https://orcid.org/0000-0003-0464-0577
Figures
Abstract
The gene SON is on human chromosome 21 (21q22.11) and is thought to be associated with hematopoietic disorders that accompany Down syndrome. Additionally, SON is an RNA splicing factor that plays a role in the transcription of leukemia-associated genes. Previously, we showed that mutations in SON cause malformations in human and zebrafish spines and brains during early embryonic development. To examine the role of SON in normal hematopoiesis, we reduced expression of the zebrafish homolog of SON in zebrafish at the single-cell developmental stage with specific morpholinos. In addition to the brain and spinal malformations we also observed abnormal blood cell levels upon son knockdown. We then investigated how blood production was altered when levels of son were reduced. Decreased levels of son resulted in lower amounts of red blood cells when visualized with lcr:GFP transgenic fish. There were also reduced thrombocytes seen with cd41:GFP fish, and myeloid cells when mpx:GFP fish were examined. We also observed a significant decrease in the quantity of T cells, visualized with lck:GFP fish. However, when we examined their hematopoietic stem and progenitor cells (HSPCs), we saw no difference in colony-forming capability. These studies indicate that son is essential for the proper differentiation of the innate and adaptive immune system, and further investigation determining the molecular pathways involved during blood development should elucidate important information about vertebrate HSPC generation, proliferation, and differentiation.
Citation: Belmonte RL, Engbretson IL, Kim J-H, Cajias I, Ahn E-YE, Stachura DL (2021) son is necessary for proper vertebrate blood development. PLoS ONE 16(2): e0247489. https://doi.org/10.1371/journal.pone.0247489
Editor: Daniel R. Larson, National Institutes of Health, UNITED STATES
Received: July 17, 2020; Accepted: February 8, 2021; Published: February 25, 2021
Copyright: © 2021 Belmonte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This research was supported by the CSU Program for Education and Research in Biotechnology (CSUPERB) (New Investigator Awards to D.L.S.), NSF MRI (proposal 1626406), a California State University Chico Internal Research Grant (to D.L.S.), Student Research and Creativity Awards from California State University, Chico (to R.B.) and the NIH (R01CA190688 to E.-Y.E.A. and R15DK114732-01 to D.L.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: D.L.S. is a scientific consultant and has received compensation from Finless Foods, Inc. and Xytogen Biotech, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors declare no competing interests.
Introduction
Blood development (hematopoiesis) is the process by which blood cells acquire specific characteristics through carefully regulated gene expression [1]. All blood cells arise from hematopoietic stem cells (HSCs), which are adult stem cells capable of self-renewal and differentiation into more developmentally restricted blood progenitor cells [2–4]. HSCs differentiate into either a common myeloid progenitor (CMP) or a common lymphoid progenitor (CLP). The CMP can produce all erythromyeloid cells, which are red blood cells, thrombocytes, granulocytes, and monocytes [5, 6]. The CLP can produce lymphoid cells, such as B cells, T cells, and NK cells [7, 8]. In essence, red blood cells (which carry oxygen to tissues), granulocytes (which fight infections), thrombocytes (which prevent bleeding), and lymphocytes (which confer adaptive immunity) are very different mature blood cells with diverse functions that all originated from HSCs. Many molecular factors affect the differentiation of blood cells by changing the gene expression of progenitor cells, and this control is essential- if the wrong numbers or wrong types of mature blood cells are produced it can have serious clinical effects on an individual. Understanding the molecular control of these processes is essential for understanding blood development and treating human diseases. With the advent of gene therapy, discovering mutant genes associated with abnormal blood development will also allow their correction, providing new treatments for blood diseases.
Our gene of interest, SON, is located on human chromosome 21 and may play a role in regulation of gene expression associated with the Down syndrome phenotype. Down syndrome patients with trisomy 21 have increased levels of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL) [9]. SON codes for a protein that forms an RNA-splicing molecule important for processing other genes during transcription, especially several involved in cell-cycle progression [10]. SON was also described as a negative regulatory element binding protein (NREBP) due to its transcriptional repression of human hepatitis B virus genes [11] and regulates the transcriptional initiation of leukemia-associated genes in human and mouse cells [12]. SON was later classified as a regulator of pre-mRNA splicing and a nuclear speckle protein [13]. In 2011, it was further shown that SON functioned in pre-mRNA splicing [10, 14]. SON also has a role in regulating the pluripotency of human embryonic stem cells [15] and is highly expressed in HSCs and downregulated during hematopoietic differentiation [16]. Previous work we performed indicates that mutations in SON are linked to spinal and brain malformations in human patients [17], and these patients also have hematologic and kidney abnormalities [18, 19]. Due to the ability of SON to regulate multiple genes, mutations seen in human patients, and its expression in HSCs, we hypothesized that SON played a role in regulating blood development and formation of the immune system.
