Developmental expression of nadph phagocytic oxidase components in mouse embryos

ABSTRACT We wished to determine when each of the four NADPH oxidase components p22 phagocytic oxidase (phox), gp91 phox, p47 phox, p67 phox is first expressed embryologically and whether the

expression pattern occurs in a consistent temporal sequence or whether the four genes are expressed simultaneously. A deficiency of any one of them results in chronic granulomatous disease

(CGD). mRNA transcripts and protein expression for p22 phox, gp91 phox, p47 phox, p67 phox was monitored in murine embryos at time of implantation (E5.5) until E 11.5, and in fetal liver,

spleen, and limb bone marrow from E 14 until term (E 19). We observed that mRNA was first expressed for p22 phox at E 5.5, for p67 phox at E 7.0 and for p47 phox at E 7.5 before the onset of

yolk sac hematopoiesis (E 8.0). gp91 Phox mRNA was first expressed at E 9.0. However, only p22 phox protein was expressed in circulating hemacytoblast by E 9.0. No other embryonic tissue

contained phox proteins either before or after the establishment of hemocytoblastic circulation. The four specific mRNA transcripts and phox proteins were expressed in nests of developing

granulocytes in liver by E 14 and the expression continued in the liver at E 16 and E 19. Spleen and limb bone marrow showed inconsistent results. Cord blood neutrophils contained all phox

proteins. These studies confirm that the four CGD-related phox mRNA components of NADPH oxidase are expressed early in embryonic development and the expression occurs in a consistent

sequential fashion but only p22 phox protein appears in embryonic hemocytoblast. SIMILAR CONTENT BEING VIEWED BY OTHERS DIFFERENTIAL REGULATION OF FETAL BONE MARROW AND LIVER HEMATOPOIESIS

BY YOLK-SAC-DERIVED MYELOID CELLS Article Open access 14 May 2025 ROBUST TEMPORAL MAP OF HUMAN IN VITRO MYELOPOIESIS USING SINGLE-CELL GENOMICS Article Open access 24 May 2022

ONTOGENETICALLY DISTINCT NEUTROPHILS DIFFER IN FUNCTION AND TRANSCRIPTIONAL PROFILE IN ZEBRAFISH Article Open access 15 August 2023 MAIN We sought to determine the developmental expression

of NADPH oxidase components in mouse embryos. In humans, failure to express any one of four components results in a heterogenous group of inherited disorders of impaired superoxide

production in phagocytes termed CGD(1,2). At least five protein components have been characterized at a molecular level in both human(3–7) and mouse phagocytes(8–11), and the gene products

are designated phox, indicating phagocytic oxidase, p22 Phox and gp91 phox form a transmembrane alpha/beta complex known as cytochrome b(12,13) that associates with three cytosolic

components, p 47 phox, p 67 phox, and p 40 phox during activation of NADPH oxidase. Rac GTP-binding proteins are also involved(14). However, only gp91 phox, p22 phox, p47 phox, and p67 phox

gene mutations have been described as causes of CGD. We wished to determine the expression pattern of these four phox components of NADPH oxidase during the entire period of embryologic

development. We examined mouse embryos from postimplantation (E 5.5 d) through term (E 19 d) for expression of specific RNA transcripts of the four CGD-associated phox genes. In addition,

the presence or absence of specific phox protein was determined at the time of and after the onset of specific gene expression. MATERIALS AND METHOD RNA was prepared from whole mouse embryo

stages E 5.5, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, 10.5, 11.5. In addition, RNA was prepared from liver, spleen, and limbs of mid- and term fetuses (stage E 14, 16, 19). Reverse transcriptase PCR

was used using primer made to known nucleotide sequences of murine gp91 phox, p22 phox, p47 phox, and p67 phox genes to identify specific transcripts in mouse embryos and fetal liver,

spleen, and limbs. The PCR products for all phox genes were confirmed by DNA sequencing. Primers selected for identification of p22 phox were: upper primer 14 mer starting at nucleotide

