the mnr gene product independent of its role in miR-144/451 up-regulation.

Together, these observations indicate a specific requirement for miR-451 (but not miR-144) in promoting erythroid maturation. The MO-451 knockdown and miR-deficient mnr phenotypes indicate a necessity for miR-451 for normal erythrocyte maturation. The partial rescue by miR-451 replenishment in mnr indicates that miR-451 is sufficient to promote erythroid maturation in this sensitized genetic background. In all these assays, miR-451 makes a much more critical contribution to erythrocyte maturation than miR-144.

We used database searching to generate lists of potential mRNA targets of miR-451 that might mediate these biologic effects.³³  miRNA target specificity is conferred by complementary base pairing of its “seed sequence” (nucleotides 2-7) to target mRNAs, and the inhibitory effect is amplified as the number of binding sites in an individual transcript increases.³⁴  As with most miRNAs, miR-451 has numerous potential targets. However, mRNAs encoding transcription factors expressed in the ICM before or during miR-144/miR-451 expression, whose 3′UTR carried 2 or more predicted miRNA binding sites, seemed reasonable candidate targets. Using these criteria, gata2, known to preserve the immaturity of hematopoietic precursor cells,³⁵  stood out among 631 predicted targets as an attractive potential target of miR-451 (Figure 4A).

The validity of this prediction was tested using a reporter assay based on monitoring GFP fluorescence in embryos microinjected with mRNA encoding GFP fused to the gata2-3′UTR (GFP-3′UTR^gata2), in the presence or absence of miRNA duplex (Figure 4B-D). In this assay, a decrease in GFP-fluorescence in the presence of duplex indicated miRNA-mediated repression. Embryos sequentially injected with GFP-3′UTR^gata2 reporter and miR-451 showed suppressed GFP-fluorescence, whereas miR-144 had no effect on fluorescence intensity (Figure 4C). This confirmed that the gata2-3′UTR was targeted by miR-451 but not by miR-144. The 2 predicted miR-451 binding sites in the gata2-3′UTR were validated as bona fide target sequences by further modifications of this assay in which (1) embryos microinjected with mRNA encoding GFP fused to each individual recognition site in triplicate (GFP-3′UTR^{3x site a} and GFP-3′UTR^{3x site b}) displayed strong miR-451–induced suppression of GFP-fluorescence (Figure 4C); (2) miR-451–mediated repression on the gata2-3′UTR was abolished when both seed sequences were mutated (GFP-3′UTR^gata2-mut), shown by comparable GFP-fluorescence of this reporter construct alone and in the presence of miR-451 (Figure 4C,D). Collectively, these data prove the capability of miR-451 to target the gata2-3′UTR via the 2 predicted recognition sequences.

We sought in vivo evidence in support of the hypothesis that miR-451 negatively regulates gata2, which predicts an inverse correlation between the presence of gata2 transcript and miR-451 activity.

First, such an inverse correlation is observed in the wild-type expression dynamics of gata2 and miR-451. In zebrafish, gata2 caudal hematopoietic expression initiates in the lateral-plate mesoderm at 10 hpf.^23,36  These cells give rise to the anterior ICM directly above the yolk extension and the posterior ICM^37,38  distal to the yolk. After 19 hpf, gata2 expression decreases substantially in the anterior ICM but is maintained in the posterior ICM.²³  The dynamics of this change in gata2 expression at 19 hpf coincides with the activation of the miR-144/451 locus in the anterior but not posterior ICM. By 24 hpf, gata2 expression is nearly completely lost in the strongly miR-451 expressing anterior ICM (comparing miR-451, Figure 1E with gata2, Figure 5A).

Second, if miR-451 were involved in the clearance of gata2 transcripts in the anterior ICM, then in a miR-451–deficient environment such as mnr or miR-451 morphant embryos, gata2 transcripts would persist beyond this transition point. Consistent with this hypothesis, although anterior ICM gata2 expression is normally nearly completely down-regulated in WT at 24 hpf, in genotyped mnr embryos it persisted to 27 hpf and in miR-451 morphants was still present at 24 hpf (Figure 5A). The negative controls (WT siblings, miR-144 morphants, and control-MO–injected embryos) all displayed comparable loss of anterior ICM gata2 expression at 24 hpf. Up until 48 hpf, erythrocyte gata2 was not reinstated in mnr, miR-451 morphants, WT, or miR-144 morphant controls (data not shown).