To understand the effects of SON in blood development, zebrafish (Danio rerio) were utilized as a model system. These experiments are not possible to conduct on humans, and zebrafish have many advantages over other animal models. Zebrafish have a well-conserved genome compared to humans, and they undergo rapid development [20, 21]. It is difficult to study blood development in mammals because they develop in utero, but zebrafish develop externally and are optically transparent at the larval stage, aiding in their visualization during embryonic development. Overall, zebrafish have been widely established as a model organism to study hematopoiesis [22, 23]. To study how SON affects blood development, we utilized transgenic zebrafish that have blood-specific gene promoters driving green fluorescent protein expression in specific blood cell types. These fluorescently labelled cells allow for easy in vivo visualization of blood in developing embryos and it allows the use of flow cytometry to interrogate and quantify the numbers of these cells present in an animal [20, 24]. To understand how each branch of hematopoiesis is affected, we used zebrafish that have fluorescently labeled red blood cells, thrombocytes, myeloid cells, and T cells.
For this investigation, we utilized previously validated morpholinos (MOs) [17, 19] to study the role of SON in blood development. While there is not a zebrafish gene annotated in the genome named “SON,” the well-conserved ortholog to human SON is on an unplaced scaffold of the zebrafish genome (NCBI Gene: LOC565999) identified as CABZ01113192.1 (www.ensembl.org, assembly GRCz11). Although the protein encoded by CABZ01113192.1 is much smaller than the human SON protein, the domains critical for SON’s function (i.e. serine/arginine-rich domain, G-patch, and double-stranded RNA-binding motif) are well conserved between these two species. As these genes are orthologous (see [17] for alignments between zebrafish and human orthologs), throughout the paper we refer to CABZ01113192.1 as son. MOs we used are specific antisense oligonucleotide that bind only to son mRNA; this binding alters its splicing, reducing the levels of normal Son protein in the developing fish [17, 19, 25]. Zebrafish are excellent model organisms to use with MOs, since they have been established as an inexpensive, effective method of knocking down levels of specific genes [26]. MOs allow researchers to reduce gene products versus causing their complete ablation, which can sometimes be lethal, especially with genes that may have pleiotropic effects. MOs also do not cause genetic compensation, an issue recently discovered with CRISPR-mediated gene knockout [27].
Overall, reducing son and investigating blood and immune cells indicates that this gene plays an important role in early hematopoietic development. After injecting single-cell embryos with son MO, we saw a significant decrease in the amount of red blood cells, thrombocytes, myeloid cells, and T cells. However, we did not see a difference in the quantity of erythromyeloid hematopoietic stem and progenitor cells (HSPCs), indicating that son is involved in proper blood maturation. While we don’t yet understand the specific molecular mechanism by which this happens, this study offers insight into an important gene regulating normal blood cell development that is altered in human diseases. Additionally, it will offer new avenues for treatment of hematologic disorders associated with dysregulated SON expression.
Materials and methods
Zebrafish husbandry and care
Zebrafish were mated, staged, and raised as described [28] and maintained in accordance with California State University (CSU), Chico Institutional Animal Care and Use Committee (IACUC) guidelines. All procedures were approved by the CSUC IACUC before being performed. Personnel were trained in animal care by taking the online Citi Program training course entitled “Working With Zebrafish (Danio rerio) in Research Settings” (https://www.citiprogram.org). Wildtype (wt) and the transgenic zebrafish lines lcr:GFP [29], mpx:EGFP [30], cd41:GFP (also known as itga2b:GFP) [31], lck:EGFP [32], and fli1a:EGFP [33] were used for these studies. Zebrafish were housed in a 700L recirculating zebrafish aquarium system (Aquatic Enterprises, Seattle, WA) regulated by a Profilux 3 Outdoor module that regulated salinity, pH, and temperature (GHL International, Kaiserslautern, Germany) 24-hours-a day. The facility was illuminated on a 14-hour light/ 10-hour dark cycle. Zebrafish were fed once a day with hatched brine shrimp (Brine Shrimp Direct, Ogden, UT) and once a day with Gemma micro 300 (Skretting, Westbrook, ME). After experiments were performed all animals were returned to the aquarium system to be used for further research.
