New Results

Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos

Hailey E. Edwards, Jaclyn P. Souder, Daniel A. Gorelick
doi: https://doi.org/10.1101/2022.01.04.474920

ABSTRACT

During embryonic development, a subset of cells in the mesoderm germ layer are specified as hemato-vascular progenitor cells, which then differentiate into endothelial cells and hematopoietic stem and progenitor cells. In zebrafish, the transcription factor npas4l, also known as cloche, is required for the specification of hemato-vascular progenitor cells. However, it is unclear if npas4l is the sole factor at the top of the hemato-vascular specification cascade. Here we show that arnt1 and arnt2 genes are required for hemato-vascular specification. We found that arnt1;arnt2 double homozygous mutant zebrafish embryos (herein called arnt1/2 mutants), but not arnt1 or arnt2 single mutants, lack blood cells and most vascular endothelial cells. arnt1/2 mutants have reduced or absent expression of etv2 and tal1, the earliest known endothelial and hematopoietic transcription factor genes. npas4l and arnt genes are PAS domain-containing bHLH transcription factors that function as dimers. We found that Npas4l binds both Arnt1 and Arnt2 proteins in vitro, consistent with the idea that PAS domain-containing bHLH transcription factors act in a multimeric complex to regulate gene expression. Our results demonstrate that npas4l, arnt1 and arnt2 act together as master regulators of endothelial and hematopoietic cell fate. Our results also demonstrate that arnt1 and arnt2 act redundantly in a transcriptional complex containing npas4l, but do not act redundantly when interacting with another PAS domain-containing bHLH transcription factor, the aryl hydrocarbon receptor. Altogether, our data enhance our understanding of hemato-vascular specification and the function of PAS domain-containing bHLH transcription factors.

INTRODUCTION

Endothelial and hematopoietic cells are derived from the mesoderm during vertebrate embryogenesis. These two cell types are closely linked and arise from a common progenitor during development (Huber et al., 2004; Kataoka et al., 2011; Liao et al., 2000). The differentiation and specification of endothelial and hematopoietic cells occurs in two waves— primitive and definitive hematopoiesis (Costa et al., 2012; Paik and Zon, 2010; Palis et al., 1999). During primitive hematopoiesis the first hemato-vascular progenitors are observed in the lateral plate mesoderm of zebrafish and in the extraembryonic yolk sac mesoderm of mice (Lee et al., 1994; Palis et al., 1999; Stainier et al., 1993). These progenitors give rise to blood islands which contain primitive erythroid cells (Palis et al., 1999). During definitive hematopoiesis in mice and zebrafish, fully multipotent hematopoietic stem cells (HSCs) are derived from specialized epithelial cells, hemogenic endothelium (Hirschi, 2012; Kobayashi et al., 2014).

In humans, the genes that drive the specification of hemato-vascular progenitors from the multipotent mesoderm are not completely understood. We have a more complete understanding of this process in other vertebrates. In zebrafish and chicken, Neuronal PAS 4-like (npas4l) is essential for the specification of hemato-vascular progenitors from multipotent mesoderm (Reischauer et al., 2016; Weng et al., 2020). Mammals do not appear to have a npas4l homolog in their genomes and the mammalian functional equivalent to zebrafish npas4l is unknown. Many genes involved in embryonic hematopoiesis are conserved between zebrafish, mice, and humans (Aplan et al., 1992; Brownlie et al., 1998; Casie Chetty et al., 2017; Dooley et al., 2005; Ferrara et al., 1996; Gong et al., 2004; Kalev-Zylinska et al., 2002b; Kataoka et al., 2011; Okuda et al., 1996; Wang et al., 1996), but npas4l appears to be an exception. Understanding the specific mechanisms of hemato-vascular specification in zebrafish may help identify functional homologs in humans and other mammals. Understanding the specific mechanisms of hemato-vascular specification may prove useful in regenerative therapies for blood and vascular diseases. Recipients of embryonic stem cell-based regenerative therapies have an increased risk of tumor formation. A promising alternative, which ameliorates the risk of tumor formation, is to produce specific multipotent progenitors from embryonic stem cells prior to transplantation (Doss et al., 2012; Gunaseeli et al., 2010).

Zebrafish embryos and larvae are an ideal organism in which to study hemato-vascular development in vivo. Zebrafish oocytes are fertilized externally and embryos are optically transparent. Zebrafish embryos can survive up to 7 days post fertilization without circulating red blood cells because oxygen can freely diffuse through the skin (Hughes et al., 2019; Pelster and Burggren, 1996). This trait allows for characterization of cardiovascular phenotypes that would be lethal in embryos from other organisms.

In zebrafish, endothelial cells and HSCs can be derived from a single hemato-vascular progenitor cell population called the hemangioblast, which is hypothesized to exist in mammals but has not been definitively confirmed (Huber et al., 2004; Moore et al., 2018; Shu et al., 2015; Zhang and Featherstone, 2020). It has been previously reported that the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor, npas4l, is the master regulator of hemato-vascular specification in zebrafish (Reischauer et al., 2016) and in chicken (Weng et al., 2020).

bHLH-PAS transcription factors are classified as Class I or Class II, which must dimerize to regulate transcription (Erbel et al., 2003; Pongratz et al., 1998)(Figure 1A). Npas4l is a Class I bHLH-PAS protein, but its binding partner during hemato-vascular progenitor specification is not known. We hypothesized that Arnt1 or Arnt2 is the dimerization partner of Npas4l in zebrafish because there are only 4 bHLH-PAS Class II proteins encoded in the zebrafish genome and arnt1 and arnt2 genes are expressed at the the same stages of embryo development as npas4l (Prasch et al., 2006; Reischauer et al., 2016; Tanguay et al., 2000).

Figure 1.
Figure 1. arnt1/2 double mutant embryos, but not arnt1 or arnt2 single mutants, lack circulating blood, have cardiac edema and atrial enlargement.

(A) bHLH-PAS transcription factors act as heterodimers, where a class I and indicated class II protein interact and bind DNA. Asterisks indicate proteins encoded by genes present in zebrafish but absent in humans. (B,C) Generation of zebrafish with mutations in arnt1 and arnt2 genes. The amino acid sequences for the zebrafish Arnt1 (B), and Arnt2 proteins (C) are represented by the black line. Basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domains, and the motif C-terminally located to the PAS (PAC) are represented by the labeled bubbles. The numbers below each domain indicate the starting and ending amino acid (AA) for each motif. The arnt1bcm1 mutation causes a premature stop codon to occur at AA139 and loss of the PAS-B, PAC, and majority of the PAS-A. The arnt1bcm2 mutation causes a premature stop codon to occur at AA250 and loss of the PAS-B and PAC domains (B). The arnt2bcm3 mutation causes a premature stop codon to occur at AA287 and loss of the PAS-B and PAC domains (C). (D-I) Live embryos from arnt1bcm2/+;arnt2bcm3/+ heterozygous parents were imaged at 2 days post fertilization (dpf) prior to genotyping. All double homozygous arnt1bcm2/bcm2; arnt2bcm3/bcm3 (arnt1/2 mutant) embryos showed cardiac edema and atrial enlargement (G and G’). The wildtype (D and D’), arnt1bcm2/bcm2 (arnt1 mutant) (E and E’), and arnt2bcm3/bcm3 (arnt2 mutant) (F and F’) siblings of the arnt1/2 mutants showed normal heart development. Black arrows in panels D’-G’ indicate the atrium and ventricle of the heart. Additionally, all siblings of arnt1/2 mutants had circulating blood (H and M1) while arnt1/2 mutants lacked visible circulting blood cells (I and M2). The white arrows in panel H indicate blood cells in the dorsal aorta. Images are representative of 7 different cluches, each cluch contained between 20-300 embryos. For all images of zebrafish embryos (D-I) anterior is towards the right and doral is towards the top of the image. The scale bar in Panel D applies to Panels D-G and is 1000 μm in lenght. The scale bar in Panel D’ apples to Panels D’-G’ and is 100 μm in length.

To test this hypothesis, we examined hemato-vascular development in zebrafish with loss-of-function mutations in arnt1 and arnt2 genes. We find that Arnt1 and Arnt2 are essential for hemato-vascular specification. Double homozygous arnt1;arnt2 zebrafish embryos phenocopy npas4l mutants (also called cloche), showing lack of circulating blood, absence of hematopoietic stem and progenitor cells, and dramatic reduction in the number of vascular endothelial cells. Additionally, we show that zebrafish Npas4l protein interacts with both zebrafish Arnt1 and zebrafish Arnt2 proteins in vitro. Together, our results suggest that Arnt1 and Arnt2 act redundantly and form a transcriptional complex with Npas4l that sits atop the hemato-vascular specification cascade.

