The transcriptional regulator GON4L is required for viability and hematopoiesis in mice
2021; Elsevier BV; Volume: 98; Linguagem: Inglês
10.1016/j.exphem.2021.04.001
ISSN1873-2399
AutoresDiana F. Colgan, Renee X. Goodfellow, John Colgan,
Tópico(s)Zebrafish Biomedical Research Applications
Resumo•Previous study of a recessive, hypomorphic point mutation in the gene Gon4l suggested a role in the early stages of B-cell development. However, the point mutation does not completely abolish Gon4l gene function.•Unlike the point mutation, conventional knockout of Gon4l revealed the gene is required for mouse viability.•Conventional knockout of Gon4l also indicated that Gon4l is required not only for B-cell development, but also for the generation and maintenance of the entire hematopoietic system.•The defects observed because of conventional knockout of Gon4l extended to definitive hematopoietic stem/progenitor cells, which are the source of all components of the hematopoietic system.•Mechanistic studies using transformed pro-B cells revealed that loss of Gon4l induced growth arrest and apoptosis, suggesting the GON4L protein is essential for cell viability. The Gon4l gene encodes a putative transcriptional regulator implicated in the control of both cell differentiation and proliferation. Previously, we described a mutant mouse strain called Justy in which splicing of pre-mRNA generated from Gon4l is disrupted. This defect severely reduces, but does not abolish, GON4L protein expression and blocks the formation of early B-lineage progenitors, suggesting Gon4l is required for B-cell development in vertebrates. Yet, mutations that disable Gon4l in zebrafish impair several facets of embryogenesis that include the initiation of primitive hematopoiesis, arguing this gene is needed for multiple vertebrate developmental pathways. To better understand the importance of Gon4l in mammals, we created mice carrying an engineered version of Gon4l that can be completely inactivated by Cre-mediated recombination. Breeding mice heterozygous for the inactivated Gon4l allele failed to yield any homozygous-null offspring, indicating Gon4l is an essential gene in mammals. Consistent with this finding, as well previously published results, cell culture studies revealed that loss of Gon4l blocks cell proliferation and compromises viability, suggesting a fundamental role in the control of cell division and survival. Studies using mixed bone marrow chimeras confirmed Gon4l is required for B-cell development but also found it is needed to maintain definitive hematopoietic stem/progenitor cells that are the source of all hematopoietic cell lineages. Our findings reveal Gon4l is an essential gene in mammals that is required to form the entire hematopoietic system. The Gon4l gene encodes a putative transcriptional regulator implicated in the control of both cell differentiation and proliferation. Previously, we described a mutant mouse strain called Justy in which splicing of pre-mRNA generated from Gon4l is disrupted. This defect severely reduces, but does not abolish, GON4L protein expression and blocks the formation of early B-lineage progenitors, suggesting Gon4l is required for B-cell development in vertebrates. Yet, mutations that disable Gon4l in zebrafish impair several facets of embryogenesis that include the initiation of primitive hematopoiesis, arguing this gene is needed for multiple vertebrate developmental pathways. To better understand the importance of Gon4l in mammals, we created mice carrying an engineered version of Gon4l that can be completely inactivated by Cre-mediated recombination. Breeding mice heterozygous for the inactivated Gon4l allele failed to yield any homozygous-null offspring, indicating Gon4l is an essential gene in mammals. Consistent with this finding, as well previously published results, cell culture studies revealed that loss of Gon4l blocks cell proliferation and compromises viability, suggesting a fundamental role in the control of cell division and survival. Studies using mixed bone marrow chimeras confirmed Gon4l is required for B-cell development but also found it is needed to maintain definitive hematopoietic stem/progenitor cells that are the source of all hematopoietic cell lineages. Our findings reveal Gon4l is an essential gene in mammals that is required to form the entire hematopoietic system. Hematopoiesis generates all components of the blood [1Jagannathan-Bogdan M Zon LI Hematopoiesis.Development. 