MO injection
To examine the specific function of son, 1nL of son MO (Gene Tool, LLC, Philomath, OR) was injected at 6.25μM into single-cell zebrafish embryos, resulting in a total injection of 6.25ng of the MO into each individual. This amount was used based on previous studies [17, 19]. The son MO sequence is 5’-TGGTCCTGGATATAACAGACAGATT-3’ [17]. Control MOs were also utilized (5’-CCTCTTACCTCAGTTACAATTTATA-3’) at the same concentration.
Rescue assays
son cDNA was subcloned into pcDNA3.1+/- and linearized with Sca1. son mRNA was generated using a mMessage T7 kit (Ambion, Austin, TX). 1nL of son MO (at 6.25μM) and 0.5nL of son mRNA (at 1μg/μL) was injected into single-cell zebrafish embryos, resulting in a total injection of 6.25ng of the MO and 500ng mRNA into each individual. son mRNA was visualized after being electrophoresed on a 1% w/v agaose gel with TAE and 1% bleach added (S1 Fig).
Microscopy
All observations were made with a Leica M165C microscope and pictures were taken with a Leica DFC295 camera. lcr:GFP, mpx:GFP, and fli1:GFP embryos were sorted at 24 hours post fertilization (hpf) and observed and imaged at 48hpf. Images of mpx:GFP embryos were taken at 48hpf and the number of fluorescent cells per embryo were enumerated. To perform this, embryo images were taken at the same exposure and every mpx:GFP+ cell was manually counted. To prevent bias, the images were coded and students counted the mpx:GFP+ blinded from the experimental variable. cd41:GFP embryos were sorted into phenotypic groups, counted, and imaged at 72hpf in a similar manner. lck:GFP embryos were observed at 5 days post fertilization (dpf) and images were taken of each embryo. For thymus quantitation, every image was taken at 80x and the focal plane was adjusted to best capture the whole thymus. The exposure was kept constant for all images to reduce variation between images. Images of lck:GFP embryos were analyzed with ImageJ (https://imagej.nih.gov/ij/) to determine the pixel density of each thymus. Briefly, a bounding box was drawn around the thymus, and ImageJ calculated the numbers of pixels present. To correct for background fluorescence, each image had an equal sized box drawn outside of the embryo’s image in a non-fluorescent region. The pixel density of this box was subtracted from the pixel density of the thymus to normalize all the images. To prevent bias, the images were coded and students analyzed the pixel density blinded from the experimental variable.
Methylcellulose assay
To determine the effect of reduced son expression on erythromyeloid HSPC colony formation, clonal methylcellulose assays were performed [24, 34–38]. Briefly, five embryos were selected at random from uninjected animals, digested, and plated in supportive media. Five animals were also randomly selected from the son MO-injected group and plated at the same time to compare the colony growth seen in uninjected versus MO-injected samples [38, 39]. The digested embryos were grown in methylcellulose media with carp serum, Gcsf, and Epo added to selectively stimulate myeloid and erythroid differentiation from HSPCs. Samples were incubated at 5% CO2 at 32°C for 7 days. Each sample was observed at 40x with an Olympus IX53 inverted microscope (Olympus Life Science, Waltham, MA) and colonies were counted from each sample. Each point shown on the graph is a biological replicate of five randomly selected embryos from uninjected versus MO-injected categories.
Reverse transcriptase PCR (RT-PCR)
RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) from uninjected embryos and those injected with son MO at 24hpf, 48hpf, and 72hpf. iScript (Bio-Rad, Hercules, CA) was used to generate cDNA from each sample. Each cDNA sample was made by pooling ten random whole embryos per condition. PCR was performed with JumpStart TAQ polymerase ready mix (Invitrogen, Carlsbad, CA) with the following primers for son: FWD: 5’-ATGGAGAAAATCCAACTGTG-3’ and REV: 5’-GACCTTAAAGAGGAAGTTC-3’. ef1α [40] was used as a reference gene.
Quantitative reverse transcriptase PCR (qRT-PCR)
RNA was extracted with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) from uninjected embryos and those injected with son MO and those injected with son MO and son mRNA at 48hpf, 72hpf, and 5dpf. iScript (Bio-Rad, Hercules, CA) was used to generate cDNA from each sample. Each cDNA sample was made by pooling ten random whole embryos per condition. Samples were then run on an Eppendorf realplex2 (Eppendorf, Hamburg, Germany) with primers for ef1α [40], band3 (also called slc4a1a) [41], cmpl [40], hbaa1 (FWD: 5’-GGACAAGGCTGTTGTTAAGG-3’, REV 5’-AGACCAGTGAGAGAAGTAG-3’), cd41 [40], mpx [42], csf3r [42], and lck [43]). Data were analyzed for relative expression change with ef1α [40] as the reference gene. ΔΔCt was calculated by comparing the expression of the injected embryos to control embryos and to the reference gene, ef1α.