MATERIALS AND METHODS

Zebrafish

Adult zebrafish were raised at 28.5°C on a 14-h light, 10-h dark cycle in the UAB Zebrafish Research Facility or the BCM Zebrafish Research Facility in an Aquaneering recirculating water system (Aquaneering, Inc., San Diego, CA) and a Tecniplast recirculating water system (Tecniplast USA, Inc., West Chester, PA). All zebrafish used for experiments were wild-type AB strain (Westerfield, 2000) and all mutant lines were generated on the AB strain. All procedures were performed in accordance with Institutional Animal Care and Use Committees (IACUC) and BCM guidelines.

Embryo collection

Adult zebrafish were allowed to spawn naturally in pairs or in groups. Embryos were collected in intervals of 20 minutes to ensure precise developmental timing or staged following collection, placed in 60 cm2 Petri dishes at a density of no more than 100 per dish in E3B media (60X E3B: 17.2g NaCl, 0.76g KCl, 2.9g CaCl2-2H2O, 2.39g MgSO4 dissolved in 1L Milli-Q water; diluted to 1X in 9L Milli-Q water plus 100 μL 0.02% methylene blue), and then stored in an incubator at 28.5°C on a 14-h light, 10-h dark cycle.

Genotyping adult zebrafish

Genomic DNA was isolated from tail biopsies from individual adult zebrafish by incubation in 50 μL ELB (10 mM Tris pH 8.3, 50 mM KCl, 0.3% Tween 20) with 0.5 μL proteinase K (800 U/ml, NEB) in 96 well plates, one sample per well, at 55°C for 8 hours (tail biopsies). Proteinase K was inactivated by incubation at 98°C for 10 minutes and DNA was stored at - 20°C. Genotyping was performed by PCR and high-resolution melting curve analysis as described (Parant et al., 2009; Romano et al., 2017). All melting curves were generated with a Bio-Rad CFX96 Real-Time System over a 70-95°C range and analyzed with the Bio-Rad CFX Manager 3.1 or the Bio-Rad CFX Maestro 4.1 software. All mutations were confirmed by TA cloning and sequencing.

CRISPR-Cas9 Mutant Generation

Cas9 mRNA and gRNAs for arnt1, and arnt2 mutants were generated as previously described (Romano et al., 2017; Souder and Gorelick, 2019). Cas9 mRNA was transcribed from a linearized pT3TS-nCas9n plasmid, Addgene #46757 (Jao et al., 2013), and purified. The target sequences were identified using CHOPCHOP (Labun et al., 2016; Labun et al., 2019). Oligonucleotides were annealed to each other and cloned into a pT7-gRNA plasmid, Addgene #46759 (Jao et al., 2013). Oligonucleotides utilized are shown in Table 1. gRNAs were synthesized from plasmids using a MEGAshortscript T7 Transcription Kit (Invitrogen AM1354) and purified.

View this table:
Table 1.

CRISPR target sequence and oligos used to generate arnt1 and arnt2 mutants. PAM sequence in CRISPR target is underlined

One-cell-stage embryos were injected using glass needles pulled on a Sutter Instruments Fleming/Brown Micropipette Puller, model P-97 and a regulated air-pressure micro-injector (Harvard Apparatus, NY, PL1–90). Each embryo was injected with a 1nL solution containing one gRNA for arnt1 and one gRNA for arnt2 (30 ng/μL per target), Cas9 mRNA (150 ng/μL), and 0.1% phenol red. Mixtures were injected into the yolk of each embryo. Approximately, 100 injected embryos per gRNA pair were raised to adulthood and crossed to AB zebrafish to generate F1 embryos. F1 offspring with heritable mutations were sequenced to identify mutations predicted to cause loss of function.

Live Imaging

Embryos were imaged with a Nikon SMZ25 microscope equipped with a Hamamatsu ORCA-Flash4.0 digital CMOS camera. Images were equally adjusted for brightness and contrast in Adobe Photoshop CC 2020. Embryos and larvae were anesthetized with 0.04% tricaine and imaged in Petri dish containing E3B. All embryos were genotyped after imaging.

Whole mount in situ hybridization

etv2, tal1, runx1, and flk1 antisense RNA probes were used as described (Moore et al., 2013; Thisse and Thisse, 2008). All DNA clones were verified by sequencing. Digoxigenin-labeled antisense RNA probes were synthesized using T7 for runx1 and flk1, or T3 for etv2. Colorimetric in situ hybridization (ISH) was performed on fixed zebrafish embryos and larvae as previously described, with 5% dextran in the hybridization buffer (Lauter et al., 2011; Thisse and Thisse, 2008). Following ISH, embryos were washed several times in 1x PBST (phosphate buffered saline, 0.01% Tween), cleared with glycerol or mounted in 3% methylcellulose on a class coverslip, and imaged on either a Nikon SMZ18 stereoscopic microscope or an inverted Nikon ECLIPSE Ti2-E microscope. Embryos were then genotyped. Genomic DNA was extracted using the HotSHOT protocol (Dobrzycki et al., 2018). Briefly, embryos were suspended in 20 μL of lysis buffer (25 mM NaOH, 0.2 mM EDTA) and incubated at 95°C for 30 minutes, cooled to 4°C, then an equal volume of neutralization buffer (40 mM Tris-HCl) was added. DNA regions of interest were amplified with JumpStart REDTaq ReadyMix (Sigma-Milipore, P0982) PCR according to manufacturer’s protocols.

Cell Culture and Transient Transfection

HEK293T cells (gift of Dr. Charles Foulds), were thawed and maintained at 37°C in humidified 5% CO2 atmosphere incubator. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, 2062564), and 1% penicillin-streptomycin (Gibco, 15140-122). After 24 hours passaging in a 100mm plate at a density of 2.2 × 106 cells per dish and reaching at least 70 confluency, the media was changed to DMEM supplemented with 1% dialyzed FBS. Cells were transiently transfected with 12 μg of total DNA and Lipofectamine 2000 according to manufacturer’s instructions (Invitrogen, 2400439) with npas4l-v5 together with either arnt1-myc or arnt2-HA plasmid DNA.

Cloning of plasmids for expression in cultured cells was performed as follows. For npas4l-V5, we took pCS2+npas4lORF (gift of Dr. Leonard Zon) that contains the open reading frame (ORF) of zebrafish npas4l downstream of the CMV promoter. This plasmid was modified to include a C-terminal V5 tag using Q5 Site-Directed Mutagenesis Kit (New England Biolabs, #E0554S). For arnt1-myc, we synthesized a plasmid containing CMV promoter driving a codon-optimized zebrafish arnt1 ORF (ENSDART00000081852.5) with a C-terminal myc tag (Twist Bioscience, San Francisco, CA). For arnt2-myc, we synthesized a gene block of the zebrafish arnt2 ORF (ENSDART00000158162.2, Integrated DNA Technologies). The gene block was integrated into a plasmid containing a CMV promoter using pcDNA3.3-TOPO TA cloning kit (Invitrogen, K8300-01) in accordance with manufacturer’s instructions. A C-terminal HA tag was added using Q5 Site-Directed Mutagenesis Kit (New England Biolabs, #E0554S).

Co-immunoprecipitation and Western blot

Transiently transfected cells were washed with ice cold PBS and lysed in 500 μL of 1x RIPA buffer containing Pierce Protease (ThermoScientific, A32955). Cells were sonicated in 6 × 30 second intervals using a Bioruptor 300 Machine (Diagenode), then centrifuged for 10 minutes at 10,000g at 4°C. The lysate (supernatant) was removed from the pellet and placed in fresh 1.5mL Eppendorf tube. Cell lysate was precleared with Protein A beads (Invitrogen #159 18-014) for 30 minutes 4°C, incubated with either 5 μg of V5 antibody (ThermoFisher Scientific R960-25) or myc antibody (SantaCruz, SC-40) diluted 1:200 for 1 hour, then immunoprecipitated with 20 μL of Protein A beads for 1 hour at 4°C. The beads were washed three times with RIPA buffer and once with PBS and finally suspended in 30μL of PBS. Equal volume of 2x Laemmli Sample Buffer (BioRad, cat# 1610737) was added to each sample and boiled for 5 minutes at 95°C then placed on ice. Samples were run on 4-20% Mini-PROTEAN TGX gels (BioRad, cat# 4561093) and transferred onto PVDF membrane using Trans-Blot Turbo Transfer System (BioRad) using the pre-defined Mixed MW program. The membranes were washed several times with 1x Tris-Buffered Saline, 0.1% Tween 20 (TBST) (GBiosciences, R043) and incubated for 1 hour with agitation in 5% milk in 1x TBST. Membranes were probed with 1:1000 V5-HRP (ThermoFisher Scientific, R960-25), 1:1000 myc-HRP (SantaCruz, SC-40), or 1:200 HA (Cell Signaling, 3724) antibodies rocking overnight at 4°C. Membranes were washed 5 times with 1xTBST on orbital rocker for 15 minutes at room temperature. Membranes probed with the anti-HA antibody were then probed with 1:10,000 IRDye 800CW donkey anti-rabbit (Licor, 926-32213) in 2.5%-5% milk in 1x TBST for 1 hour and then washed multiple times with 1xTBST on orbital rocker for 5 minutes prior to imaging. Membranes probed with HRP conjugated antibodies were developed using Clarity™ Western ECL Substrate (BioRad, 170-5061). Membranes were imaged using BioRad ChemiDoc™ MP Imaging System.