2013; 140: 2463-2467Crossref PubMed Scopus (181) Google Scholar]. In vertebrates, bone marrow is the main site of hematopoiesis after birth. Here, rare, self-renewing hematopoietic stem cells (HSCs) respond to environmental cues and produce multipotent progenitors (MPPs). These cell populations differentiate and commit to a particular fate, leading to the generation of erythroid, myeloid, and lymphoid cells. Hematopoiesis is largely controlled by transcription factor networks that enforce widespread remodeling of gene expression and promote differentiation into specific cell lineages [2Menegatti S de Kruijf M Garcia-Alegria E Lacaud G Kouskoff V Transcriptional control of blood cell emergence.FEBS Lett. 2019; 593: 3304-3315Crossref PubMed Scopus (12) Google Scholar]. Progression of hematopoiesis is also coordinated with bursts of cell proliferation [3Hao S Chen C Cheng T Cell cycle regulation of hematopoietic stem or progenitor cells.Int J Hematol. 2016; 103: 487-497Crossref PubMed Scopus (37) Google Scholar]. Although much has been learned about the factors that guide hematopoiesis, it is likely that additional proteins important for this process remain to be identified.GON4L is a 250-kDa nuclear protein predicted to contain domains characteristic of transcriptional regulators; these include a highly acidic region with homology to nucleoplasmin histone-binding domains, two paired amphipathic helix repeats, and a SANT-L domain [4Ohtomo T Horii T Nomizu M Suga T Yamada J Molecular cloning of a structural homolog of YY1AP, a coactivator of the multifunctional transcription factor YY1.Amino Acids. 2007; 33: 645-652Crossref PubMed Scopus (14) Google Scholar,5Lu P Hankel IL Hostager BS et al.The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.J Biol Chem. 2011; 286: 18311-18319Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. GON4L forms complexes with the transcriptional regulators YY1, SIN3A, and HDAC1 [5Lu P Hankel IL Hostager BS et al.The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.J Biol Chem. 2011; 286: 18311-18319Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar] and also interacts with NPAT, a transcriptional coactivator important for replication-dependent histone gene expression [6Zhao J Kennedy BK Lawrence BD et al.NPAT links cyclin E-Cdk2 to the regulation of replication-dependent histone gene transcription.Genes Dev. 2000; 14: 2283-2297Crossref PubMed Scopus (268) Google Scholar,7Yang XC Sabath I Kunduru L van Wijnen AJ Marzluff WF Dominski Z A conserved interaction that is essential for the biogenesis of histone locus bodies.J Biol Chem. 2014; 289: 33767-33782Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. GON4L appears to have a key role in regulating the expression of genes that guide specific developmental pathways [8Liu Y Du L Osato M et al.The zebrafish udu gene encodes a novel nuclear factor and is essential for primitive erythroid cell development.Blood. 2007; 110: 99-106Crossref PubMed Scopus (28) Google Scholar, 9Bulchand S Menon SD George SE Chia W Muscle wasted: a novel component of the Drosophila histone locus body required for muscle integrity.J Cell Sci. 2010; 123: 2697-2707Crossref PubMed Scopus (28) Google Scholar, 10Lu P Hankel IL Knisz J et al.The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis.J Exp Med. 2010; 207: 1359-1367Crossref PubMed Scopus (19) Google Scholar, 11Williams MLK Sawada A Budine T Yin C Gontarz P Solnica-Krezel L Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes.Nat Commun. 