Flow cytometry
To enumerate the percentage of RBCs in an embryo, we used transgenic lcr:GFP [29] zebrafish in combination with flow cytometry. 48 hpf transgenic embryos were grouped in samples of ten and washed 3x with Dulbecco’s phosphate-buffered saline (DPBS) containing Ca2+ and Mg2+. After the last wash, 500 μL of DPBS and 5 μL of 5 mg/mL (26U/mL) Liberase TM (Roche, Upper Bavaria, Germany) were added. Samples were incubated at 37°C on a horizontal orbital shaker at 180 rpm for 60 mins. Samples were than triturated with a P-1000 to ensure proper dissociation and transferred to a 5 mL polystyrene round bottom tube with cell strainer cap. Samples were strained, centrifuged, and resuspended in 100 μL of DPBS and 1 μL of SytoxRed (ThermoFisher Scientific, Waltham, MA) was added to each sample to label dead cells. Samples were run through a BD FACSAria Fusion flow cytometer (BD Biosciences, San Jose, CA) and enumerated. Data were analyzed using FloJo software (FloJo LLC, Ashland, Oregon) to quantitate total percentage of positive fluorescent cells.
Statistical methods
Statistical analyses were performed in Microsoft Excel. To discern statistical difference, data were analyzed using an unpaired two-tailed Student’s T test assuming unequal variance. All raw data from these studies are supplied in S1 Data.
Results
To elucidate the role of son in blood development, we used a son-specific MO [17, 19] to reduce protein levels. Injection of the MO at the single-cell stage of development and examination of son mRNA levels at 24, 48, and 72 hpf indicated a reduction in full-length son, likely caused by mis-splicing and nonsense-mediated decay (S2 Fig). These injections allowed us to then observe phenotypic changes caused by a reduction of son levels. After seeing no blood-specific phenotypic differences between embryos injected with the control MO and uninjected embryos (data not shown, [17, 38, 39], S3 and S4 Figs), we used uninjected embryos as the control for the remaining experiments. We first performed experiments in a transgenic zebrafish line that has the alpha globin locus control region (lcr) driving GFP expression; these lcr:GFP transgenic zebrafish only express GFP in red blood cells (RBCs) [29]. Injection of son MO into embryos caused several phenotypes; the majority of injected embryos either had no circulating RBCs or reduced levels of visible RBCs present at 48hpf (Fig 1A–1E). About 10% of injected fish had no discernible RBCs at all, while less than 10% were phenotypically normal (Fig 1A–1E). To confirm these studies, we quantitated RBC numbers in son morphants with flow cytometry. Embryos injected with son MOs had reduced levels of RBCs when compared to embryos injected with control MOs (S3A and S3B Fig), and this was rescued by the addition of son mRNA (S3C Fig). Quantitation of these data can be seen in S3D Fig. Additionally, qRT-PCR showed a significant decrease in the expression of band3 (slc4a1a) and hbaa1, both specific markers of mature erythrocytes, when son MO was injected compared to control embryos (Fig 1F). Importantly, rescuing son reduction with the addition of son mRNA rescued RBC loss (Fig 1E) and restored the expression of band3 and hbaa1 (Fig 1F). Together, these data indicate that decreased son impairs successful erythropoiesis.
Representative images of 48hpf lcr:GFP embryos injected with son MO at the one-cell-stage of development show four common phenotypic categories associated with son MO injection. Category titles are listed to the left of each image. Brightfield images are shown in top left (40x), and fluorescent images are shown in top right (40x). Zoomed in fluorescent images are shown below (80x); each green dot in the animal is a labelled RBC. Fluorescent “trails” of green are cells moving in the vasculature. Red arrows indicate RBCs flowing though the dorsal aorta, and blue arrows indicate RBCs flowing back to the heart through the caudal vein. Red arrowheads indicate large numbers of RBCs pooled in the ducts of Cuvier, located on the yolk ball. (A) Representative images of normal RBC numbers and blood flow. (B) Representative animals with no RBCs (no blood), (C) no RBCs circulating in the animal (no circulation), and (D) reduced numbers of RBCs, some of which are circulating (little blood). (E) Quantitation of phenotypes shown. Number of embryos analyzed is shown above the chart. (F) qRT-PCR for slc4a1a (left) and hbaa1 (right) was performed. Each point represents ten embryos randomly selected from uninjected, MO-injected (son MO), or MO-injected with son RNA (rescue) conditions that were analyzed by qRT-PCR. Middle lines represent mean and error bars represent SD. * represents p = 0.04, ** represents p = 0.02, *** represents p = 0.004, **** represents p < 0.001, N.S. represents no significance.