Third, we hypothesized that if mnr erythroid maturation is delayed because of persistent gata2 expression, then gata2 knockdown should rescue the maturation defect of mnr (Figure 5B). Strikingly, MO knockdown of gata2 translation²¹  accelerated erythroid maturation in mnr both morphologically and by a significant reduction in N:C ratio (Figure 5C; Figure S11).

Finally, we sought to determine whether gata2 was the sole miR-451 target responsible for the erythrocyte maturation delay of mnr. This was achieved by using the newly described target-blocking MOs,³⁹  which we submitted to a rigorous efficacy and specificity evaluation. MOs complementary to each of the gata2-3′UTR miR-451 binding sites (sites a or b as in Figures 4A and 6A; designated MO-TB site a and MO-TB site b) were designed to selectively block endogenous miR-451 binding to the gata2-3′UTR, without interfering with the interaction of miR-451 with other target 3′UTRs. The efficacy of these MOs at protecting their respective miR-451 binding site was demonstrated in a set of appropriately designed GFP-fluorescence reporter assays (Figure 6B-D). As a baseline, embryos injected with GFP-3′UTR^gata2 reporter and then miR-451 showed suppressed GFP-fluorescence as previously demonstrated (Figures 4C,6B). In contrast, in the presence of both target blocking MOs (MO-TB-sites a and b), GFP-fluorescence was not suppressed by the presence of miR-451, as expected from blocking the miR-451/gata2-3′UTR interaction (Figure 6B). This protection was not due simply to the MO-TBs binding to the gata2-3′UTR and thereby stabilizing the transcript, because the MO-TB control (designed to bind to the gata2-3′UTR between the miR-451 binding sites) did not block the miR-451 suppression of GFP-fluorescence. Further demonstration of the sequence specificity of the MO-TBs and of their inability to bind miR-451 duplexes themselves came from assays in which the GFP-3′UTR^{3x site a} sensor mRNA was protected only by MO-TB site a but not MO-TB site b, and vice versa (Figure 6C). Together, these evaluations demonstrate that the 2 MO-TBs together block the miR-451/gata2-3′UTR interaction in a sequence-specific manner.

Wild-type embryos microinjected with MO-TB sites a and b together showed prolonged gata2 expression at 24hpf in the anterior ICM (ie, replicating the mnr gata2 phenotype; Figure 6E), providing evidence both for their functionality in vivo and supporting a direct interaction between endogenous miR-451 and gata2 in vivo. However, these embryos did not show altered erythrocyte morphology or N:C ratio compared with control embryos (Figure 6F; Figure S12). This suggests that in mnr and miR-451 morphant embryos, at least one other miR-451 target needs to be dysregulated to impair erythroid maturation.

The specific biologic functions of individual miRNAs are now emerging through reverse genetic studies, revealing important roles in development, physiology and disease, including hematopoiesis.^3,40  Several murine miRNA loci have recently been disrupted by gene targeting with resultant hematopoietic phenotypes (eg, mice lacking miR-155, a lymphoid-restricted miRNA, have defective immune responses^41,42 ; overexpression of miR-155 in murine bone marrow cells drives a myeloproliferative syndrome⁴³ ; mice lacking miR-223, a granulocyte-lineage-restricted miRNA, have granulocytosis and impaired granulocyte function⁴⁴ ).

Zebrafish offer several advantages over mice for the study of miRNA function, particularly in developmental processes. In zebrafish, loss- and gain-of-function studies can be undertaken quickly using transient genetic approaches: overexpression by microinjecting a synthetic RNA duplex and miRNA knockdown by injecting an MO antagonist. Predicted miRNA/transcript interactions can be evaluated using fluorescent reporter assays. Interactions between endogenous miRNAs and their targets can be specifically interrupted by target-blocking MOs. Because these approaches are transient, the efficacy and specificity of reagents can be rigorously evaluated quickly and efficiently in vivo in a manner not possible in mice.

However, in wild-type backgrounds, miRNA knockdown/overexpression in zebrafish appears only occasionally to result in any phenotype,^29,45  and this approach can be technically challenging, particularly to adequately control for spurious effects. A relevant mutant, particularly those that are not epistatic to the pathway of interest, offers advantages such as genetic stability over these transient approaches (eg, the utility of the miRNA biogenesis mutant dicer in elucidating the role of miR-430 in early development).^22,34  However, biogenesis mutants such as this lack numerous miRNAs and have complex later phenotypes^22,46  that complicate discerning the specific roles of later-expressed miRNAs. A mutant selectively deficient in a particular miRNA but retaining the cell type in which it is expressed would be particularly useful.