2018; 9: 1319Crossref PubMed Scopus (19) Google Scholar, 12Budine TE de Sena-Tomas C Williams MLK Sepich DS Targoff KL Solnica-Krezel L Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.Dev Biol. 2020; 262: 223-234Crossref Scopus (5) Google Scholar], as well as those important for cell proliferation [12Budine TE de Sena-Tomas C Williams MLK Sepich DS Targoff KL Solnica-Krezel L Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.Dev Biol. 2020; 262: 223-234Crossref Scopus (5) Google Scholar,13Barr JY Goodfellow RX Colgan DF Colgan JD Early B cell progenitors deficient for GON4L fail to differentiate due to a block in mitotic cell division.J Immunol. 2017; 198: 3978-3988Crossref PubMed Scopus (5) Google Scholar]. Further supporting a role in gene regulation, GON4L associates with sites throughout the zebrafish genome via some unknown mechanism, and its loss broadly disrupts gene expression in zebrafish embryos [11Williams MLK Sawada A Budine T Yin C Gontarz P Solnica-Krezel L Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes.Nat Commun. 2018; 9: 1319Crossref PubMed Scopus (19) Google Scholar].Genetic studies involving various species have suggested Gon4l orthologs are important for multiple developmental pathways. In Caenorhabditis elegans, Gon4l is needed to generate somatic gonadal tissues [14Friedman L Santa Anna-Arriola S Hodgkin J Kimble J gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans.Dev Biol. 2000; 228: 350-362Crossref PubMed Scopus (32) Google Scholar], whereas in Drosophila, the gene is required for muscle cell differentiation in developing embryos [9Bulchand S Menon SD George SE Chia W Muscle wasted: a novel component of the Drosophila histone locus body required for muscle integrity.J Cell Sci. 2010; 123: 2697-2707Crossref PubMed Scopus (28) Google Scholar]. Mutations affecting Gon4l in zebrafish disrupt several embryonic developmental pathways, including anteroposterior axis extension, somite formation, heart development, and the onset of primitive hematopoiesis [8Liu Y Du L Osato M et al.The zebrafish udu gene encodes a novel nuclear factor and is essential for primitive erythroid cell development.Blood. 2007; 110: 99-106Crossref PubMed Scopus (28) Google Scholar,11Williams MLK Sawada A Budine T Yin C Gontarz P Solnica-Krezel L Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes.Nat Commun. 2018; 9: 1319Crossref PubMed Scopus (19) Google Scholar,12Budine TE de Sena-Tomas C Williams MLK Sepich DS Targoff KL Solnica-Krezel L Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.Dev Biol. 2020; 262: 223-234Crossref Scopus (5) Google Scholar,15Hammerschmidt M Pelegri F Mullins MC et al.Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.Development. 1996; 123: 143-151Crossref PubMed Google Scholar,16Lim CH Chong SW Jiang YJ Udu deficiency activates DNA damage checkpoint.Mol Biol Cell. 2009; 20: 4183-4193Crossref PubMed Google Scholar]. We have studied a mouse strain named Justy, which bears a recessive mutation that severely reduces Gon4l mRNA expression in B-cell progenitors and blocks B-cell development [10Lu P Hankel IL Knisz J et al.The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis.J Exp Med. 2010; 207: 1359-1367Crossref PubMed Scopus (19) Google Scholar,13Barr JY Goodfellow RX Colgan DF Colgan JD Early B cell progenitors deficient for GON4L fail to differentiate due to a block in mitotic cell division.J Immunol. 2017; 198: 3978-3988Crossref PubMed Scopus (5) Google Scholar]. Although these findings suggest Gon4l function differs between species, a common theme among them is that loss of Gon4l function affects gene regulation and cell proliferation.Gon4l mutations cause distinct defects in two vertebrate species, zebrafish and mice. Given this difference, we sought to further characterize the role of Gon4l in mice using a model in which the gene can be completely disabled rather than partially as the Justy mutation does. We therefore created mice carrying a floxed Gon4l allele that can undergo Cre-mediated recombination in a drug-inducible or tissue-specific manner, resulting in complete loss of function. Using this model, we found Gon4l is an essential gene in mice. We confirmed Gon4l was necessary for B-cell development, but also found this gene is needed to sustain definitive hematopoietic stem/progenitor cells that give rise to all hematopoietic cell lineages. Together, our data indicate that as in zebrafish, Gon4l has roles in several developmental pathways in mammals and is critical for hematopoiesis.MethodsMiceWild-type (WT) C57BL/6J (B6) mice, WT CD45.1 B6 mice, those bearing the FLPe cDNA inserted into the ROSA26 (R26) locus [17Farley FW Soriano P Steffen LS Dymecki SM Widespread recombinase expression using FLPeR (flipper) mice.Genesis. 