We next examined thrombocytes, cells responsible for blood clotting, by utilizing cd41:GFP transgenic animals that have GFP+ thrombocytes [31] (Fig 2). These embryos showed a significant decrease in cd41:GFP+ thrombocytes at 72hpf, with most injected animals showing no thrombocytes at all (Fig 2A–2E). Over 15% of embryos had less thrombocytes than uninjected controls, close to 10% had no thrombocytes in circulation, and very few had normal, circulating cells (Fig 2A–2E). We also performed qRT-PCR on these embryos at 72hpf and observed a significant decrease in the thrombocytic markers cmpl and cd41 in injected embryos when compared to the control embryos (Fig 2F). Again, rescuing the loss of son expression with mRNA restored levels of thrombocytes (Fig 2E) and thrombocytic genes (Fig 2F). Together, these data indicate that son is also important for normal thrombocyte production in the embryo.
Representative images of 72hpf cd41:GFP embryos injected with son MO at the one-cell-stage of development show four common phenotypic categories associated with MO injection. Category titles are listed to the left of each image. Brightfield images are shown in top left (40x), and fluorescent images are shown in top right (40x). Zoomed in fluorescent images are shown below (80x); each green dot in the animal is a labelled thrombocyte. Red arrows indicate fluorescent “trails” made by moving thrombocytes flowing though the vasculature. Red brackets indicate the caudal hematopoietic region where the majority of thrombocytes reside at this time. (A) Representative images of normal thrombocyte numbers and circulating thrombocytes. (B) Representative animals with no thrombocytes, (C) no thrombocytes circulating in the animal (no circulation), and (D) reduced numbers of thrombocytes, some of which are circulating (little blood). (E) Quantitation of phenotypes shown. Number of embryos analyzed is shown above the chart. (F) qRT-PCR for cmpl (left) and cd41 (right) was performed. Each point represents ten embryos randomly selected from uninjected, MO-injected (son MO), or MO-injected with son RNA (rescue) conditions that were analyzed by qRT-PCR. Middle lines represent mean and error bars represent SD. * represents p = 0.04, ** represents p = 0.006, *** represents p = 0.002, **** represents p = 0.001, N.S. represents no significance.
Next, we investigated the effects of son reduction on myeloid cells using mpx:GFP embryos, which label neutrophils [30]. 48hpf embryos from the uninjected and son MO-injected groups (Fig 3A) were imaged. Then, the number of fluorescent myeloid cells per embryo were enumerated (Fig 3B). The control embryos had a significantly higher number of myeloid cells compared to embryos injected with son MO (Fig 3B). We also performed qRT-PCR on these embryos at 48hpf and observed a significant decrease in the myeloid markers mpx and csf3r in MO-injected embryos when compared to controls (Fig 3C). Once again, son mRNA rescued these phenotypic reductions in neutrophils (Fig 3B and 3C). These data indicate that son is also crucial for proper myeloid cell development.
(A) Representative images of mpx:GFP embryos at 48hpf that were uninjected (left) or MO-injected (son MO, right). Images are taken at 40x, and every green dot is a labelled neutrophil; these cells are in tissues and not circulating. (B) Each point represents the number of GFP+ myeloid cells present in an individual uninjected, MO-injected (son MO), or MO-injected with son RNA (rescue) embryo. Images were taken and GFP+ cells were manually enumerated. Middle lines represent mean and error bars represent SD. (C) qRT-PCR for mpx (left) and csf3r (right) was performed. Each point represents ten embryos randomly selected from uninjected, MO-injected (son MO), or MO-injected with son RNA (rescue) conditions that were analyzed by qRT-PCR. Middle lines represent mean and error bars represent SD. * represents p = 0.02, ** represents p = 0.01, *** represents p < 0.001, N.S. represents no significance.
Next, we investigated if son perturbation negatively affected the number of T cells with lck:GFP embryos [32]. Since T cells develop in the thymus, we imaged each fluorescent thymus from individuals at the same exposure time from both the control and son MO-injected groups at 5dpf, the time when T cells arise (Fig 4A). We maintained a consistent focal plane to minimize outside effects on the magnitude of fluorescence detected and used ImageJ to calculate the pixel density of each thymus. The control embryos had a higher average thymus pixel density than the son MO-injected embryos (Fig 4B). qRT-PCR of these animals at 5dpf for lck, a T cell specific marker, also was decreased in son morphants (Fig 4C). Again, we observed that addition of son mRNA rescued these reductions (Fig 4B and 4C), indicating that son is also necessary for proper T cell development.