TCDD exposure

At 1-day post fertilization (dpf) embryos were exposed to 10 ng/mL 2,3,7,8-tetrachlorodibenzodioxin (TCDD, AccuStandard Inc, D404N) or vehicle (0.1% DMSO). All treatments were performed on a single clutch of at least 20 embryos; embryos treated with vehicle were from the same clutches as embryos treated with TCDD. Chemical exposures were performed on at least 2 clutches per genotype. Embryos were raised at 28.5°C on a 14-h light, 10-h dark cycle until 3 dpf, when they were imaged and then genotyped.

RESULTS

arnt1/2 homozygous mutants lack circulating blood

To determine if arnt1 or arnt2 is required for hemato-vascular development, we generated heritable mutant alleles for arnt1 (arnt1bcm1 and arnt1bcm2) and arnt2 (arnt2bcm3) using CRISPR/Cas9. For arnt1bcm1, we generated zebrafish with a 5 bp insertion in exon 5, which results in a frameshift at amino acid (AA) 126 and a premature stop codon in the first PAS domain at AA139 (Fig. 1B). For arnt1bcm2, we generated zebrafish with an 11 bp deletion in exon 10, which results in a frameshift at AA230 and a premature stop codon between the two PAS domains at AA250 (Fig. 1B). We did not observe any phenotypic differences between homozygous arnt1bcm/bcm1 and arnt1bcm2/bcm2 mutant embryos. These mutants are morphologically indistinct from wildtype during development and homozygous adults are viable and fertile, which is consistent with previously published arnt1 mutants (Marchi et al., 2020). We also generated maternal zygotic arnt1 mutants (derived from homozygous bcm1 or bcm2 parents) and these were morphologically indistinct from wildtype. For arnt2bcm3, we generated zebrafish with a 3 bp deletion and 1 bp insertion in exon 9, which resulted in a missense mutation at AA286 and a premature stop codon between the two PAS domains at AA287 (Fig. 1C). Homozygous arnt2 mutants (arnt2bcm3/bcm3) were viable at 5 days post fertilization (dpf) and grossly morphologically normal, however they did not survive to adulthood. Our observations are consistent with independently generated arnt2 mutant zebrafish lines (Hill et al., 2009; Lohr et al., 2009).

We crossed arnt1bcm2/+; arnt2bcm3/+ adult fish to each other and analyzed the phenotype and genotype of the resulting embryos. We observed all genotypes at the expected Mendelian ratios (Table 1). The single mutant larvae, arnt1bcm2/bcm2, herein called arnt1 mutants (Fig. 1D and D’), and arnt2bcm3/bcm3, herein called arnt2 mutants (Fig. 1E and E’), are phenotypically indistinguishable from their wildtype siblings by gross morphology (Fig. 1C and C’). However, arnt1bcm2/bcm2;arnt2bcm3/bcm3 larvae, herein called arnt1/2 mutants, showed a clear phenotype (Fig. 1F). These larvae lack all circulating blood (Fig. 1H, M3 and M4). All other intermediate arnt1;arnt2 genotypes, e.g., arnt1bcm2/+;arnt2bcm3/bcm3, appeared phenotypically wildtype (Fig. 1G, and data not shown). These results suggest that arnt1 and arnt2 function redundantly, because only the arnt1/2 double homozygous mutants show a phenotype. Additionally, the arnt1/2 mutant phenotype appears identical to the previously described npas4l mutant phenotype (also known as cloche) (Reischauer et al., 2016; Stainier et al., 1995a). Like npas4l mutants, arnt1/2 mutants exhibit cardiac edema with enlarged heart atrium, likely a secondary effect of the lack of blood and endocardial cells (Fig 1G). Taken together, these data suggest that arnt1 and arnt2 have a similar function as npas4l.

arnt1/2 mutants have reduced expression of hematopoietic and endothelial cell markers at 2 dpf

We hypothesized that the Arnt proteins are dimerization partners with Npas4l and that arnt1/2 mutants would exhibit the same phenotypes as npas4l mutants. Therefore, we examined arnt1/2 mutant embryos for additional phenotypes found in npas4l mutants. Previous studies determined that npas4l mutants lack almost all endothelial cells, except some cells located in the lower trunk and caudal tail of the larvae (Liao et al., 1997; Stainier et al., 1995a). To determine if our arnt1/2 mutants showed a similar reduction in endothelial cells, we performed whole-mount in situ hybridization (WISH) on 2 dpf embryos for flk-1 (also known as kdrl). flk-1, a widely used marker of endothelial cells in blood vessels, is essential for the development of blood vessels in mice and zebrafish (Dumont et al., 1995; Liao et al., 1997; Shalaby et al., 1995). We found that maternal zygotic arnt1 mutants (MZarnt1) and arnt2 mutants showed clear expression of flk-1 in the vasculature, similar to wild type (Fig 2B-C). In contrast, arnt1/2 mutants had a reduced number of flk-1-positive cells (Fig. 2D and D’). The flk-1-positive cells present in the arnt1/2 mutant were in the caudal tail and likely comprised a portion of the caudal vein (Fig. 2D’), similar to the flk-1 expression pattern seen in npas4l mutants (Liao et al., 1997). This suggests that npas4l and arnt1 or arnt2 are required for the development of most blood vessels.

Figure 2.
Figure 2. arnt1/2 double homozygous mutants show reduced levels of flk-1-positive endothelial cells and runx-1-positive cells in blood island (A-H).

Embryos were probed for flk-1 (A-D) or runx-1 (E-H) using whole-mount in situ hybridization at 2 days post fertilization (dpf). Wild-type embryos were derived from an in-cross of laboratory strain AB parents (A and E). arnt1 mutant embryos were derived from arnt1;arnt2 heterozygous parents or are maternal zygotic derived from arnt1 homozygous parents (MZarnt1). arnt2 mutant embryos were derived from heterozygous parents. arnt1/2 double homozygous mutant embryos (D and H) were derived from arnt1;arnt2 double heterozygous parents and identified by cardiovascular phenotype prior to fixation. Panels A’-D’ are enlarged images of each embryo’s trunk near the end of the yolk extension (area indicated by red box in panels A-D). (A’-D’) Black arrows indicate flk-1 staining in the dorsal longitudinal anastomotic vessels (DLAV), white arrows indicate the dorsal aorta and caudal vein (DA/CV), arrowheads indicate inter-segmental vessels. (E-H) runx-1 labeling near the end of the yolk extension, arrows indicate blood island region. n=2-5 clutches for each genotype, 10-100 embryos per clutch. All embryos are oriented with anterior to the right and dorsal towards the top. The scale bar in D applies to, the scale bar in D’ applies to A’-D’, and the scale bar in H applies to E-H. All scale bars are 10 μm.

In addition to a significant reduction of endothelial cells, npas4l mutants show a reduction in the number of hematopoietic stem cells (HSC) (Stainier et al., 1995a; Thompson et al., 1998). To determine if arnt1/2 mutants contain HSCs, we examined runx1 expression by WISH. runx1 is a marker of HSCs and is required for definitive hematopoiesis in zebrafish (Kalev-Zylinska et al., 2002a). By 48 hpf, runx1-positive HSCs are localized to the area between the dorsal aorta and the posterior caudal vein (Lam et al., 2009). At 2 dpf, wildtype, arnt1, and arnt2 single mutants all contain runx1-positive cells in the blood island region (Fig 2E-G), indicating the presence of HSCs. However, arnt1/2 mutants lack runx1-positive cells in this region (Fig. 2H). This suggests that arnt1 or arnt2 is necessary for hematopoietic cell development during embryogenesis, similar to npas4l (Liao et al., 1998; Stainier et al., 1995a; Thompson et al., 1998).