2000; 28: 106-110Crossref PubMed Scopus (483) Google Scholar]), those carrying the EIIa-cre transgene [18Lakso M Pichel JG Gorman JR et al.Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.Proc Natl Acad Sci USA. 1996; 93: 5860-5865Crossref PubMed Scopus (899) Google Scholar], those bearing the Vav-cre transgene [19Georgiades P Ogilvy S Duval H et al.VavCre transgenic mice: a tool for mutagenesis in hematopoietic and endothelial lineages.Genesis. 2002; 34: 251-256Crossref PubMed Scopus (149) Google Scholar], and those carrying the CreERT2 cDNA inserted into the R26 locus [20Ventura A Kirsch DG McLaughlin ME et al.Restoration of p53 function leads to tumour regression in vivo.Nature. 2007; 445: 661-665Crossref PubMed Scopus (1359) Google Scholar] were obtained from the Jackson Laboratory (Bar Harbor, ME). Embryonic stem (ES) cells of the C57BL/6N background bearing an engineered Gon4l allele were obtained from the International Mouse Phenotyping Consortium. Mice of both sexes were used for experiments. All mouse procedures were approved by the University of Iowa Institutional Care and Use Committee and adhere to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.Generation of pro-B cellsThe generation of pro-B cells has been described [13Barr JY Goodfellow RX Colgan DF Colgan JD Early B cell progenitors deficient for GON4L fail to differentiate due to a block in mitotic cell division.J Immunol. 2017; 198: 3978-3988Crossref PubMed Scopus (5) Google Scholar]. Pro-B cells were transduced with a retroviral vector that expresses the BCR-ABL p210 oncoprotein and green fluorescent protein (GFP) (Addgene, Watertown, MA). The BCR-ABL vector and the plasmid pCL-Eco [21Naviaux RK Costanzi E Haas M Verma IM The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.J Virol. 1996; 70: 5701-5705Crossref PubMed Google Scholar] were transfected into 293T cells. After 24 hours, culture supernatants were collected. One million pro-B cells in RPMI (RPMI-1640, Thermo Fisher, Waltham, MA) supplemented with 10% fetal bovine serum, 1% L-glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin and 50 μmol/L β-mercaptoethanol) containing interleukin (IL)-7 were added to wells in a 24-well plate coated with OP9 stromal cells. The next day, 1 mL of culture supernatant containing retroviral particles was added, and the plates were centrifuged at room temperature for 2 hours at 800g. Transduction was confirmed by analysis of GFP expression.Gon4l inactivation in pro-B cellsOne million pro-B cells in RPMI containing IL-7 were added to wells in a 24-well plate. The drug 4-Hydroxytamoxifen (4-OHT, Sigma Aldrich, St. Louis, MO) dissolved in ethanol was added to 2 μM, after which cells were cultured for up to 5 days. DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Protein lysates were prepared by resuspending cells in RIPA buffer containing protease inhibitors (Roche Life Sciences, Indianapolis, IN), incubating on ice for 10 min, and then centrifuging to remove insoluble material. Cell proliferation was assayed by harvesting cells on day 3 of culture, staining with trypan blue, and counting live cells. Cell survival was tracked by harvesting cells on days 1 through 5 of culture and identifying live cells by flow cytometry and staining with Hoechst 33258 (Sigma Aldrich). Levels of activated caspases 3 and 7 were assayed as described [22Gorman JV Starbeck-Miller G Pham NL et al.Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection.J Immunol. 