(A) Representative images of lck:GFP embryos at 5dpf that were uninjected (control; left) or injected with MO at the one-cell-stage of development (son MO, right). Images were taken at 80x. Individual GFP+ T cells are located in the thymi (red dashed oval); these cells are not circulating. * denotes background fluorescence present in the yolk ball due to refraction of light from lipids present in the yolk. (B) Images (like shown in A) were subjected to analysis with ImageJ to determine the pixel density of uninjected, MO-injected (son MO), or MO-injected with son RNA (rescue) thymi. (C) qRT-PCR for lck was performed. Each point represents ten embryos randomly selected from uninjected, MO-injected (son MO), or MO-injected with son RNA (rescue) conditions that were analyzed by qRT-PCR. Middle lines represent mean and error bars represent SD. * represents p = 0.02, ** represents p = 0.01, *** represents p < 0.001, N.S. represents no significance.
After observing a decrease in mature blood cell types, we turned our attention to enumerating erythromyeloid HSPCs, which give rise to those blood lineages. We performed these experiments to examine if these key players in hematopoiesis were also reduced or if the loss of blood cells was somehow caused by a failure of HSPC differentiation. To perform these experiments, we used a methylcellulose assay to enumerate the amount of these HSPCs in each embryo at 48hpf [38]. With this assay, either five random uninjected embryos or five random son MO-injected embryos were enzymatically digested in separate tubes. The digested embryos were then plated in methylcellulose medium along with hematopoietic-supportive cytokines and growth factors. Under these conditions, HSPCs divide and proliferate, but the medium does not allow the cells to migrate throughout the plate, so each colony that is seen represents one HSPC that was present in the original embryo (Fig 5A). The numbers of colonies were then counted under a microscope to determine the colony forming units (CFUs) present in individual embryos. Interestingly, we saw no significant difference between the number of CFUs from control and son MO-injected embryos (Fig 5B). These data indicate that son is not crucial for HSPC generation in the 48hpf embryo.
(A) Experimental procedure. (B) wt embryos uninjected (left) or injected with son MO at the one-cell-stage of development (son MO, right) were dissociated at 48hpf and plated in methylcellulose media with Epo, Gcsf, and carp serum. Fold difference in colony forming units (CFUs) is shown. Each point represents ten embryos randomly selected from the respective conditions that were dissociated and plated. Middle lines represent mean and error bars represent SD. N.S. represents no significance.
Finally, we wanted to ensure that progenitors were not reduced due to a defect in the vascular system of the developing embryo, as HSCs arise from hemogenic endothelium in the dorsal aorta between 36-52hpf [44, 45]. We also wanted to confirm that the vasculature was not negatively affected in embryos that have severe bending of the spine and other defects associated with son reduction [17–19]. Imaging the vasculature of son MO-injected fli1:GFP transgenic embryos at 48hpf showed no discernible defects in the dorsal aorta, even if fish were severely bent, a phenotype associated with son reduction (S4 Fig). Together, these data point to son regulating HSPCs without negatively affecting the formation of mesoderm that gives rise to those progenitors.
Discussion
We previously showed that zebrafish are an effective model organism for investigating the effects of son knockdown with a specific MO and that son is necessary for proper brain [17], skeletal [17], and kidney formation [19]. Here we report that son is necessary for proper blood formation through an analysis of mature blood cells and embryonic HSPCs. We saw a decrease in the number of RBCs, thrombocytes, myeloid cells, and T cells; however, we did not see a change in the number of erythromyeloid HSPCs. Further studies are warranted to establish the molecular mechanism by which son (and its human ortholog SON), directs and manages blood cell differentiation.
In our studies, we show that mature RBCs are reduced in number at 48hpf when measured by fluorescent microscopy, flow cytometry, and qRT-PCR analysis. While we observed many fish with no RBCs and reduced numbers of RBCs, we also saw fish with less RBCs in circulation. It is interesting to note that the morphant fish do not have vascular defects; imaging of fli1:GFP embryos indicated no issue with vessel integrity or formation, and visible liquid could be seen circulating through the vessels. We also saw a reduction in thrombocytes at 72hpf when measured by fluorescence microscopy and qRT-PCR. Again, in addition to fish with no thrombocytes and reduced numbers of thrombocytes, we observed fish with no thrombocytes in circulation. Examination of non-circulating RBCs indicated that many of them were lodged in the ducts of Cuvier, while the thrombocytes were mostly located in the caudal hematopoietic tissue (CHT). It is unclear why these cells never entered circulation; it did not appear to be a developmental delay, as the cells were still not circulating when examined at 5dpf. Homing is not completely defective in son morphants, however. Even though we observed decreased numbers of neutrophils in mpx:GFP fish, they still migrated throughout the embryo similar to uninjected controls. Additionally, lck:GFP T cells were decreased in morphants, but still were properly located in the thymus. Examination of son’s role in homing or trafficking cells may be warranted in the future to see why these cells were specifically affected in this way.