Hematopoietic and endothelial progenitor populations are reduced in arnt1/2 mutants

Hematopoietic cells and endothelial cells arise from a common progenitor population in zebrafish (Vogeli et al., 2006). To determine if arnt1/2 mutants have a reduction of hemato-vascular progenitor cells, we performed WISH on embryos at the 4-6 somite stage and examined expression of the earliest known regulators of endothelial and hematopoietic cell differentiation: etv2 and tal1, both of which are expressed in the posterior lateral plate mesoderm (PLPM) during early somitogenesis stages (Qian et al., 2005; Reischauer et al., 2016; Stainier et al., 1995a; Thompson et al., 1998). We bred arnt1bcm2/+; arnt2bcm3/+ adults to each other to generate arnt1/2 mutant, arnt1 mutant, arnt2 mutant, and wildtype embryos. We then performed WISH to examine the expression of etv2 (Fig. 3A and B, n=516 embryos from 5 clutches) or tal1 (Fig. 3C and D, n=665 embryos from 3 clutches). 8% and 6% of embryos showed reduced expression of etv2 and tal1, respectively, neither of which is statistically significantly different from the expected Mendelian ratio of 6.25% double homozygous mutant embryos (binomial test, etv2 n=516 p>0.05; tal1 n=665 p>0.05). To confirm that the observed reduction of etv2 and tal1 in these embryos correlates with the arnt1/2 double homozygous mutant genotype, following WISH we genotyped 24 of the embryos with absent or reduced expression of etv2 and 17 with absent or reduced expression of tal1. 100% of the low/no expressing etv2 embryos were arnt1/2 double homozygous mutants, while 83% of the low/no expressing tal1 embryos were arnt1/2 double homozygous mutants, demonstrating a strong correlation between the reduced expression of etv2 and tal1 and the arnt1/2 double homozygous genotype. Additionally, we genotyped 26 of the embryos with normal expression of etv2 and 7 with normal expression of tal1. 7.7% of the normal expressing etv2 embryos were arnt1/2 mutants (genotypes of the 26 embryos: 2 arnt1/2 mutants, 1 wildtype, 3 arnt1bcm2/bcm2, 4 arnt1bcm2/bcm2;arnt2bcm3/+, 4 arnt1bcm2/+, 5 arnt1bcm2/+;arnt2bcm3/+, 4 arnt1bcm2/+;arnt2bcm3/bcm3, 1 arnt2bcm3/bcm3, and 3 arnt2bcm3/+), while 0% of the normal expressing tal1 embryos were arnt1/2 mutants (genotypes of the 7 embryos: 1 arnt1bcm2/+;arnt2bcm3/+, 1 arnt1bcm2/+;arnt2bcm3bcm3, 1 arnt1bcm2/+, 1 arnt2bcm3/+, and 3 arnt1bcm2/bcm2;arnt2bcm3/+). We conclude that arnt1 and arnt2 are required for hemato-vascular progenitor specification. Previous findings demonstrated that npas4l is a master regulator of hemato-vascular progenitor specification (Reischauer et al., 2016). Together, these results demonstrate that npas4l, arnt1 and arnt2 are co-master regulators of hemato-vascular specification.

Figure 3.
Figure 3. arnt1/2 mutants show decreased etv2 and tal1 expression in the posterior lateral plate mesoderm (PLPM) at 4-6 somite stages.

Whole-mount in situ hybridization for etv2 and tal1 expression was performed on 4-6 somite stage embryos which were offspring of arnt1bcm2/+; arnt2bcm3/+ adult zebrafish. (A) Expression of etv2 was observed in the PLPM in 92% of embryos (n=477 of 516 total embryos). (B) Limited or no expression of etv2 was observed in 8% of embryos (n=39 of 516), consistent with the expected Mendelian ratios for arnt1/2 mutants (6.25%). 24 of the 39 presumptive arnt1/2 mutants were genotyped and 24/24 embryos were confirmed arnt1/2 mutants. (C) Expression of tal1 was observed in 94% of embryos (n=624 of 665 total embryos).(D) Limited or no expression of tal1 was observed in 6% of embryos (n=41 of 665), consistent with the expected Mendelian ratios for arnt1/2 mutants (6.25%). 17 of the 41 presumptive arnt1/2 mutants were genotyped and 14/17 embryos were confirmed arnt1/2 mutants. All embryos are oriented with dorsal towards the top of the image and posterior is closest to the reader. Scale bar is 10 μm and applies to all images.

Npas4l interacts with both Arnt1 and Arnt2

Npas4l is a Class I bHLH-PAS domain transcription factor (Reischauer et al., 2016), and as such, must heterodimerize with Class II bHLH-PAS transcription factors, like Arnt1 and Arnt2, to initiate gene transcription. To determine if Npas4l forms a complex with either Arnt1 or Arnt2, we performed a coimmunoprecipitation experiment. Due to the absence of commercially available antibodies that recognize zebrafish Npas4l, Arnt1 or Arnt2 proteins, we co-expressed tagged versions of zebrafish npas4l (Npas4l-V5), arnt1 (Arnt1-myc), and arnt2 (Arnt2-HA) in HEK293T cells. We performed co-immunoprecipitation studies and found that Arnt1-myc interacts with Npas4l-V5 and that Arnt2-HA interacts with Npas4l-V5 (Fig. 4A, B). These data suggest that Npas4l uses either Arnt1 or Arnt2 or both as a binding partner. Furthermore, these data indicate that Arnt1 and Arnt2 may be functionally interchangeable and redundant, at least in the case of Npas4l.

Figure 4.
Figure 4. Arnt1 and Arnt2 co-immunoprecipitate with Npas4l.

(A) HEK293T cells were transfected with zebrafish Npas4l-V5 and Arnt1-myc. Cells were homogenized (input) and immunoprecipitated (IP) with antibodies aganist V5 or an immunoglobulin G control (IgG). Samples were run on an immunoblot and probed using myc antibodies (anti-myc) to detect the presence of Arnt1-myc (top) or samples were probed using V5 antibodies (anti-V5) to verify sucessful pulldown of Npas4l-V5 (bottom). (B) HEK293T cells were transfected with zebrafish Npas4l-V5 and Arnt2-HA. Cells were homogenized and immunoprecipitated with antibodies aganist HA or an IgG control. Samples were run on an immunoblot and probed using V5 antibodies (anti-V5) to detect the presence of Npas4l-V5 (top) or samples were probed using HA antibodies (anti-HA) to verify sucessful pulldown of Arnt2-HA (bottom).

Arnt1 and Arnt2 cannot functionally compensate for one another in the case of Ahr2

Our results demonstrate that Arnt1 and Arnt2 are functionally interchangeable and redundant when it comes to interacting with Npas4l in the context of hemato-vascular development. It is unclear if Arnt1 and Arnt2 are functionally interchangeable with all Class I bHLH-PAS domain transcription factors or just with Npas4l. To determine if arnt1/arnt2 redundancy is unique to Npas4l, we examined if arnt1 and arnt2 are interchangeable in the case of a different class I bHLH-PAS transcription factor, the aryl hydrocarbon receptor 2 (Ahr2). Previous studies demonstrate that Ahr2 can bind DNA in the presence of either Arnt1 or Arnt2 in vitro (Andreasen et al., 2002; Hirose et al., 1996; Tanguay et al., 2000), but whether Arnt1 and Arnt2 bind Ahr2 interchangeably in vivo is not known. Ahr2 is a ligand-dependent transcription factor. A known ligand of Ahr2 is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which we have previously shown induces cardiotoxicity in zebrafish larvae and requires wild-type ahr2 (Souder and Gorelick, 2019). We asked whether TCDD causes cardiotoxicity in arnt1 or arnt2 single mutants, reasoning that if Ahr2 interacts with Arnt1 or Arnt2 interchangeably, then only arnt1/2 double mutants, but not single mutants, would be resistant to TCDD exposure. We treated wildtype, arnt1 mutants, and arnt2 mutants with either vehicle (0.1% DMSO) (Fig. 5A, B, and C) or with 10 ng/mL TCDD (Fig. 5D, E, and F) from 1 dpf to 3 dpf. Wildtype and arnt2 mutants treated with TCDD exhibit cardiac edema and the heart fails to loop properly (Fig. 5D, and F), unlike vehicle treated siblings (Fig. 5A and C). In contrast, arnt1 mutants treated with TCDD showed normal heart looping and lack cardiac edema (Fig. 5E), like their vehicle treated siblings (Fig. 5B). This suggests that arnt1, but not arnt2, is required for Ahr2-dependent TCDD toxicity. This also shows that arnt1 and arnt2 are not always redundant in vivo and that the Class I dimerization partner may influence whether the Class II dimerization partners are redundant.

Figure 5.
Figure 5. Mutation in arnt1, but not arnt2, prevents AHR2-dependent TCDD toxicity.