position 89 of exon 2,5′-GGGGCATCGTGGCT-3′, lower primer 14 mer starting at nucleotide position, 431 of exon 3,5′-GGGGTGGGGGGTTG-3′. Optimal annealing temperature 48°C with 30 cycles yielded

a product length of 404 base pairs. Primers selected for identification of gp91 phox were: upper primer 16 mer starting at nucleotide position 986 murine cDNA 5′-GCCCCAAGGTATCCAA-3′, lower

primer 17 mer starting at nucleotide position 1524, murine cDNA 5′-GCCGTCCATACAGAGTC-3′. Optimal annealing temperature 50°C with 30 cycles yielded a product length of 555 base pairs. Primers

selected for identification of p47 phox were: upper primer 24 mer starting at exon 6,5′-ACATCACAGGCCCCATCATCCTTC-3′, lower primer 20 mer at exon 7,5′-ACCCACCTCGCTTTGTCTTC-3′. Optimal

annealing temperature 60°C with 35 cycles yielded a product length of 162 base pairs. Primers selected for identification of p67 phox were: upper primer 24 mer starting at exon

10,5′-TGGACTTCGGATTCACCCTCAGTC-3′, lower primer 24 mer starting at exon 135′-CACCTTGAGCATGTAAGGCATAGG-3′ Optimal annealing temperature 50°C with 30 cycles yielded a product length of 205

base pairs. The four primer sets were equally sensitive and detected mRNA in E 19 liver samples diluted 1:100 in E 5.0 which did not contain detectable mRNA for any of the phox genes. Serial

sections of embryos E 5.5, 6.5, 8.0, 9.0, 10.0 and sections of liver, spleen, and limbs containing bone marrow from fetuses stage E 14, 16, 19 were prepared for immunoperoxidase staining

using Zymed Histostain-SP and rabbit polyclonal anti-p22 phox antibody (gift from Dr. Mary Dinauer) and rabbit polyclonal anti-gp91 phox, anti-p47 phox, anti-gp40 phox and anti-p67 phox

(gift from Dr. John Curnutte, Genetech, San Francisco, CA). The presence of tissue and umbilical cord blood neutrophils was determined histologically employing rabbit polyclonal

anti-lactoferrin and anti-muramidase (Zymed Labs. Inc., South San Francisco, CA 94080). RESULTS Transcripts to 22 kD phox were consistently identified in whole embryos starting at E 5.5

(Fig. 1). The onset of yolk sac hematopoiesis commences about stage E 8.0 to 8.5 in murine embryos(15). In contrast, transcripts to gp91 kD phox were not found in embryo stages E 5.5, 6.5,

8.0, 8.5, but were found in stages E 9 and all later stage embryos tested (up to E 11.5) (Fig. 2). Transcripts to p22 phox were identified in liver, limbs, and spleen at fetal stages E 14,

E16, and E19 and transcripts to gp91 phox were identified in liver but usually not in spleen and marrow at stages E 14, E 16, and E 19 (Fig. 3). Expression of p47 phox transcripts was not

detected in embryos at stages E 6.0 and E 7.0 but was found in all embryos tested between stages E 7.5 and E 11.5 (Fig. 4). In contrast, p67 phox transcripts were expressed in all embryos

between stages E 7.0 and E 11.5 but were not present at stage E 6.0 (Fig. 5). Transcripts to p47 phox and p67 phox were consistently expressed in fetal liver at stages E 14, E 16, and E 19.

Spleen and marrow inconsistently expressed modest levels of transcripts specific for p47 phox and p67 phox at all three stages of fetal organ development (data not shown). Immunoperoxidase

histochemistry of umbilical blood neutrophils from newborn mice identified each of the phox proteins of the NADPH oxidase (Fig. 6). Clusters of cells in the liver of E 14, 16, and 19 fetuses

were identified as neutrophils by positive staining for muramidase and lactoferrin. Similar clusters of cells also were positive for gp91 phox, p22 phox, p47 phox, p40 phox, and p 67 phox.