2014; 192: 3133-3142Crossref PubMed Scopus (59) Google Scholar].Mixed bone marrow chimerasWild-type CD45.1 B6 mice were used as bone marrow transplant recipients; these were irradiated with 1100 rad and injected with donor bone marrow 1 hour later. Donor bone marrow was obtained by flushing bones with phosphate-buffered saline (PBS) containing 3% fetal bovine serum (FBS) and depleting red blood cells using Pharm Lyse (BD Biosciences, San Diego, CA). Ten million donor bone marrow cells were injected intravenously into recipients, which were given drinking water containing ampicillin (2mg/mL) for 2 weeks and allowed to reconstitute for an additional 8 weeks before use. Mixed bone marrow chimeras (MBMCs) were injected intraperitoneally with 40 mg/kg tamoxifen dissolved in corn oil (both from Sigma Aldrich) for 5 consecutive days and analyzed 3 weeks later.Flow cytometric analysisFlow cytometric analysis was performed as described [10Lu P Hankel IL Knisz J et al.The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis.J Exp Med. 2010; 207: 1359-1367Crossref PubMed Scopus (19) Google Scholar]. Data were collected using an LSR II (BD Biosciences) and analyzed using FlowJo software (BD Biosciences). Antibodies were obtained from BD Biosciences, BioLegend (San Diego, CA), and eBioscience (San Diego, CA). Most antibody clones used have been described [13Barr JY Goodfellow RX Colgan DF Colgan JD Early B cell progenitors deficient for GON4L fail to differentiate due to a block in mitotic cell division.J Immunol. 2017; 198: 3978-3988Crossref PubMed Scopus (5) Google Scholar]. Additional clones used were CD45.1 (A20), CD45.2 (104), c-KIT (2B8), and SCA-1 (E13-161.7). GON4L antibodies were described previously [10Lu P Hankel IL Knisz J et al.The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis.J Exp Med. 2010; 207: 1359-1367Crossref PubMed Scopus (19) Google Scholar]. β-Actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Streptavidin Qdot 605 was obtained from BD Biosciences.Statistical analysisData were analyzed using the unpaired, two-tailed Student t test in Prism software (GraphPad, San Diego, CA).ResultsCreation of mice bearing a floxed Gon4l alleleTo begin our studies, we generated mice carrying a floxed Gon4l allele that can be inactivated by excision of sequences by the Cre recombinase (Supplementary Figure E1, online only, available at www.exphem). A gene-targeting vector was constructed in which exon 4 of Gon4l was floxed or flanked by LoxP sites that serve as Cre recognition elements. Cre-mediated recombination removes exon 4 of Gon4l, creating an allele that produces mRNA in which exon 5 onward is out of frame.The gene-targeting vector was electroporated into mouse ES cells, and neomycin-resistant clones that had undergone homologous recombination between the vector and an endogenous copy of Gon4l were isolated. These were used to generate chimeric mice, which were mated to produce progeny carrying the engineered Gon4l allele. These offspring were mated with mice expressing the FLPe recombinase to delete sequences encoding the LacZ and neomycin resistance genes. Removal of these sequences generated mice carrying a floxed Gon4l allele. Mice homozygous for this version of Gon4l are fertile and live a normal life span.The floxed Gon4l allele is inactivated by Cre-mediated recombinationWe next sought to confirm that the floxed Gon4l allele was rendered inactive by Cre-mediated recombination. We first generated Gon4lfl/fl/R26CreERT mice, which are homozygous for both the floxed Gon4l allele and a modified R26 gene that expresses the CreERT fusion protein. In the presence of tamoxifen or its derivatives, CreERT migrates from the cytoplasm to the nucleus and recombines floxed genes.We used MPPs from Gon4lfl/fl/R26CreERT mice to generate pro-B cells, which were then transduced with a retrovirus expressing the BCR-ABL p210 oncoprotein (BCR-ABL) to promote growth and survival. These cells were cultured with either vehicle or vehicle containing the tamoxifen derivative 4-OHT to induce CreERT-mediated recombination of the floxed Gon4l allele. On day 3 of culture, pro-B cells were harvested to prepare genomic DNA and protein lysates. Polymerase chain reaction (PCR) amplification of