Wild type or mutant larvae were exposed to 0.1% DMSO (A, B, C), or 2,3,7,8-tetrachlorodibenzodioxin (TCDD) (D, E, F) from 1-3 dpf and imaged at 3 dpf. Wildtype embryos exposed to TCDD (D) lack an S-shaped cardiac loop and exhibit cardiac edema, unlike their vehicle treated siblings (A). arnt1 homozygous mutant larvae (arnt1bcm2/bcm2) treated with vehicle and TCDD (B,E) show normal heart looping and do not have cardiac edema. arnt2 homozyous mutant larvae (arnt1bcm3/bcm3) treated with vehicle (C) show expected heart looping similar to wild type siblings, but arnt2 homozyous mutant larvae treated with TCDD (F) lack an S-shaped cardiac loop similar to wild type and exhibit cardiac edema similar to TCDD treated wild-type embryos. Black arrows indicate location of the heart. Embryos were genotyped following imaging and representative images are shown. TCDD treated wildtype siblings of arnt1 mutant embryos, n= 9 embryos across 3 clutches; TCDD treated arnt1 mutant larvae, n=21 larvae across 3 clutches. TCDD treated wildtype siblings of arnt2 larvae, n=19 embryos across 3 cluches; TCDD treated arnt2 mutant larvae, n=22 embryos across 4 cluches. All embryos are oriented with anterior to the right and dorsal towards the top of the image. Scale bar is 100 μm.

DISCUSSION

Class I bHLH-PAS proteins must dimerize with Class II bHLH-PAS proteins to transcribe their target genes (Ema et al., 1996; Erbel et al., 2003; Jiang et al., 1996; Lees and Whitelaw, 1999; Michaud et al., 2000; Wu and Rastinejad, 2017). It has previously been established that Npas4l, a Class I bHLH-PAS transcription factor, is required for hemato-vascular development in zebrafish (Reischauer et al., 2016). To determine the Class II bHLH-PAS binding partner of Npas4l, we generated arnt1 mutant and arnt2 mutant zebrafish and expected one of these mutants to mimic the npas4l mutant phenotype. However, both arnt1 mutant and arnt2 mutant larvae are viable to 5dpf and are indistinguishable from wildtype embryos by eye. Our results demonstrate that double homozygous arnt1/2 mutant embryos have a similar phenotype to npas4l mutants (Reischauer et al., 2016; Stainier et al., 1995b; Thompson et al., 1998) and that Npas4l can function with either Arnt1 or Arnt2. Thus, Arnt1 and Arnt2 act redundantly to specify hemato-vascular progenitor cells.

Endothelial cell development and hematopoietic cell development are closely related during embryogenesis. Both cell types primarily arise from the lateral plate mesoderm (LPM) and share a common progenitor (Qian et al., 2005; Reischauer et al., 2016; Stainier et al., 1995a; Thompson et al., 1998). Additionally, during definitive hematopoiesis, some HSCs are derived from specialized endothelial cells, called hemogenic endothelium (Boisset et al., 2010; Kissa and Herbomel, 2010). Both npas4l and arnt1/2 mutants lack all blood cells but do have a small population of endothelial cells, which is restricted to the most posterior region of the animal (Figure 2 and (Reischauer et al., 2016)). In zebrafish, there is a population of endothelial cells in the caudal region of the embryo that are not derived from the LPM and differentiate in the absence of npas4l expression (Pak et al., 2020). We hypothesize that arnt1 and arnt2 are required for the differentiation of endothelial cells from the LPM but are not required for the differentiation of endothelial cells, such as the caudal population, from outside the LPM. The transcriptomic signature of the caudal population of endothelial cells was distinct from endothelial cells that require npas4l for differentiation, with genes for somitogenesis and neurogenesis highly enriched compared to LPM-derived endothelial cells (Pak et al., 2020). Based on this transcriptome analysis, we speculate that the caudal population of endothelial cells fails to express genes required for hemogenic endothelium and that no blood cells are derived from the caudal population of endothelial cells. This is consistent with the observation that the caudal population of endothelial cells, but not blood cells, are present in npas4l or arnt1/2 mutants.

Our results show that either Arnt1 or Arnt2, Class II bHLH-PAS proteins, can act as a required co-factor for Npas4l-dependent mesodermal specification to hematopoietic and endothelial progenitors. Likewise, Arnt1 is essential for adult hematopoietic stem cells in mice and is required for survival of hematopoietic progenitor cells in the fetal liver (Krock et al., 2015). Homozygous Arnt1 mutant mice are not viable beyond E10.5 and show severe defects in vascularization, suggesting that Arnt1 is essential for normal vascular development (Kozak et al., 1997; Maltepe et al., 1997). The role of Arnt1 during mammalian embryonic hematopoiesis remains unclear.

Based on these data we speculate that the mammalian equivalent to zebrafish Npas4l is likely to be a Class I bHLH-PAS protein that heterodimerizes with ARNT1. Zebrafish Npas4l shows the most sequence homology to mouse Npas4 (Reischauer et al., 2016). Although Npas4 is known to dimerize with Arnt1 and Arnt2 in vivo (Brigidi et al., 2019), mammalian Npas4 is unlikely to be the functional equivalent to zebrafish Npas4l because homozygous Npas4 mutant mice are viable to adulthood and do not show any overt vascular or hematopoietic defects (Bloodgood et al., 2013).

Similarly, we speculate that Npas1, 2, and 3 are unlikely to be the mammalian equivalent to Npas4l, as mice mutant for these genes are viable to adulthood and no endothelial or hematopoietic phenotype has been reported (Dudley et al., 2003; Erbel-Sieler et al., 2004). Additionally, we think that the Sim (Sim1/2) and Circadian Locomotor Output Cycles Kaput (CLOCK) genes are unlikely to the be the master regulators of hemato-vascular specification in mice or humans because mutant mice appear to have normal blood cells and vasculature (DeBruyne et al., 2007; Epstein et al., 2000; Holder et al., 2004). We cannot exclude the possibility that Npas1, 2, 3 and 4 proteins act redundantly to specify hemato-vascular progenitors in mammalian embryos because mice with mutations in multiple Npas genes have not been reported.

Three other Class I bHLH-PAS proteins, Ahr, Hif1α, and Hif2α, are known to be involved in mammalian hematopoiesis and vascularization. Lack of Ahr in vitro and in vivo promotes adult hematopoietic progenitor expansion and resulted in aberrant ratios and functionality of some blood cell populations, suggesting that Ahr could be functionally relevant during definitive hematopoiesis (Singh et al., 2011; Smith et al., 2013). However, there is no evidence that Ahr is expressed during primitive hematopoiesis in mice and Ahr mutant mice are viable to adulthood and do not show any vascular defects (Singh et al., 2011).

Hif1α and Hif2α are involved in mouse vascular development and hematopoiesis (Iyer et al., 1998; Krock et al., 2015; Peng et al., 2000; Scortegagna et al., 2003; Takubo et al., 2010). However, the role of Hif1α and Hif2α during early mouse embryonic hematopoiesis and endothelial development is not well characterized. Hif1α is required for cell cycle quiescence of adult hematopoietic stem cells (Takubo et al., 2010). Hif1α mRNA is detected during mouse primitive hematopoiesis and Hif1α homozygous mutant mice die in utero before E11.5 (Iyer et al., 1998; Kotch et al., 1999). The Hif1α mutant mice are reported to have defects in blood vessels, increased death of vascular-supporting mesenchymal cells, and cardiovascular defects (Iyer et al., 1998; Kotch et al., 1999). In mice, Hif2α mRNA is expressed during primary hematopoiesis specifically within the yolk sac, where primary hematopoiesis originates (Flamme et al., 1997). Global knockout of Hif2α is reported to be embryonic or postnatal lethal depending upon the mutation and strain of mouse used (Compernolle et al., 2002; Peng et al., 2000; Tian et al., 1998). Embryonic lethal Hif2α mutants showed severe vascular disorganization and increased frequency of hemorrhaging in utero (Compernolle et al., 2002; Peng et al., 2000). Both Hif1α and Hif2α mutants have endothelial and blood cells, thus neither mutation mimics the zebrafish npas4l phenotype. But both Hif1α and Hif2α mutant mice do have a vascular phenotype (Compernolle et al., 2002; Compernolle et al., 2003; Iyer et al., 1998; Peng et al., 2000; Ryan et al., 1998). Although neither Hif1α nor Hif2α single mutant mice recapitulate the phenotype observed in zebrafish cloche or arnt1;arnt2 mutants, the HIFs are strong candidates to be the functional equivalent of Npas4l.