We exploited zebrafish reverse genetic techniques to examine the function of the erythroid-restricted miRNAs miR-144 and miR-451, showing that miR-451 accelerated the rate of erythrocyte maturation, an action mediated in part by repression of gata2. Our studies were aided by the mutant mnr, which dissociated the otherwise invariable link between the presence of erythrocytes and the presence of these 2 miRNAs. mnr provided an miR-144/451–deficient model that could independently corroborate observations from other loss-of-function studies and provided a genetically stable miR-144/451–deficient background “sensitized” for the study of the function of these miRNAs. Indeed, the subtle and somewhat variable effects of miR-451 MO knockdown on erythrocyte maturation were strongly corroborated by the stronger, more stable, immature erythrocyte phenotype of mnr, and the biologic effects of miR-451 overexpression were discernible only on the mnr background. mnr is not a general miRNA biogenesis mutant, nor is it a mutation of the miR-144/451 locus itself; exactly how the mnr gene product acts as a strong positive regulator of miR144/451 is unknown and will require the identification of the mnr mutation.

A recently published study demonstrated that miR-144/451 expression is directly controlled by GATA1, and this was interpreted as GATA1-dependence, based on a comprehensive analysis of the murine locus promoter, and on the loss of miR-144/451 expression in the zebrafish gata1 mutant vlad tepes.⁹  However, vlad tepes has markedly defective precirculation ICM erythropoiesis with abundant cell death and is “bloodless” at the onset of circulation^32,47 ; hence, we interpret its loss of miR-144/451 expression to largely reflect the loss of the cells expressing these 2 miRNAs. In contrast, mnr expresses ICM gata1 normally and is erythrocyte-replete but miR-144/451–deficient, thus severing the epistatic relationship between gata1-dependent erythrocyte production and miR-144/451 expression. Furthermore, mnr indicates that Gata1 requires at least one other gene product, mnr, to activate the miR-144/451 promoter. The genetic location of mnr on chromosome (Chr) 14 precludes mnr from being either gata1 itself (Chr 11) or several other known or potential components of GATA1-containing transcriptional complexes^30,48 : zfpm1/fog1 (Friend-of-gata-1), Chr 18; klf4 (Krüppel-like factor 4) Chr 2; and lmo2 (Lim domain only 2) Chr 18.

Our data are consistent with the previous interpretation of miR-451 MO knockdown experiments in zebrafish (ie, that miR-451 is not required for erythroid specification).⁹  However, in contrast to this previous study, we did not observe severe anemia in miR-451 morphant embryos at the onset of circulation or at 48 hpf. The late-onset anemia observed in this other study was interpreted as indicating a requirement for miR-451 in erythrocyte maturation or survival. The reasons for these different outcomes are unclear, particularly because the miR-451 MO sequences were identical, but may reflect subtle differences in MO dose, preparations, or other technical differences. However, our MO knockdown observations are independently corroborated by the erythroid phenotype of mnr, which does not show postcirculation erythroid failure, but only delayed erythroid maturation.

Maturation of a population of cells could result either from the progressive maturation of a synchronized cohort of cells, or alternately from the progressive addition of newly produced cells of increasing maturity. Cell survival studies demonstrated that the first cohort of ICM-derived erythrocytes was undiluted by newly produced erythrocytes until 4 dpf³² ; hence, we hypothesize that miR-451–dependent promotion of erythrocyte maturation up to 48 hpf is a cell autonomous effect driving the maturation of individual cells.

In mammalian hematopoiesis, persistent production of GATA2 maintains a stem cell phenotype and needs to be down-regulated to allow erythropoiesis to proceed normally.³⁵  One known mechanism for down-regulating GATA2 is by GATA1 directly binding to and repressing its promoter.⁴⁹  An important outcome of our studies was the identification of a mechanism for miR-451 promotion of erythroid maturation in zebrafish: the clearance of gata2 transcripts. To prove that the prolonged expression of gata2 in mnr and miR-451-morphant erythrocytes was not merely secondary to erythroid immaturity, but directly due to miR-451 loss, we evaluated the gata2-3′UTR/miR-451 interaction outside the context of erythrocyte maturation in a direct in vivo reporter assay. This confirmed that a functional interaction occurred. We also evaluated the cause/effect biologic consequences of perturbing this interaction: rerepressing gata2 in miR-451–deficient mnr with MO-gata2 and relieving gata2 alone of miR-repression in WT with the target-blocking MOs.