It is important to note that we reduced the levels of Son in this study with a son-specific MO, which is a splice-blocking MO that significantly reduced the amount of full length son mRNA present in the embryo. To examine the effect of son MO on blood cells, we examined the time at which they arose during development. Definitive RBCs and myeloid cells are first detected at 48hpf. Thrombocytes are first seen at 72hpf, and T cells arise at 5dpf (as reviewed in [46]). All images were taken and qRT-PCR was performed when mature blood cells were first seen; we did follow son morphants up to 5dpf, but we never saw a recovery in the number of blood cells. It is not useful to follow the fish for much longer than this, as MOs appear to start losing their effectiveness as early as 4dpf [47]. While it is difficult to say whether or not fish would recover normal blood cell counts after this time period, these data indicate that the blood reduction observed is not just a delay in the development of the hematopoietic system caused by MO injection. Observation of embryos injected with control MOs showed no blood defects, confirming these findings.
All of the cells that we examined are derived from definitive HSPCs during development, meaning that they differentiate from upstream progenitor cells. While RBCs are present before 48hpf, they are mostly primitive RBCs that do not derive from a definitive precursor like an erythromyeloid progenitor (EMP) or HSC [48]. Thrombocytes observed at 72hpf and neutrophils imaged at 48hpf also are derived from either EMPs or HSCs [48]. T cells, on the other hand, are derived from the differentiation of HSCs; they are lymphoid cells that cannot be generated by EMPs [48]. To examine if HSPCs were affected, we used an in vitro methylcellulose assay that can quantitate differences between normal and morphant fish. While other experimental assays exist to study and measure HSPCs, they all have their limitations. Microscopy of labelled cells can indicate putative progenitors, but one must follow these cells over time to see where they go and what they become in the developing embryo, which is challenging and requires specialized microscopy equipment. Transplantation experiments also exist, but are costly and time consuming. Transplantation is also usually used to prove long-term engraftment, so it is more useful for proving the existence of long-lived HSCs versus downstream short-lived HSPCs like CMPs, GMPs, and CLPs. In essence, methylcellulose assays are an alternative to quickly and efficiently determine if there is an imbalance in the number of HSPCs present in an organism. However, methylcellulose assays also have deficiencies; namely that they depend on the cytokines added to the medium for encouraging what cells will survive and proliferate. Additionally, you cannot detect T cells, which require a thymus to develop.
In our studies, the numbers of HSPCs grown with Epo and Gcsf were similar between the morphant and uninjected fish. This indicates that erythroid and myeloid differentiation is similar in the morphant animals when compared to uninjected animals. Importantly, human patients with SON deficiency have recurrent infections, low immunoglobulin levels, and clotting issues [18], suggesting that these patients have issues with hematopoiesis progressing properly from HSCs to definitive progenitors to mature blood cells. Human SON is located on human chromosome 21 and triplicated in Down syndrome, and these patients have elevated rates of AMKL, TMD, and other leukemias primarily caused by defects in the differentiation of HSPCs. Clearly further studies are warranted, possibly in another model system; CMPs, GMPs, and CLPs are speculated to exist in the zebrafish, but no transgenics or good assays exist to examine these cell types. There should also be attention given to blood cell types that were not addressed in our study, such as B cells, NK cells, dendritic cells, and basophils. All of these cells exist in zebrafish but are difficult to assay. Finally, it could be that son has an effect on the actual formation of the hematopoietic system, which derives from similar progenitors responsible for vasculogenesis. While the vasculature marker fli1 appears to be completely unaffected in morphants, there may be some interest in understanding the biology of this gene regulation in the cardiovascular system, especially on the role it plays in the formation of blood. Previously we showed that son plays a role in kidney development [19]. As the main site of hematopoiesis in teleosts, it will be of interest to see if this delay in kidney development is involved in aberrant hematopoietic development. Overall, these studies indicate that there should be further examination of the mechanism by which son affects HSPC formation and differentiation.