A positive pattern is shown for p22 phox and for granule lactoferrin and muramidase in Figure 7. E 14, E 16, and E 19 spleen and limb marrow did not contain identifiable cells containing

phox proteins, lactoferrin, or muramidase. No specific phox protein was identified in embryos before onset of vascularization and hematopoiesis, which occurs at approximately stage E 8. In

particular, despite the early expression of p22 phox transcripts at E 5.5, p22 phox was only identified in circulating hemocytoblasts at E 8.0, E 9.0, and E 10.0 (Fig. 8). These mononuclear

cells were negative for lactoferrin and muramidase. No other specific phox proteins were identified in circulating hemocytoblasts examined at E 8.0, E 9.0, and E 10.0. DISCUSSION NAPDH

oxidase is a unique complex of proteins highly expressed in blood phagocytes of all mammalian species and is responsible for the respiratory burst required for normal killing of bacteria and

fungi(1,2). The complex consists of a 91-kD glycoprotein (gp91 phox) and a 22-kD protein (p22 phox) that form an alpha-beta dimer constitutively expressed in plasma membranes and specific

granule membranes of human and murine blood neutrophils(16,17). CGD results from mutations in either gp91 phox, p22 phox, p47 phox, or p67 phox genes. The genes for phox proteins have been

mapped and sequenced in both humans(3,4,10,18) and mice(7–9,11). We wished to study the developmental expression of the four CGD-related components of the murine NADPH oxidase by employing

an _in vivo_ embryonic mouse system that monitors the time of onset of gene expression and the localization of expressed protein. Embryonic and fetal hematopoiesis has been well

characterized in the mouse(15,19). The phenomenon is first extraembryonic, occurring in blood islands in the yolk sac at stage E 8. The precursors of these islands known as hemangioblasts

migrate from the primitive streak region of blastoderm by invaginating through the primitive streak spreading laterally to produce the mesodermal layer. Hemangioblasts differentiate in two

directions, one forming angioblasts more peripherally and the other forming hemoblasts centrally. Gross and microscopic evidence of an embryonic red blush due to circulating nucleated

hemocytoblasts was observed by us between E 8 and E 9. Liver hemopoiesis is detectable in the mouse by d 10 (E 10). The liver then remains the major hemopoietic organ during fetal life.

Splenic hemopoiesis is initiated by about d 15(E 15) and continues throughout fetal life. The bone marrow is the final hemopoietic site during fetal development with hemopoiesis commencing

between E 17 and E 18. The early expression of transcription factors GATA-1, GATA-3, and vav are required for initiation of hemopoiesis(20–23). The initial development of hemocytoblasts

occurs independent of bone marrow growth factors but by E 10 differentiation along either erythroid or granulocyte-macrophage lineage does require specific growth factors. Although we found

p22 phox protein in the circulating hemocytoblasts of E 8.0, E 9.0, and E 10.0, the cells were negative as expected for lactoferrin and muramidase, two markers of differentiated myeloid

cells(24). These hemocytoblasts were also negative for the other phox proteins to the limits of detection as determined by standard immunoperoxidase histochemistry although RNA transcripts

for p47 phox and p67 phox were found in embryos at these stages of development. The detection of p22 phox without the other components is also consistent with the previous observation that

its expression, unlike the other oxidase genes, is not lineage specific(13). Transcripts for all CGD-associated NADPH oxidase components were found in the liver of fetuses at E 14, E 16, and

E 19. Nests of cells strongly positive for lactoferrin and muramidase confirmed the presence of granulopoiesis in fetal liver at each of these stages. Similar nests of cells in the liver of