Repression of GATA2 by this miRNA locus may be conserved in mammals, although it may be achieved through miR-144 rather than miR-451, because human, murine, and rat GATA2-3′UTRs all contain several predicted miR-144 binding sites but no predicted miR-451 sites (Table S2). The in vivo GFP reporter assay provides a method for evaluating the functional validity of these potential miRNA/transcript interactions.

Gata1 displaces Gata2 from the murine miR-144/451 promoter to activate promoter function.⁹  Hence, in zebrafish, at least, there is scope for a 2-pronged Gata1-driven regulatory mechanism for relaxing Gata2 repression of erythroid maturation; Gata1 turns off further gata2 transcription and activates transcription of a miRNA locus that down-regulates extant gata2 transcripts.

We interpret the failure of MOs exclusively interfering with the miR-451/gata2 interaction to repress erythrocyte maturation to indicate that miR-451 targets other than gata2 are also important in erythrocyte maturation. This indicates that there is redundancy and robustness in miR-451–driven erythroid maturation. Reporter assays and mnr-based functional assays provide a basis for evaluating other candidate target transcripts carrying putative miR-451 binding sites for their role in erythrocyte maturation. An alternate hypothesis is that the target blocking MOs alter the dose of Gata2 but not as much as in mnr or miR-451 morphants.

Together, our results assemble a new genetic pathway that regulates the pace of erythroid maturation: mnr activating miR-451, which in turn represses gata2 (Figure 7A). This genetic pathway informs a biologic model (Figure 7B) consistent with our experimental observations. In general, once committed to an erythroid fate, cells require mnr to use miR-451 to down-regulate repressors of erythroid maturation, gata2 being one of them. miR-451 deficiency (either MO-451 morphants or mnr mutants) retards the pace of erythroid maturation by permitting gata2 and other target transcripts to persist longer than usual. In mnr, normalizing levels either of miR-451 alone or of its target gata2 (by restoring miR-451 levels or by gata2 knockdown) is sufficient to partially rescue the rate of red cell maturation. However, because gata2 target-blocking MOs are insufficient to retard erythrocyte maturation in wild-type embryos, there must be other miR-451 targets that need to be codysregulated to result in the erythrocyte immaturity of mnr or miR-451 morphants. Finally, these experiments exemplify an hypothesized miRNA role in sharpening developmental transitions,^34,50  in this case from a hematopoietic progenitor cell into a maturing erythrocyte.

We thank the following for helpful discussions, encouragement, support, and/or technical help: Warren Alexander, Tony Burgess, Stephen Cody, David Curtis, John Hayman, Joan Heath, Ben Hogan, Stephen Jane, Luke Kapitany, Anne Lagendijk, Nick Nicola, Sony Varma, and colleagues in the Colon Molecular and Cell Biology Group at the Ludwig Institute and the Bone Marrow Research Laboratory (Royal Melbourne Hospital). We particularly thank Belinda Phipson and Gordon Smyth for statistical advice and Stephen Renshaw for supplying the Tg(mpx:EGFP) line. We thank several anonymous reviewers for constructive criticism and suggestions that improved the manuscript.

This work was supported in part by grants from the National Institutes of Health (Bethesda, MD; R01-HL079545) and National Health and Medical Research Council (NHMRC 461222 and 461208) to G.L. L.P. was supported by an Australian Postgraduate Award and CSIRO enhancement stipend and received travel support from the ARC/NHMRC Network in Genes and Environment in Development (NGED).

National Institutes of Health

Contribution: L.P. designed, conducted, and interpreted the experiments and wrote the manuscript; J.E.L. generated the mutant meunier and initiated its mapping; W.P.K. performed miR-451 and miR-206 in situ hybridizations; D.C. performed some in situ hybridizations and made early contributions to mnr mapping and erythrocyte morphometrics; P.M.W. helped plan the studies and cosupervised L.P.; and G.J.L. designed and interpreted the experiments, supervised L.P., and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Dr Graham J. Lieschke, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, Australia 3050; e-mail: lieschke@wehi.edu.au.