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Expression pattern of macrophage migration inhibitory factor during embryogenesis
Mechanisms of Development 84 (1999) 153±156
Gene expression pattern
Expression pattern of macrophage migration inhibitory factor during embryogenesis Shigetoshi Kobayashi a, Kazuhito Satomura b, Jeffrey M. Levsky a, Taduru Sreenath a, Graeme J. Wistow c, Ichiro Semba d, Lillian Shum d, Harold C. Slavkin d, Ashok B. Kulkarni a,* b
a Functional Genomics Unit and Gene Targeting Facility, Bethesda, MD 20892, USA Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, Building 30, Room 529, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892-4326, USA c Section on Molecular Structure and Function, National Eye Institute, Bethesda, MD 20892, USA d Craniofacial Development Section, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Received 13 January 1999; received in revised form 8 March 1999; accepted 9 March 1999
Abstract Although macrophage migration inhibitory factor (MIF) was originally identi®ed as a lymphokine that inhibits the migration of macrophages, its ubiquitous expression suggests it may have a role beyond the immune system. Here we report a detailed characterization of MIF expression during mouse embryogenesis. The MIF expression pattern was found to parallel tissues speci®cation and organogenesis. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Macrophage migration inhibitory factor; Cytokine; Embryogenesis
1. Results 1.1. Macrophage migration inhibitory factor is expressed in multiple tissues during organogenesis Macrophage migration inhibitory factor (MIF) is a multifunctional molecule which has been associated with many biological activities as a cytokine, as a hormone (Bucala, 1994), and as an enzyme (Rosengren et al., 1996). MIF expression has been reported in several adult tissues (Wistow et al., 1993; Nishino et al., 1995; Imamura et al., 1996; Nishibori et al., 1996), suggesting broader functions. Although MIF expression is identi®ed in the early stages of murine embryos by RT-PCR (Suzuki et al., 1996) and in the brain of rat embryos (Suzuki et al., 1999), its spatial and temporal expression pattern during embryogenesis has not been well studied. We report here a detailed characterization of MIF expression pattern during embryogenesis which suggests the involvement of MIF in the development of various tissues. MIF mRNA was detected in somites, precartilage primordia in ribs and vertebrae, and branchial * Corresponding author. Tel.: 1 1-301-435-2887; fax: 1 1-301-4352888. E-mail address: [email protected] (A.B. Kulkarni)
arches (Fig. 1a±e,g,i±k). Somitogenesis commences at E8 and continues to E14, following a rostral-to-caudal gradient of differentiation. MIF expression was observed in somites by whole-mount in situ hybridization from E8.5 (Fig. 1a± e,i±k). As the number of somites increased, MIF expression was more prominent in caudal somites. Mesenchymal cells of ventral medial cell origin initiate differentiation at E11± E11.5 and ultimately form the cartilage of vertebrae and ribs. MIF was identi®ed in primordia of these cartilages at E11.5 and E12.5 (Fig. 1g,s). MIF expression began at E9.5 in branchial arches with uniform distribution (Fig. 1b). At E10.5, stronger expression was seen on the oral region of the maxillary and mandibular components of ®rst branchial arch, and this pattern became more prominent at E11.5 (Fig. 1e,g). MIF expression began to appear in limb buds with uniform distribution when mesenchymal cells accumulated to form the primordium of limb buds at E9.5±E10.5 (Fig. 1b,e). MIF expression was also observed in neural tissues, including forebrain, midbrain, hindbrain, neural tube, cranial ganglia, and dorsal root ganglia during all of the embryonic stages (Fig. 1a±c,e±g,i,j,l±r). MIF expression in dorsal root ganglia progressed in a rostal-to-caudal pattern at E10.5±E11.5 similar to the expression seen in somites. Optic and otic vesicles also expressed MIF from E9.5 (Fig. 1b,e,f,g).