We have now shown that son affects brain [17], spine [17], kidney [19], and blood development in zebrafish. It is particularly interesting that both brain and blood development are affected by son since the brain develops from ectoderm while blood develops from mesoderm. One hypothesis for how one gene can affect both endoderm and mesoderm derived tissues is through niche signaling from the ectoderm to the mesoderm during HSPC development. Research suggests that ectoderm-derived neural crest cells contribute to the HSC niche, impacting HSC development [49]. Trunk neural crest cells, which become sympathetic nervous system neurons, physically associate with the dorsal aorta, the location of primary blood development, prior to hematopoietic initiation. Any disruption of this interaction results in impaired hematopoiesis [49]. Further examination of this is warranted- although son reduction does not reduce the number of HSPCs detected in our experiments, it may reduce the number of HSPCs that mature properly to give rise to mature blood. It may also play a role in their release from the niche to seed hematopoietic organs later in development or influence skewing of HSCs into CMPs, CLPs, or other downstream HSPCs. Another possibility is that son is regulating brain, spinal, and blood development through alternative mRNA splicing in these different tissues. son may operate pleiotropically, regulating distinct genes in different tissue types or by regulating different genes at different developmental time points. Differential gene regulation in this way could explain how SON is able to impact a variety of cell types. A closer look into the specific genes that son regulates may shed light on how one gene can affect the formation and homeostasis of many diverse tissues.
It is worth noting that zebrafish Son and human SON are not exactly the same, which may limit the use of zebrafish to understand certain human phenotypes related to specific mutations in SON, especially in a repetitive amino acid region that is lacking in the zebrafish ortholog. Son and SON share only 47% sequence identity, but do have a sequence identity of 48% in the RS domain (Ser/Arg-rich domain), 79% in the G-patch domain (an RNA binding domain), and 67% in the DSRM (double stranded RNA-binding motif). Importantly, the total homology between the proteins from the RS domain to the C terminus is 59% [17]. While these domains are essential for the function of Son and SON, not all human patients had mutations in these regions. In fact, 13/27 patients identified in our previous studies had mutations in a repetitive amino acid region upstream of the RS domain [17, 19]. While the zebrafish loss-of-function phenotypes seem to recapitulate the brain [17], spine [17], kidney [19], and blood phenotypes seen in human patients, it will be difficult to study specific mutations located in the repeat regions with the zebrafish model.
Overall, these studies indicate that son is involved in normal blood cell differentiation in a vertebrate model system. Additional research on the role of son in mammals such as mice and humans will help gain a clearer understanding of how son affects blood development and homeostasis, and how its perturbation plays a role in blood diseases.
Supporting information
S1 Fig. son mRNA shows two distinct sizes.
Full length son mRNA produced by the mMachine kit has two sizes: the top band is mRNA produced from circular son plasmid, and the bottom band is from linearized son plasmid.
https://doi.org/10.1371/journal.pone.0247489.s001
(TIF)
S2 Fig. son MO reduces full length son transcript.
10 random embryos either uninjected or injected with son MO at the one-cell-stage of development (son MO) collected at 24hpf (left), 48hpf (middle), and 72hpf (right) were subjected to RT-PCR for son (top) and ef1α (bottom) transcripts.
https://doi.org/10.1371/journal.pone.0247489.s002
(TIF)
S3 Fig. son MO reduces RBC numbers.
Flow cytometry of lcr:GFP+ embryos at 48hpf either injected with control MO (A), son MO (B), or MO-injected with son RNA (rescue) (C). lcr:GFP is shown along the x-axis; y-axis is the red fluorescence channel, examined at the same time to gate out auto-fluorescent and light-refractive cells in the developing embryo. Each plot represents ten embryos randomly selected from control MO, MO-injected (son MO), and MO-injected with son RNA (rescue) conditions that were enzymatically dissociated and analyzed with a flow cytometer. The numbers in the gates are the percentage of GFP+ RBCs present. (D) Data presented in graphical format. Middle lines represent mean and error bars represent SD. * represents p = 0.05, ** represents p < 0.001, N.S. represents no significance.
https://doi.org/10.1371/journal.pone.0247489.s003
(TIFF)
S4 Fig. son knockdown does not negatively affect vasculature formation.
Representative images of 48hpf fli1:GFP embryos injected with control MO (top) or son MO (bottom) at the one-cell-stage of development show no significant differences in vessel formation or integrity associated with son MO injection. Brightfield images are shown in top left (40x), and fluorescent images are shown in top right (40x). Zoomed in fluorescent images are shown below (80x). Red arrows and brackets indicate the dorsal aorta, the site of HSC formation.
https://doi.org/10.1371/journal.pone.0247489.s004
(TIF)
Acknowledgments
We thank Betsey Tamietti for excellent laboratory management and Kathy Johns for administrative assistance.
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