genomic DNA revealed that both floxed Gon4l alleles had undergone recombination in cells treated with to 4-OHT, resulting in excision of exon 4 (Figure 1A,B). Western blot analysis of protein lysates using an antibody raised against the C-terminal 514 amino acids of GON4L [10Lu P Hankel IL Knisz J et al.The Justy mutation identifies Gon4-like as a gene that is essential for B lymphopoiesis.J Exp Med. 2010; 207: 1359-1367Crossref PubMed Scopus (19) Google Scholar] confirmed deletion of exon 4-abolished expression of full-length GON4L protein (Figure 1C). These data illustrate that Cre-mediated recombination inactivates the floxed version of Gon4l, creating a null allele.Gon4l inactivation in pro-B cells arrests growth and causes cell deathThe Justy mutation in Gon4l impairs the proliferation and survival of early B-cell progenitors [13Barr JY Goodfellow RX Colgan DF Colgan JD Early B cell progenitors deficient for GON4L fail to differentiate due to a block in mitotic cell division.J Immunol. 2017; 198: 3978-3988Crossref PubMed Scopus (5) Google Scholar]. To test whether Gon4l inactivation by CreERT elicits the same effects, Gon4lfl/fl/R26CreERT pro-B cells were cultured in medium containing vehicle or 4-OHT. After 3 days, cells were harvested and viable cells counted (Figure 2A). As expected, cells treated with vehicle proliferated, increasing in number by approximately fourfold. In contrast, cells treated with 4-OHT slightly decreased in number, suggesting their proliferation was blocked. Flow cytometry was used to determine the frequencies of live pro-B cells over the course of 5 days in culture (Figure 2B,C). For cells treated with vehicle, the frequencies of live cells remained relatively constant. In contrast, the frequencies of live cells treated with 4-OHT declined dramatically over time. Staining with the vital dye Hoechst confirmed that 4-OHT-treated cells died during the culture period (Figure 2D). Additionally, cells treated with 4-OHT contained higher levels of the active forms of the proapoptotic caspases 3 and 7 (Figure 2E,F). These data indicate Gon4l inactivation blocks pro-B cell proliferation and induces cell death via apoptosis.Figure 2Gon4l inactivation greatly impairs pro-B-cell growth and survival. BCR-ABL-expressing Gon4lf/f/R26CreERT pro-B cells were cultured in medium containing either vehicle or vehicle plus 4-OHT and harvested for analysis. (A) Yields of live (trypan blue-negative) pro-B cells harvested on day 3 of culture. The dotted line represents the number of cells (1 × 106) added to start the cultures. (B) Flow cytometric analysis of cell viability based on forward (FSC-A) and side (SSC-A) scatter. Numbers are the frequencies of cells in the gate. (C) Graph of the frequencies of viable cells detected when harvested on the indicated days of culture. Data were generated by the analysis illustrated in (B). Values are relative to those obtained on day 0, which were set at 1. The y-axis represents a log 2 scale. (D) Histograms of uptake of the vital dye Hoechst by cells treated with vehicle or vehicle plus 4-OHT. Numbers are the frequencies of cells in the gate, which are those that excluded Hoechst and are thus viable. (E) Analysis of the levels of activated caspases 3 and 7 in cells treated with vehicle or vehicle plus 4-OHT. (F) Graph of the frequencies of cells staining positive for activated caspases 3 and 7. Data were generated by the analysis shown in (E). Histograms in (B), (D), and (E) are representative of three independent experiments. The bar graphs in (A), (C), and (F) illustrate the average and SEM of values obtained from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gon4l is essential for mouse viabilityStudies by others have found that Gon4l is required for zebrafish embryogenesis and is thus an essential gene [8Liu Y Du L Osato M et al.The zebrafish udu gene encodes a novel nuclear factor and is essential for primitive erythroid cell development.Blood. 2007; 110: 99-106Crossref PubMed Scopus (28) Google Scholar,11Williams MLK Sawada A Budine T Yin C Gontarz P Solnica-Krezel L Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes.Nat Commun. 