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00057-X
154
S. Kobayashi et al. / Mechanisms of Development 84 (1999) 153±156
Fig. 1. Analysis of MIF expression during embryogenesis by whole-mount in situ hybridization and immunohistochemistry. Whole-mount MIF mRNA distribution at various embryonic stages: (a) E8.5, (b) E9.5, (c) and (d) higher magni®cation of E9.5, (e) E10.5, (f) MIF expression in neural tube and cranial ganglia at E10.5, (g) E11.5, and (h) control sample using sense probe at E10.5. Sections of embryos hybridized in whole-mount: (i) somites and neural tube at E8.5, (j) somites and neural tube at E10.5, (k) somites in tail region at E11.5, (l) neural fold at E8.0, (m) neural tube at E9.5, (n) and (o) neural tube and dorsal root ganglia at E11.5, and (q) forebrain. Immunohistochemical localization of MIF: (p) dorsal root ganglia at E11.5, (r) forebrain at E11.5, and (s) precartilage primordium of rib at E12.5. (1) ®rst branchial arch, (1a) maxillar component of ®rst branchial arch, (1b) mandibular component of ®rst branchial arch, (2) second branchial arch, (3) third branchial arch, (C) cranial ganglion, (D) dorsal root ganglion, (F) forebrain, (Fl) forelimb bud, (Fp) frontal prominence, (H) hindbrain, (Hl) hindlimb bud, (M) midbrain, (N) neural tube, (Nf) neural fold, (Np) nasal prominence, (Op) optic vesicle, (Ot) otic vesicle (Pl) primordiurn of limb bud, (Pr) precartilage primordiurn, (So) somite.
S. Kobayashi et al. / Mechanisms of Development 84 (1999) 153±156
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Fig. 2. MIF expression in different tissues during development. Developmental pro®le of MIF expression analyzed by immunohistochemistry in (a) liver, (b) kidney, and (c) testis. Developmental expression of MIF in muscle (d±f): (d) MIF expression in E9.5-adult cardiac muscle analyzed by immunohistochemistry. (e) MIF expression in E9.5±12 cardiac muscle by whole-mount in situ hybridization, including section of the heart at E10.5. (f) MIF expression in smooth muscle of artery and in skeletal muscle at E16.5. (Ca) common atrium, (Cv) common ventricle, (Cve) central vein, (G) glomeruli, (Lv) left ventricle, (Met) mesonephric tubule, (Mt) metanephric tubule, (Mv) metaphephric vesicle, (Ra) right atrium, (Rv) right ventricle, (S) seminiferous epithelium, (Se) Sertoli cell, (Sk) skeletal muscle, (Sm) smooth muscle, (U) urinary tubule.
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S. Kobayashi et al. / Mechanisms of Development 84 (1999) 153±156
1.2. Onset of MIF expression coincides with the speci®cation of tissues MIF expression was apparent in many embryonic tissues with a few exceptions of lung, spleen and thymus. The onset of MIF expression coincides with the speci®cation of tissues during organogenesis and continued through adulthood. From E9.5 to E11.5, the liver primordium consists of loose, undifferentiated mesenchymal tissue. At E10.5 invasion of the mesenchyme by the hepatic cells of ventral endoderm and hematopoietic activity are initiated. Overt MIF expression was not detectable in E9.5±E11.5 day embryos (Fig. 2a). MIF expression became apparent in a few cells at E12.5, when functional hepatic parenchymal cells appear in this region and steadily increased with developmental stages (Fig. 2a). MIF-expressing cells are square-shaped with large cytoplasm, suggesting these cells are hepatocytes. MIF expression began to appear in metanephric tubules when tubulogenesis was initiated at E14.5 (Fig. 2b). After E15 day, epithelial cells in convoluted tubules continuously expressed MIF. However, MIF was not detected in glomeruli in embryos or in adults. MIF began to appear in seminiferous epithelium at E12.5 when mesonephros is replaced by testicular tissue and was produced continuously during subsequent embryonic stages (Fig. 2c). Such expression pattern is also seen in skin, intestine, adrenal gland, pancreas, salivary gland and nervous system (data not shown). The comparison of expression pro®les generated using immunohistochemistry with those of in situ hybridization showed similar expression pattern. 1.3. MIF is expressed during myogenesis MIF expression was observed in all muscle cell types, including cardiac, smooth, and skeletal muscle, during embryonic development (Fig. 2d±f). Continuous expression of MIF from E9.5 till birth was seen in cardiac muscle and a relatively low level was observed in 2-week-old mice (Fig. 2d,e). Similarly, MIF was expressed prominently in skeletal and smooth muscle during embryogenesis (Fig. 2f) and relatively lower expression was observed in adult (data not shown).