One reason we may not see an Npas4l-like phenotype in Hif1α or Hif2α single mutant mice could be because of genetic compensation. Genetic compensation occurs in both zebrafish and mice (Bunton-Stasyshyn et al., 2019; Rossi et al., 2015). There is evidence in zebrafish and mice of Hif1α and Hif2α functionally compensating for one another. Zebrafish with reduced expression of all 4 Hif1 and Hif2 transcripts (hif1aa, hif1ab, hif2aa, hif2ab) lack hemogenic endothelium, suggesting that at least one HIF gene is required for the specification of hemogenic endothelium (Gerri et al., 2018). These results also indicate that hif1α and hif2α are functionally redundant for the generation of hemogenic endothelium (Gerri et al., 2018). Global knockout of mouse Hif1α is embryonic lethal (Compernolle et al., 2003; Iyer et al., 1998; Ryan et al., 1998) and global knockout of mouse Hif2α is embryonic or postnatal lethal depending upon the strain of mouse used (Compernolle et al., 2002; Peng et al., 2000; Tian et al., 1998).

To our knowledge, no one has generated a mouse in which both Hif1α and Hif2α are globally knocked-out. Future studies could examine if global or mesodermal-specific knockout of both Hif1α and Hif2α genes in mouse results in a phenotype similar to knockout of both arnt1 and arnt2 in zebrafish. Our results demonstrate that under some conditions, Arnt1 and Arnt2 act redundantly. In other contexts, Arnt1 is the preferred binding protein for a class I PAS transcription factor. The extent to which this occurs during different developmental stages in vivo, or in different cell types, warrants further study.

Acknowledgements

We thank Lauren Pandolfo and the staff of the Baylor College of Medicine aquatics facility for taking care of our zebrafish colony. This work funded by NIH grants R01ES026337 and P30ES030285.