E 14, E 16, and E 19 also were strongly positive for gp91 phox, p47 phox, and p67 phox proteins. Spleens and limb marrow from E 14, E 16, and E 19 did not contain cells with identifiable

phox proteins which may reflect the delay in onset of granulopoiesis in these fetal tissues and the predominance of liver as the primary site of granulopoiesis in the fetal mouse. The

expression of RNA transcripts of p22 phox in embryos as early as E 5.5 remains unexplained. It was not associated with any identifiable p22 phox protein in serial whole mount sections

through the entire embryo. Since gp91 phox transcripts and protein were not found in these early stage embryos, the lack of p22 phox could be explained by the known requirement of gp91 phox

for the stabilization of p22 phox in cells expressing these proteins(12,17,25). Furthermore, the expression of transcripts for p47 phox and p67 phox also occurred before the onset of

hemopoiesis (E 7.0 and E 7.5, respectively) but again, serial sections of whole mount embryos failed to reveal either p47 phox or p67 phox protein. Finally, even when gp91 phox transcripts

were expressed at E 9.0, no gp91 phox protein was found in hemocytoblasts expressing p22 phox protein within the sensitivity limits of our assay. The _in vivo_ temporal sequential pattern

expression of mRNA and proteins required for activation of a complex biologic system has been studied previously. The major cytoskeletal proteins α-spectrin, β-spectrin, and ankyrin are

synthesized and assembled into a supportive membrane skeleton during murine erythroid differentiation initially in embryonic yolk sac hemoblast and later in fetal liver cells(26).

Disaggregated embryonic yolk sac cells and circulating blood cells from E 9 embryos before formation of fetal liver are capable of colonizing E 11 to E 15 fetal liver to initiate hemopoiesis

supporting the notion that embryonic shifts in hemopoietic sites occurs via the fetal circulation(27). We observed a consistent temporal sequential pattern expression for the four

CGD-related NADPH oxidase components during murine embryonic and fetal development rather than an abrupt synchronous expression of all components at one stage of development. p22 Phox mRNA

is first expressed at E 5.5, the expression of p47 phox mRNA occurs at E 7.0 and the expression of p67 phox mRNA occurs at E 7.5. All stages are before the onset of hemopoiesis whereas gp91

phox mRNA first occurs at E 9.0. However, none of the phox proteins (including p40 phox and myeloid markers lactoferrin and muramidase) except p22 phox were found in circulating

hemocytoblasts. The fetal liver at stages E 14, E 16, and E 19 contains nests of cells that contain lactoferrin, muramidase, and all phox proteins indicating full maturity of the NADPH

oxidase in fetal liver myeloid cells. Indeed, umbilical cord blood neutrophils of newborn mice also contain all phox proteins. This observation is consistent with results of fetal human

granulocytes and prenatal studies on human fetuses evaluated for CGD. Both studies reported normal functional phagocytic oxidase activity of fetal blood neutrophils including two potentially

affected males(28,29). ABBREVIATIONS * CGD: chronic granulomatous disease * E+ number: refers to specific embryonic stage of development from day of fertilization * phox: phagocytic oxidase

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granulomatous disease. _Am J Med Genet_ 17: 731–739 Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank Drs. Sharon H. Jackson and Steven M. Holland for

providing PCR primer pair information for mouse p47 phox and mouse p67 phox. We also thank Drs. Mary Dinauer and John Curnutte for providing antibodies to the mouse phox proteins. I am

grateful to Drs. Hal Slavkin and Charles Shuler for allowing me (RLB) to work in their laboratory. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pediatrics, School of Medicine,

School of Dentistry, University of Southern California, Los Angeles, California Robert L Baehner * Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern

California, Los Angeles, California Sharon Millar-Groff & Pablo Bringas Authors * Robert L Baehner View author publications You can also search for this author inPubMed Google Scholar *

Sharon Millar-Groff View author publications You can also search for this author inPubMed Google Scholar * Pablo Bringas View author publications You can also search for this author inPubMed

 Google Scholar ADDITIONAL INFORMATION Supported in part by CCMB National Institutes of Health Center Grant NIDR P50DE09166-10.Results presented in part at the Annual Meetings of the

American Society of Hematology, December 1995 and 1996, Seattle WA and Orlando FL. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Baehner, R.,

Millar-Groff, S. & Bringas, P. Developmental Expression of NADPH Phagocytic Oxidase Components in Mouse Embryos. _Pediatr Res_ 46, 152–157 (1999).

https://doi.org/10.1203/00006450-199908000-00004 Download citation * Received: 02 October 1998 * Accepted: 26 April 1999 * Issue Date: 01 August 1999 * DOI:

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