2. Materials and methods 2.1. Immunohistochemical analysis of MIF Immunohisto- and immunocytochemical analyses were carried out as described (Satornura et al., 1991). 2.2. In situ hybridization for MIF mRNA In situ hybridization was performed using the SureSitewII system Kit (Novagen, Madison, WI) following manufacturer's instructions. Whole-mount in situ analysis was carried out as described (Riddle et al., 1993). Embryos were frozen in OCT compound and sectioned at 15±50 mm thickness. References Bucala, R., 1994. Identi®cation of MIF as a new pituitary hormone and macrophage cytokine and its role in endotoxic shock. Immunol. Lett. 43, 23±-26. Imamura, K., Nishihira, J., Suzuki, M., Yasuda, K., Sasaki, S., Kusunoki, Y., Tochimaru, H., Takekoshi, Y., 1996. Identi®cation and immunohistochemical localization of macrophage migration inhibitory factor in human kidney. Biochem. Mol. Biol. Int. 40, 1233±-1242. Nishibori, M., Nakaya, N., Tahara, A., Kawabata, M., Mon, S., Saeki, K., 1996. Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain. Neurosci. Lett. 213, 193±-196. Nishino, T., Bernhagen, J., Shiiki, H., Calandra, T., Dohi, K., Bucala, R., 1995. Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland. Mol. Med. 1, 781±-788. Riddle, R.D., Johnson, R.L., Laufer, E., Tabin, C., 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401±-1416. Rosengren, E., Bucala, R., Aman, P., Jacobsson, L., Odh, G., Metz, C.N., Rorsman, H., 1996. The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol. Med. 2, 143±-149. Satornura, K., Hiraiwa, K., Nagayama, M., 1991. Mineralized nodule formation in rat bone marrow stromal cell culture without beta-glycerophosphate. Bone Miner. 14, 41±-54. Suzuki, H., Kanagawa, H., Nishihira, J., 1996. Evidence for the presence of macrophage migration inhibitory factor in murine reproductive organs and early embryos. Immunol. Lett. 51, 141±-147. Suzuki, T., Ogata, A., Tashiro, K., Nagashima, K., Tamura, M., Nishihira, J., 1999. Augmented expression of macrophage migration inhibitory factor (MIF) in the telencephalon of the developing rat brain. Brain Res. 816, 457±-462. Wistow, G.J., Shaughnessy, M.P., Lee, D.C., Hodin, J., Zelenka, P.S., 1993. A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc. Natl. Acad. Sci. USA 90, 1272±-1275.
Gene expression pattern
Expression pattern of macrophage migration inhibitory factor during embryogenesis Shigetoshi Kobayashi a, Kazuhito Satomura b, Jeffrey M. Levsky a, Taduru Sreenath a, Graeme J. Wistow c, Ichiro Semba d, Lillian Shum d, Harold C. Slavkin d, Ashok B. Kulkarni a,* b
a Functional Genomics Unit and Gene Targeting Facility, Bethesda, MD 20892, USA Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, Building 30, Room 529, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892-4326, USA c Section on Molecular Structure and Function, National Eye Institute, Bethesda, MD 20892, USA d Craniofacial Development Section, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
Received 13 January 1999; received in revised form 8 March 1999; accepted 9 March 1999
Abstract Although macrophage migration inhibitory factor (MIF) was originally identi®ed as a lymphokine that inhibits the migration of macrophages, its ubiquitous expression suggests it may have a role beyond the immune system. Here we report a detailed characterization of MIF expression during mouse embryogenesis. The MIF expression pattern was found to parallel tissues speci®cation and organogenesis. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Macrophage migration inhibitory factor; Cytokine; Embryogenesis
1. Results 1.1. Macrophage migration inhibitory factor is expressed in multiple tissues during organogenesis Macrophage migration inhibitory factor (MIF) is a multifunctional molecule which has been associated with many biological activities as a cytokine, as a hormone (Bucala, 1994), and as an enzyme (Rosengren et al., 1996). MIF expression has been reported in several adult tissues (Wistow et al., 1993; Nishino et al., 1995; Imamura et al., 1996; Nishibori et al., 1996), suggesting broader functions. Although MIF expression is identi®ed in the early stages of murine embryos by RT-PCR (Suzuki et al., 1996) and in the brain of rat embryos (Suzuki et al., 1999), its spatial and temporal expression pattern during embryogenesis has not been well studied. We report here a detailed characterization of MIF expression pattern during embryogenesis which suggests the involvement of MIF in the development of various tissues. MIF mRNA was detected in somites, precartilage primordia in ribs and vertebrae, and branchial * Corresponding author. Tel.: 1 1-301-435-2887; fax: 1 1-301-4352888. E-mail address: [email protected] (A.B. Kulkarni)