References

  1. ↵
    Andreasen, E.A., Hahn, M.E., Heideman, W., Peterson, R.E., Tanguay, R.L., 2002. The zebrafish (Danio rerio) aryl hydrocarbon receptor type 1 is a novel vertebrate receptor. Molecular pharmacology 62, 234–249.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    Aplan, P.D., Nakahara, K., Orkin, S.H., Kirsch, I.R., 1992. The SCL gene product: a positive regulator of erythroid differentiation. The EMBO journal 11, 4073–4081.
    OpenUrlPubMedWeb of Science
  3. ↵
    Bloodgood, B.L., Sharma, N., Browne, H.A., Trepman, A.Z., Greenberg, M.E., 2013. The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition. Nature 503, 121–125.
    OpenUrlCrossRefPubMedWeb of Science
  4. ↵
    Boisset, J.C., van Cappellen, W., Andrieu-Soler, C., Galjart, N., Dzierzak, E., Robin, C., 2010. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120.
    OpenUrlCrossRefPubMedWeb of Science
  5. ↵
    Brigidi, G.S., Hayes, M.G.B., Delos Santos, N.P., Hartzell, A.L., Texari, L., Lin, P.A., Bartlett, A., Ecker, J.R., Benner, C., Heinz, S., Bloodgood, B.L., 2019. Genomic Decoding of Neuronal Depolarization by Stimulus-Specific NPAS4 Heterodimers. Cell 179, 373–391 e327.
    OpenUrlCrossRef
  6. ↵
    Brownlie, A., Donovan, A., Pratt, S.J., Paw, B.H., Oates, A.C., Brugnara, C., Witkowska, H.E., Sassa, S., Zon, L.I., 1998. Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia. Nat Genet 20, 244–250.
    OpenUrlCrossRefPubMedWeb of Science
  7. ↵
    Bunton-Stasyshyn, R.K.A., Wells, S., Teboul, L., 2019. When all is not lost: considering genetic compensation in laboratory animals. Lab Anim (NY) 48, 282–284.
    OpenUrlCrossRef
  8. ↵
    Casie Chetty, S., Rost, M.S., Enriquez, J.R., Schumacher, J.A., Baltrunaite, K., Rossi, A., Stainier, D.Y., Sumanas, S., 2017. Vegf signaling promotes vascular endothelial differentiation by modulating etv2 expression. Dev Biol 424, 147–161.
    OpenUrlCrossRef
  9. ↵
    Compernolle, V., Brusselmans, K., Acker, T., Hoet, P., Tjwa, M., Beck, H., Plaisance, S., Dor, Y., Keshet, E., Lupu, F., Nemery, B., Dewerchin, M., Van Veldhoven, P., Plate, K., Moons, L., Collen, D., Carmeliet, P., 2002. Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 8, 702–710.
    OpenUrlCrossRefPubMedWeb of Science
  10. ↵
    Compernolle, V., Brusselmans, K., Franco, D., Moorman, A., Dewerchin, M., Collen, D., Carmeliet, P., 2003. Cardia bifida, defective heart development and abnormal neural crest migration in embryos lacking hypoxia-inducible factor-1alpha. Cardiovasc Res 60, 569–579.
    OpenUrlCrossRefPubMed
  11. ↵
    Costa, G., Kouskoff, V., Lacaud, G., 2012. Origin of blood cells and HSC production in the embryo. Trends Immunol 33, 215–223.
    OpenUrlCrossRefPubMedWeb of Science
  12. ↵
    DeBruyne, J.P., Weaver, D.R., Reppert, S.M., 2007. CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock. Nat Neurosci 10, 543–545.
    OpenUrlCrossRefPubMedWeb of Science
  13. ↵
    Dobrzycki, T., Krecsmarik, M., Bonkhofer, F., Patient, R., Monteiro, R., 2018. An optimised pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridisation. Biol Open 7.
  14. ↵
    Dooley, K.A., Davidson, A.J., Zon, L.I., 2005. Zebrafish scl functions independently in hematopoietic and endothelial development. Dev Biol 277, 522–536.
    OpenUrlCrossRefPubMed
  15. ↵
    Doss, M.X., Gaspar, J.A., Winkler, J., Hescheler, J., Schulz, H., Sachinidis, A., 2012. Specific gene signatures and pathways in mesodermal cells and their derivatives derived from embryonic stem cells. Stem Cell Rev Rep 8, 43–54.
    OpenUrl
  16. ↵
    Dudley, C.A., Erbel-Sieler, C., Estill, S.J., Reick, M., Franken, P., Pitts, S., McKnight, S.L., 2003. Altered patterns of sleep and behavioral adaptability in NPAS2-deficient mice. Science 301, 379–383.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Dumont, D.J., Fong, G.H., Puri, M.C., Gradwohl, G., Alitalo, K., Breitman, M.L., 1995. Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development. Dev Dyn 203, 80–92.
    OpenUrlCrossRefPubMedWeb of Science
  18. ↵
    Ema, M., Morita, M., Ikawa, S., Tanaka, M., Matsuda, Y., Gotoh, O., Saijoh, Y., Fujii, H., Hamada, H., Kikuchi, Y., Fujii-Kuriyama, Y., 1996. Two new members of the murine Sim gene family are transcriptional repressors and show different expression patterns during mouse embryogenesis. Molecular and cellular biology 16, 5865–5875.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Epstein, D.J., Martinu, L., Michaud, J.L., Losos, K.M., Fan, C., Joyner, A.L., 2000. Members of the bHLH-PAS family regulate Shh transcription in forebrain regions of the mouse CNS. Development 127, 4701–4709.
    OpenUrlAbstract
  20. ↵
    Erbel, P.J., Card, P.B., Karakuzu, O., Bruick, R.K., Gardner, K.H., 2003. Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor. Proc Natl Acad Sci U S A 100, 15504–15509.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Erbel-Sieler, C., Dudley, C., Zhou, Y., Wu, X., Estill, S.J., Han, T., Diaz-Arrastia, R., Brunskill, E.W., Potter, S.S., McKnight, S.L., 2004. Behavioral and regulatory abnormalities in mice deficient in the NPAS1 and NPAS3 transcription factors. Proc Natl Acad Sci U S A 101, 13648–13653.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O’Shea, K.S., Powell-Braxton, L., Hillan, K.J., Moore, M.W., 1996. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439–442.
    OpenUrlCrossRefPubMedWeb of Science
  23. ↵
    Flamme, I., Frohlich, T., von Reutern, M., Kappel, A., Damert, A., Risau, W., 1997. HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 alpha and developmentally expressed in blood vessels. Mech Dev 63, 51–60.
    OpenUrlCrossRefPubMedWeb of Science
  24. ↵
    Gerri, C., Marass, M., Rossi, A., Stainier, D.Y.R., 2018. Hif-1alpha and Hif-2alpha regulate hemogenic endothelium and hematopoietic stem cell formation in zebrafish. Blood 131, 963–973.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Gong, B., Liang, D., Chew, T.G., Ge, R., 2004. Characterization of the zebrafish vascular endothelial growth factor A gene: comparison with vegf-A genes in mammals and Fugu. Biochim Biophys Acta 1676, 33–40.
    OpenUrlPubMed
  26. ↵
    Gunaseeli, I., Doss, M.X., Antzelevitch, C., Hescheler, J., Sachinidis, A., 2010. Induced pluripotent stem cells as a model for accelerated patient- and disease-specific drug discovery. Curr Med Chem 17, 759–766.
    OpenUrlCrossRefPubMed
  27. ↵
    Hill, A.J., Heiden, T.C., Heideman, W., Peterson, R.E., 2009. Potential roles of Arnt2 in zebrafish larval development. Zebrafish 6, 79–91.
    OpenUrlCrossRefPubMedWeb of Science
  28. ↵
    Hirose, K., Morita, M., Ema, M., Mimura, J., Hamada, H., Fujii, H., Saijo, Y., Gotoh, O., Sogawa, K., Fujii-Kuriyama, Y., 1996. cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt). Molecular and cellular biology 16, 1706–1713.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Hirschi, K.K., 2012. Hemogenic endothelium during development and beyond. Blood 119, 4823–4827.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Holder, J.L., Jr.., Zhang, L., Kublaoui, B.M., DiLeone, R.J., Oz, O.K., Bair, C.H., Lee, Y.H., Zinn, A.R., 2004. Sim1 gene dosage modulates the homeostatic feeding response to increased dietary fat in mice. American journal of physiology. Endocrinology and metabolism 287, E105–113.
    OpenUrlCrossRefPubMedWeb of Science
  31. ↵
    Huber, T.L., Kouskoff, V., Fehling, H.J., Palis, J., Keller, G., 2004. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432, 625–630.
    OpenUrlCrossRefPubMedWeb of Science
  32. ↵
    Hughes, M.C., Zimmer, A.M., Perry, S.F., 2019. Role of internal convection in respiratory gas transfer and aerobic metabolism in larval zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 316, R255–R264.
    OpenUrlCrossRef
  33. ↵
    Iyer, N.V., Kotch, L.E., Agani, F., Leung, S.W., Laughner, E., Wenger, R.H., Gassmann, M., Gearhart, J.D., Lawler, A.M., Yu, A.Y., Semenza, G.L., 1998. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes & development 12, 149–162.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Jao, L.E., Wente, S.R., Chen, W., 2013. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A 110, 13904–13909.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Jiang, B.H., Rue, E., Wang, G.L., Roe, R., Semenza, G.L., 1996. Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 271, 17771–17778.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Kalev-Zylinska, M.L., Horsfield, J.A., Flores, M.V., Postlethwait, J.H., Vitas, M.R., Baas, A.M., Crosier, P.S., Crosier, K.E., 2002a. Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis. Development 129, 2015–2030.
    OpenUrlCrossRefPubMedWeb of Science
  37. ↵
    Kalev-Zylinska, M.L., Horsfield, J.A., Flores, M.V.C., Postlethwait, J.H., Vitas, M.R., Baas, A.M., Crosier, P.S., Crosier, K.E., 2002b. Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis. Development 129, 2015–2030.
    OpenUrlCrossRefPubMedWeb of Science
  38. ↵
    Kataoka, H., Hayashi, M., Nakagawa, R., Tanaka, Y., Izumi, N., Nishikawa, S., Jakt, M.L., Tarui, H., Nishikawa, S., 2011. Etv2/ER71 induces vascular mesoderm from Flk1+PDGFRalpha+ primitive mesoderm. Blood 118, 6975–6986.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Kissa, K., Herbomel, P., 2010. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115.
    OpenUrlCrossRefPubMedWeb of Science
  40. ↵
    Kobayashi, I., Kobayashi-Sun, J., Kim, A.D., Pouget, C., Fujita, N., Suda, T., Traver, D., 2014. Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling. Nature 512, 319–323.
    OpenUrlCrossRefPubMed
  41. ↵
    Kotch, L.E., Iyer, N.V., Laughner, E., Semenza, G.L., 1999. Defective vascularization of HIF-1alpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death. Dev Biol 209, 254–267.
    OpenUrlCrossRefPubMedWeb of Science
  42. ↵
    Kozak, K.R., Abbott, B., Hankinson, O., 1997. ARNT-deficient mice and placental differentiation. Dev Biol 191, 297–305.
    OpenUrlCrossRefPubMedWeb of Science
  43. ↵
    Krock, B.L., Eisinger-Mathason, T.S., Giannoukos, D.N., Shay, J.E., Gohil, M., Lee, D.S., Nakazawa, M.S., Sesen, J., Skuli, N., Simon, M.C., 2015. The aryl hydrocarbon receptor nuclear translocator is an essential regulator of murine hematopoietic stem cell viability. Blood 125, 3263–3272.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    Labun, K., Montague, T.G., Gagnon, J.A., Thyme, S.B., Valen, E., 2016. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44, W272–276.
    OpenUrlCrossRefPubMed
  45. ↵
    Labun, K., Montague, T.G., Krause, M., Torres Cleuren, Y.N., Tjeldnes, H., Valen, E., 2019. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47, W171–W174.
    OpenUrlCrossRefPubMed
  46. ↵