2018; 9: 1319Crossref PubMed Scopus (19) Google Scholar,12Budine TE de Sena-Tomas C Williams MLK Sepich DS Targoff KL Solnica-Krezel L Gon4l/Udu regulates cardiomyocyte proliferation and maintenance of ventricular chamber identity during zebrafish development.Dev Biol. 2020; 262: 223-234Crossref Scopus (5) Google Scholar,15Hammerschmidt M Pelegri F Mullins MC et al.Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.Development. 1996; 123: 143-151Crossref PubMed Google Scholar,16Lim CH Chong SW Jiang YJ Udu deficiency activates DNA damage checkpoint.Mol Biol Cell. 2009; 20: 4183-4193Crossref PubMed Google Scholar]. To determine whether Gon4l is essential in mice, Gon4lfl/fl mice were mated with those bearing the EIIa-cre transgene, which expresses Cre in early mouse embryos and directs recombination in the germ line. The resulting offspring were bred to create mice that lacked EIIa-cre and were heterozygous (Gon4l+/–) for the inactivated Gon4l allele. These mice were then intercrossed. Genotyping of 62 progeny indicated that Gon4l+/+ and Gon4l+/– mice were recovered, but Gon4l–/– mice were not. These results indicate that Gon4l is an essential gene in mice.Gon4l is required to maintain bone marrowIn zebrafish, mutation of Gon4l blocks the initiation of hematopoiesis [8Liu Y Du L Osato M et al.The zebrafish udu gene encodes a novel nuclear factor and is essential for primitive erythroid cell development.Blood. 2007; 110: 99-106Crossref PubMed Scopus (28) Google Scholar], so we sought to determine whether Gon4l is needed for hematopoiesis in mice. For this purpose, we first attempted to generate Gon4lfl/fl mice bearing the Vav-cre transgene, which is active in hematopoietic stem/progenitor cells [19Georgiades P Ogilvy S Duval H et al.VavCre transgenic mice: a tool for mutagenesis in hematopoietic and endothelial lineages.Genesis. 2002; 34: 251-256Crossref PubMed Scopus (149) Google Scholar]. Despite extensive breeding, we were unable to obtain any viable Gon4lfl/fl/Vav-cre mice (data not shown). These findings suggested that Gon4l is required for primitive and/or definitive hematopoiesis in mice.To circumvent the problems encountered with the Vav-cre mouse model described above, MBMCs were made and analyzed as outlined in Figure 3A. This strategy allowed us to maintain hematopoietic development from one population of transplanted bone marrow cells (the CD45.1+ fraction) while testing how deletion of Gon4l affected hematopoiesis from the other (the CD45.2+ fraction). Further, this strategy allowed us to rule out effects of Gon4l deletion on other cell types, such as stromal cells that support hematopoiesis within bone marrow.Figure 3Inactivation of Gon4l gene results in loss of bone marrow cells. MBMCs were created and injected with tamoxifen. Bone marrow was analyzed 3 weeks later. (A) Schematic depicting how control and experimental MBMCs were generated and the time course for hematopoietic reconstitution, tamoxifen administration, and analysis of bone marrow. (B) Total bone marrow cell yields from the indicated MBMCs. (C) Representative flow cytometric analysis of bone marrow from the indicated MBMCs. (D) Yields of CD45.2+ bone marrow cells from the indicated MBMCs. Bar graphs in panels (B) and (D) illustrate the mean and SEM of values obtained from three independent experiments in which three control and three experimental MBMCs were analyzed (n = 9 MBMCs for each group). ****p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To generate control MBMCs, bone marrow from WT CD45.1 mice was mixed 1:1 with that from CD45.2 mice that were Gon4l+/+/R26CreERT+/+ (i.e., homozygous WT for Gon4l). To create experimental MBMCs, bone marrow from CD45.1 WT mice was mixed 1:1 with that from CD45.2 mice that were Gon4lf/f/R26CreERT+/+ (i.e., homozygous for the floxed Gon4l allele). Flow cytometric analysis confirmed bone marrow from reconstituted MBMCs contained roughly a 1:1 mix of cells from each donor (data not shown).To confirm tamox
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