arches (Fig. 1a±e,g,i±k). Somitogenesis commences at E8 and continues to E14, following a rostral-to-caudal gradient of differentiation. MIF expression was observed in somites by whole-mount in situ hybridization from E8.5 (Fig. 1a± e,i±k). As the number of somites increased, MIF expression was more prominent in caudal somites. Mesenchymal cells of ventral medial cell origin initiate differentiation at E11± E11.5 and ultimately form the cartilage of vertebrae and ribs. MIF was identi®ed in primordia of these cartilages at E11.5 and E12.5 (Fig. 1g,s). MIF expression began at E9.5 in branchial arches with uniform distribution (Fig. 1b). At E10.5, stronger expression was seen on the oral region of the maxillary and mandibular components of ®rst branchial arch, and this pattern became more prominent at E11.5 (Fig. 1e,g). MIF expression began to appear in limb buds with uniform distribution when mesenchymal cells accumulated to form the primordium of limb buds at E9.5±E10.5 (Fig. 1b,e). MIF expression was also observed in neural tissues, including forebrain, midbrain, hindbrain, neural tube, cranial ganglia, and dorsal root ganglia during all of the embryonic stages (Fig. 1a±c,e±g,i,j,l±r). MIF expression in dorsal root ganglia progressed in a rostal-to-caudal pattern at E10.5±E11.5 similar to the expression seen in somites. Optic and otic vesicles also expressed MIF from E9.5 (Fig. 1b,e,f,g).
0925-4773/99/$ - see front matterq 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00057-X
154
S. Kobayashi et al. / Mechanisms of Development 84 (1999) 153±156
Fig. 1. Analysis of MIF expression during embryogenesis by whole-mount in situ hybridization and immunohistochemistry. Whole-mount MIF mRNA distribution at various embryonic stages: (a) E8.5, (b) E9.5, (c) and (d) higher magni®cation of E9.5, (e) E10.5, (f) MIF expression in neural tube and cranial ganglia at E10.5, (g) E11.5, and (h) control sample using sense probe at E10.5. Sections of embryos hybridized in whole-mount: (i) somites and neural tube at E8.5, (j) somites and neural tube at E10.5, (k) somites in tail region at E11.5, (l) neural fold at E8.0, (m) neural tube at E9.5, (n) and (o) neural tube and dorsal root ganglia at E11.5, and (q) forebrain. Immunohistochemical localization of MIF: (p) dorsal root ganglia at E11.5, (r) forebrain at E11.5, and (s) precartilage primordium of rib at E12.5. (1) ®rst branchial arch, (1a) maxillar component of ®rst branchial arch, (1b) mandibular component of ®rst branchial arch, (2) second branchial arch, (3) third branchial arch, (C) cranial ganglion, (D) dorsal root ganglion, (F) forebrain, (Fl) forelimb bud, (Fp) frontal prominence, (H) hindbrain, (Hl) hindlimb bud, (M) midbrain, (N) neural tube, (Nf) neural fold, (Np) nasal prominence, (Op) optic vesicle, (Ot) otic vesicle (Pl) primordiurn of limb bud, (Pr) precartilage primordiurn, (So) somite.
S. Kobayashi et al. / Mechanisms of Development 84 (1999) 153±156
155
Fig. 2. MIF expression in different tissues during development. Developmental pro®le of MIF expression analyzed by immunohistochemistry in (a) liver, (b) kidney, and (c) testis. Developmental expression of MIF in muscle (d±f): (d) MIF expression in E9.5-adult cardiac muscle analyzed by immunohistochemistry. (e) MIF expression in E9.5±12 cardiac muscle by whole-mount in situ hybridization, including section of the heart at E10.5. (f) MIF expression in smooth muscle of artery and in skeletal muscle at E16.5. (Ca) common atrium, (Cv) common ventricle, (Cve) central vein, (G) glomeruli, (Lv) left ventricle, (Met) mesonephric tubule, (Mt) metanephric tubule, (Mv) metaphephric vesicle, (Ra) right atrium, (Rv) right ventricle, (S) seminiferous epithelium, (Se) Sertoli cell, (Sk) skeletal muscle, (Sm) smooth muscle, (U) urinary tubule.