    Lam, E.Y., Chau, J.Y., Kalev-Zylinska, M.L., Fountaine, T.M., Mead, R.S., Hall, C.J., Crosier, P.S., Crosier, K.E., Flores, M.V., 2009. Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors. Blood 113, 1241–1249.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Lauter, G., Soll, I., Hauptmann, G., 2011. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Neural Dev 6, 10.
    OpenUrl
  48. ↵
    Lee, R.K., Stainier, D.Y., Weinstein, B.M., Fishman, M.C., 1994. Cardiovascular development in the zebrafish. II. Endocardial progenitors are sequestered within the heart field. Development 120, 3361–3366.
    OpenUrlAbstract
  49. ↵
    Lees, M.J., Whitelaw, M.L., 1999. Multiple roles of ligand in transforming the dioxin receptor to an active basic helix-loop-helix/PAS transcription factor complex with the nuclear protein Arnt. Molecular and cellular biology 19, 5811–5822.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Liao, E.C., Paw, B.H., Oates, A.C., Pratt, S.J., Postlethwait, J.H., Zon, L.I., 1998. SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes & development 12, 621–626.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Liao, W., Bisgrove, B.W., Sawyer, H., Hug, B., Bell, B., Peters, K., Grunwald, D.J., Stainier, D.Y., 1997. The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation. Development 124, 381–389.
    OpenUrlAbstract
  52. ↵
    Liao, W., Ho, C.Y., Yan, Y.L., Postlethwait, J., Stainier, D.Y., 2000. Hhex and scl function in parallel to regulate early endothelial and blood differentiation in zebrafish. Development 127, 4303–4313.
    OpenUrlAbstract
  53. ↵
    Lohr, H., Ryu, S., Driever, W., 2009. Zebrafish diencephalic A11-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages. Development 136, 1007–1017.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Maltepe, E., Schmidt, J.V., Baunoch, D., Bradfield, C.A., Simon, M.C., 1997. Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 386, 403–407.
    OpenUrlCrossRefPubMedWeb of Science
  55. ↵
    Marchi, D., Santhakumar, K., Markham, E., Li, N., Storbeck, K.H., Krone, N., Cunliffe, V.T., van Eeden, F.J.M., 2020. Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae. PLoS Genet 16, e1008757.
    OpenUrl
  56. ↵
    Michaud, J.L., DeRossi, C., May, N.R., Holdener, B.C., Fan, C.M., 2000. ARNT2 acts as the dimerization partner of SIM1 for the development of the hypothalamus. Mech Dev 90, 253–261.
    OpenUrlCrossRefPubMedWeb of Science
  57. ↵
    Moore, C., Richens, J.L., Hough, Y., Ucanok, D., Malla, S., Sang, F., Chen, Y., Elworthy, S., Wilkinson, R.N., Gering, M., 2018. Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo. Blood Adv 2, 2589–2606.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    Moore, J.C., Sheppard-Tindell, S., Shestopalov, I.A., Yamazoe, S., Chen, J.K., Lawson, N.D., 2013. Post-transcriptional mechanisms contribute to Etv2 repression during vascular development. Dev Biol 384, 128–140.
    OpenUrlCrossRefPubMed
  59. ↵
    Okuda, T., van Deursen, J., Hiebert, S.W., Grosveld, G., Downing, J.R., 1996. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84, 321–330.
    OpenUrlCrossRefPubMedWeb of Science
  60. ↵
    Paik, E.J., Zon, L.I., 2010. Hematopoietic development in the zebrafish. Int J Dev Biol 54, 1127–1137.
    OpenUrlCrossRefPubMed
  61. ↵
    Pak, B., Schmitt, C.E., Choi, W., Kim, J.D., Han, O., Alsio, J., Jung, D.W., Williams, D.R., Coppieters, W., Stainier, D.Y.R., Jin, S.W., 2020. Analyses of Avascular Mutants Reveal Unique Transcriptomic Signature of Non-conventional Endothelial Cells. Front Cell Dev Biol 8, 589717.
    OpenUrl
  62. ↵
    Palis, J., Robertson, S., Kennedy, M., Wall, C., Keller, G., 1999. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073–5084.
    OpenUrlAbstract
  63. ↵
    Parant, J.M., George, S.A., Pryor, R., Wittwer, C.T., Yost, H.J., 2009. A rapid and efficient method of genotyping zebrafish mutants. Dev Dyn 238, 3168–3174.
    OpenUrlCrossRefPubMed
  64. ↵
    Pelster, B., Burggren, W.W., 1996. Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio). Circulation Research 79, 358–362.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    Peng, J., Zhang, L., Drysdale, L., Fong, G.H., 2000. The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodeling. Proc Natl Acad Sci U S A 97, 8386–8391.
    OpenUrlAbstract/FREE Full Text
  66. ↵
    Pongratz, I., Antonsson, C., Whitelaw, M.L., Poellinger, L., 1998. Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor. Mol Cell Biol 18.
  67. ↵
    Prasch, A.L., Tanguay, R.L., Mehta, V., Heideman, W., Peterson, R.E., 2006. Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1. Molecular pharmacology 69, 776–787.
    OpenUrlAbstract/FREE Full Text
  68. ↵
    Qian, F., Zhen, F., Ong, C., Jin, S.W., Meng Soo, H., Stainier, D.Y., Lin, S., Peng, J., Wen, Z., 2005. Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes. Dev Dyn 233, 1163–1172.
    OpenUrlCrossRefPubMedWeb of Science
  69. ↵
    Reischauer, S., Stone, O.A., Villasenor, A., Chi, N., Jin, S.W., Martin, M., Lee, M.T., Fukuda, N., Marass, M., Witty, A., Fiddes, I., Kuo, T., Chung, W.S., Salek, S., Lerrigo, R., Alsio, J., Luo, S., Tworus, D., Augustine, S.M., Mucenieks, S., Nystedt, B., Giraldez, A.J., Schroth, G.P., Andersson, O., Stainier, D.Y., 2016. Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification. Nature 535, 294–298.
    OpenUrlCrossRefPubMed
  70. ↵
    Romano, S.N., Edwards, H.E., Souder, J.P., Ryan, K.J., Cui, X., Gorelick, D.A., 2017. G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish. PLoS Genet 13, e1007069.
    OpenUrl
  71. ↵
    Rossi, A., Kontarakis, Z., Gerri, C., Nolte, H., Holper, S., Kruger, M., Stainier, D.Y., 2015. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524, 230–233.
    OpenUrlCrossRefPubMed
  72. ↵
    Ryan, H.E., Lo, J., Johnson, R.S., 1998. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. The EMBO journal 17, 3005–3015.
    OpenUrlAbstract/FREE Full Text
  73. ↵
    Scortegagna, M., Morris, M.A., Oktay, Y., Bennett, M., Garcia, J.A., 2003. The HIF family member EPAS1/HIF-2alpha is required for normal hematopoiesis in mice. Blood 102, 1634–1640.
    OpenUrlAbstract/FREE Full Text
  74. ↵
    Shalaby, F., Rossant, J., Yamaguchi, T.P., Gertsenstein, M., Wu, X.F., Breitman, M.L., Schuh, A.C., 1995. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62–66.
    OpenUrlCrossRefPubMedWeb of Science
  75. ↵
    Shu, L.P., Zhou, Z.W., Zhou, T., Deng, M., Dong, M., Chen, Y., Fu, Y.F., Jin, Y., Zhou, S.F., He, Z.X., 2015. Ectopic expression of Hoxb4a in hemangioblasts promotes hematopoietic development in early embryogenesis of zebrafish. Clinical and experimental pharmacology & physiology 42, 1275–1286.
    OpenUrl
  76. ↵
    Singh, K.P., Garrett, R.W., Casado, F.L., Gasiewicz, T.A., 2011. Aryl Hydrocarbon Receptor-Null Allele Mice Have Hematopoietic Stem/Progenitor Cells with Abnormal Characteristics and Functions. Stem Cells and Development 20, 769–784.
    OpenUrlCrossRefPubMedWeb of Science
  77. ↵
    Smith, B.W., Rozelle, S.S., Leung, A., Ubellacker, J., Parks, A., Nah, S.K., French, D., Gadue, P., Monti, S., Chui, D.H., Steinberg, M.H., Frelinger, A.L., Michelson, A.D., Theberge, R., McComb, M.E., Costello, C.E., Kotton, D.N., Mostoslavsky, G., Sherr, D.H., Murphy, G.J., 2013. The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation. Blood 122, 376–385.
    OpenUrlAbstract/FREE Full Text
  78. ↵
    Souder, J.P., Gorelick, D.A., 2019. ahr2, But Not ahr1a or ahr1b, Is Required for Craniofacial and Fin Development and TCDD-dependent Cardiotoxicity in Zebrafish. Toxicol Sci 170, 25–44.
    OpenUrl
  79. ↵
    Stainier, D.Y., Lee, R.K., Fishman, M.C., 1993. Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development 119, 31–40.
    OpenUrl
  80. ↵
    Stainier, D.Y., Weinstein, B.M., Detrich, H.W., 3rd., Zon, L.I., Fishman, M.C., 1995a. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development 121, 3141–3150.
    OpenUrlAbstract
  81. ↵
    Stainier, D.Y., Weinstein, B.M., Detrich, H.W., Zon, L.I., Fishman, M.C., 1995b. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development 121, 3141–3150.
    OpenUrlAbstract
  82. ↵
    Takubo, K., Goda, N., Yamada, W., Iriuchishima, H., Ikeda, E., Kubota, Y., Shima, H., Johnson, R.S., Hirao, A., Suematsu, M., Suda, T., 2010. Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell stem cell 7, 391–402.
    OpenUrlCrossRefPubMedWeb of Science
  83. ↵
    Tanguay, R.L., Andreasen, E., Heideman, W., Peterson, R.E., 2000. Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions. Biochim Biophys Acta 1494, 117–128.
    OpenUrlPubMedWeb of Science
  84. ↵
    Thisse, C., Thisse, B., 2008. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 3, 59–69.
    OpenUrlCrossRefPubMedWeb of Science
  85. ↵
    Thompson, M.A., Ransom, D.G., Pratt, S.J., MacLennan, H., Kieran, M.W., Detrich, H.W., 3rd., Vail, B., Huber, T.L., Paw, B., Brownlie, A.J., Oates, A.C., Fritz, A., Gates, M.A., Amores, A., Bahary, N., Talbot, W.S., Her, H., Beier, D.R., Postlethwait, J.H., Zon, L.I., 1998. The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev Biol 197, 248–269.
    OpenUrlCrossRefPubMedWeb of Science
  86. ↵
    Tian, H., Hammer, R.E., Matsumoto, A.M., Russell, D.W., McKnight, S.L., 1998. The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development. Genes & development 12, 3320–3324.
    OpenUrlAbstract/FREE Full Text
  87. ↵
    Vogeli, K.M., Jin, S.W., Martin, G.R., Stainier, D.Y., 2006. A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula. Nature 443, 337–339.
    OpenUrlCrossRefPubMed
  88. ↵
    Wang, Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A.H., Speck, N.A., 1996. Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proceedings of the National Academy of Sciences of the United States of America 93, 3444–3449.
    OpenUrlAbstract/FREE Full Text
  89. ↵
    Weng, W., Nagai, H., Hamidi, S., Sheng, G., 2020. NPAS4L is involved in avian hemangioblast specification. Haematologica.
  90. ↵
    Westerfield, M., 2000. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio Rerio). 4th ed. University of Oregon Press, Eugene, OR.
  91. ↵
    Wu, D., Rastinejad, F., 2017. Structural characterization of mammalian bHLH-PAS transcription factors. Curr Opin Struct Biol 43, 1–9.
    OpenUrl
  92. ↵
    Zhang, C., Featherstone, M., 2020. A zebrafish hox gene acts before gastrulation to specify the hemangioblast. Genesis, e23363.
Back to top
Posted January 04, 2022.
Download PDF
Email
Hemato-vascular specification requires arnt1 and arnt2 genes in zebrafish embryos
Hailey E. Edwards, Jaclyn P. Souder, Daniel A. Gorelick
bioRxiv 2022.01.04.474920; doi: https://doi.org/10.1101/2022.01.04.474920
Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area