156
S. Kobayashi et al. / Mechanisms of Development 84 (1999) 153±156
1.2. Onset of MIF expression coincides with the speci®cation of tissues MIF expression was apparent in many embryonic tissues with a few exceptions of lung, spleen and thymus. The onset of MIF expression coincides with the speci®cation of tissues during organogenesis and continued through adulthood. From E9.5 to E11.5, the liver primordium consists of loose, undifferentiated mesenchymal tissue. At E10.5 invasion of the mesenchyme by the hepatic cells of ventral endoderm and hematopoietic activity are initiated. Overt MIF expression was not detectable in E9.5±E11.5 day embryos (Fig. 2a). MIF expression became apparent in a few cells at E12.5, when functional hepatic parenchymal cells appear in this region and steadily increased with developmental stages (Fig. 2a). MIF-expressing cells are square-shaped with large cytoplasm, suggesting these cells are hepatocytes. MIF expression began to appear in metanephric tubules when tubulogenesis was initiated at E14.5 (Fig. 2b). After E15 day, epithelial cells in convoluted tubules continuously expressed MIF. However, MIF was not detected in glomeruli in embryos or in adults. MIF began to appear in seminiferous epithelium at E12.5 when mesonephros is replaced by testicular tissue and was produced continuously during subsequent embryonic stages (Fig. 2c). Such expression pattern is also seen in skin, intestine, adrenal gland, pancreas, salivary gland and nervous system (data not shown). The comparison of expression pro®les generated using immunohistochemistry with those of in situ hybridization showed similar expression pattern. 1.3. MIF is expressed during myogenesis MIF expression was observed in all muscle cell types, including cardiac, smooth, and skeletal muscle, during embryonic development (Fig. 2d±f). Continuous expression of MIF from E9.5 till birth was seen in cardiac muscle and a relatively low level was observed in 2-week-old mice (Fig. 2d,e). Similarly, MIF was expressed prominently in skeletal and smooth muscle during embryogenesis (Fig. 2f) and relatively lower expression was observed in adult (data not shown).
2. Materials and methods 2.1. Immunohistochemical analysis of MIF Immunohisto- and immunocytochemical analyses were carried out as described (Satornura et al., 1991). 2.2. In situ hybridization for MIF mRNA In situ hybridization was performed using the SureSitewII system Kit (Novagen, Madison, WI) following manufacturer's instructions. Whole-mount in situ analysis was carried out as described (Riddle et al., 1993). Embryos were frozen in OCT compound and sectioned at 15±50 mm thickness. References Bucala, R., 1994. Identi®cation of MIF as a new pituitary hormone and macrophage cytokine and its role in endotoxic shock. Immunol. Lett. 43, 23±-26. Imamura, K., Nishihira, J., Suzuki, M., Yasuda, K., Sasaki, S., Kusunoki, Y., Tochimaru, H., Takekoshi, Y., 1996. Identi®cation and immunohistochemical localization of macrophage migration inhibitory factor in human kidney. Biochem. Mol. Biol. Int. 40, 1233±-1242. Nishibori, M., Nakaya, N., Tahara, A., Kawabata, M., Mon, S., Saeki, K., 1996. Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain. Neurosci. Lett. 213, 193±-196. Nishino, T., Bernhagen, J., Shiiki, H., Calandra, T., Dohi, K., Bucala, R., 1995. Localization of macrophage migration inhibitory factor (MIF) to secretory granules within the corticotrophic and thyrotrophic cells of the pituitary gland. Mol. Med. 1, 781±-788. Riddle, R.D., Johnson, R.L., Laufer, E., Tabin, C., 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401±-1416. Rosengren, E., Bucala, R., Aman, P., Jacobsson, L., Odh, G., Metz, C.N., Rorsman, H., 1996. The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction. Mol. Med. 2, 143±-149. Satornura, K., Hiraiwa, K., Nagayama, M., 1991. Mineralized nodule formation in rat bone marrow stromal cell culture without beta-glycerophosphate. Bone Miner. 14, 41±-54. Suzuki, H., Kanagawa, H., Nishihira, J., 1996. Evidence for the presence of macrophage migration inhibitory factor in murine reproductive organs and early embryos. Immunol. Lett. 51, 141±-147. Suzuki, T., Ogata, A., Tashiro, K., Nagashima, K., Tamura, M., Nishihira, J., 1999. Augmented expression of macrophage migration inhibitory factor (MIF) in the telencephalon of the developing rat brain. Brain Res. 816, 457±-462. Wistow, G.J., Shaughnessy, M.P., Lee, D.C., Hodin, J., Zelenka, P.S., 1993. A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc. Natl. Acad. Sci. USA 90, 1272±-1275.