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cerebral cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in embryo and adult
Mechanisms of Development 117 (2002) 363–367 www.elsevier.com/locate/modo
Gene expression pattern
Krit1/cerebral cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in embryo and adult C. Denier a,b, J.-M. Gasc c, F. Chapon d, V. Domenga a, C. Lescoat a,b, A. Joutel a,b, E. Tournier-Lasserve a,b,* a
INSERM EMI 99-21, Faculte´ de Me´decine Lariboisie`re, 10, avenue de Verdun, 75010 Paris, France b Laboratoire de Cytoge´ne´tique, AP-HP, Hoˆpital Lariboisie`re, Paris, France c INSERM U36, Colle`ge de France, Paris, France d Service d’Anatomopathologie, CHU de Caen, Caen, France Received 24 May 2002; received in revised form 17 June 2002; accepted 18 June 2002
Abstract Cavernous malformations are capillaro-venous lesions mostly located within the central nervous system (CCM/OMIM#116860) and occasionally within the skin and/or retina. They occur as a sporadic or hereditary condition. Three CCM loci have been mapped, and the sole gene identified so far, CCM1, has been shown to encode KRIT1, a protein of unknown function. In an attempt to get some insight on the relationship between KRIT1 mutations and CCM lesions, we investigated Krit1 mRNA expression during mouse development from E7.5 to E20.5 and in adult tissues, of both mouse and human origin. A ubiquitous Krit1 mRNA expression was detected from E7.5 up to E9.5. Then, it became progressively restricted from E10.5 to E12.5, to become detectable later essentially in the nervous system and various epithelia. Strong labelling was observed in neurons in the brain, cerebellum, spinal cord, retina and dorsal root ganglia. In epithelia, Krit1 mRNA expression was detected in differentiating epidermal, digestive, respiratory, uterine and urinary epithelia. A similar pattern of expression persisted in mouse and man adult nervous system and epithelia. Unexpectedly, in vascular tissues, expression of Krit1 was detected only in large blood vessels of the embryo. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Krit1; Cerebral cavernous malformations; Cavernous angiomas; In situ hybridisation
1. Introduction Cerebral cavernous malformations (CCM/ OMIM#116860) are vascular anomalies mostly located within the central nervous system (CNS) and characterised by abnormally enlarged, endothelial lined, vascular sinusoid cavities, without intervening brain parenchyma (Russel and Rubinstein, 1989). Clinical features include seizures and/or focal neurological deficits mostly due to cerebral haemorrhages. Mean age of clinical onset is around 20–30 years old. CCM prevalence has been estimated to 0.1–0.5% (Otten et al., 1989). They can occur as a sporadic or familial, autosomal dominant, condition. Familial CCM are characterised by the presence of multiple cerebral lesions whose number is strongly correlated to patient age, suggesting a dynamic nature of this disease (Labauge et al., 1998).
Hereditary CCM are occasionally associated with retinal and/or cutaneous vascular malformations (Labauge et al., 1999; Eerola et al., 2000). Three CCM loci have been mapped (Dubovsky et al., 1995; Craig et al., 1998). CCM1 was identified as coding KRIT1 (Laberge-le Couteulx et al., 1999; Sahoo et al., 1999), a protein of unknown function, previously shown to interact in vitro with Rap1A, a small Ras like GTPase (Serebriiskii et al., 1997). KRIT1 also interacts with ICAP1, a modulator of b1 integrin signal transduction, whose murine ortholog gene is Bodenin (Zhang et al., 2001; Zawistowski et al., 2002; Faisst and Gruss, 1998). Pathophysiological mechanisms underlying CCM are unknown. As a first step towards the investigation of CCM mechanisms, we studied Krit1 expression by in situ hybridisation during C57BL/6 mouse development (E7.5– E20.5) and in normal adult mouse and human tissues.
* Corresponding author. Tel.: 133-1-44-89-77-50; fax: 133-1-44-89-7755. E-mail address: [email protected] (E. TournierLasserve). 0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00209-5
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networks, including those of the developing brain and eye (Fig. 2A–C). 2.2. Developing and adult nervous system and eye Neural tube labelling was homogeneous from E7.5 to E11.5 (Figs. 1 and 2A–C). By E12.5, Krit1 predominated in proliferating neural cells: maximal expression was
Fig. 1. Krit1 mRNA expression in early mouse embryos. Krit1 is ubiquitously detected in embryos at E7.5 (A–C), E8.5 (D–F) and E9.5 (G–I). First column, brightfield views for histology; second column, darkfield views of the same sections with antisense probe; third column, darkfield views with Krit1 sense probe. Scale bar, 120 mm.
2. mRNA Krit1 expression in embryo and adult mouse 2.1. Early embryonic development From E7.5 to E9.5, a ubiquitous embryonic Krit1 expression was detected in primordial layers, contrasting with the weak signal observed in the adjacent uterus (Fig. 1). At E10.5, E11.5 and E12.5, Krit1 transcripts were widely expressed in the neural tube (Fig. 2A–B) and in various epithelia including coelomic and intestinal ones. Comparatively, only moderate labelling was observed in adjacent mesenchyma, limb buds, dermomyotome and scleromyotome. Weak Krit1 labelling was observed in vascular structures such as heart and large vessels (aorta, lateral vascular network, cardinal and head veins, and umbilical vessels), whereas there was no difference between sense and antisense labelled sections when examining small capillary
Fig. 2. Krit1 mRNA expression in central and peripheral nervous system of embryo and adult mouse. (A–C) E10.5 axial sections. (D–F) E14.5 brainstem sagittal section with adjacent trigeminal ganglia (tg) and internal carotid (ic). (G–I) E14.5 sagittal thoracic section. (J–L) E20.5 axial thoracic section with the spinal cord and adjacent dorsal root (drg) and sympathetic ganglia (syg). (M–O) E20.5 and (P–R) adult brain cortical sections. (S–U) Adult sagittal brain sections showing hippocampus (h) and subjacent corpus callosum (cc). (V–X) Adult cerebellum sections. (Y–ZZ) adult retinal section. Arrow-heads point to capillaries; no labelling was detected in small blood vessels. Choroid and red blood cells exhibited spontaneous auto-fluorescence in darkfield, also seen with the sense probe. Abbreviations: nt, neural tube; ao, aorta; cv, cardinal vein; tg, trigeminal ganglia; bst, brainstem; ic, internal carotid; drg, dorsal root ganglia; sc, spinal cord; ve, vertebrae; hea, heart; thy, thymus; syg, sympathetic ganglia; v, meningeal vessel; hip, hippocampus; cc, corpus callosum; pkj, Purkinje cells; gr, granular cells; ch, choroid. Organisation of the pictures like Fig. 1. Scale bar, 120 mm.
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observed at E14.5, notably in subventricular cell layers, in the ganglionic eminence projecting into the third ventricle, in the thalamus, brainstem and trigeminal ganglia (Fig. 2D– F). During late embryogenesis, at E16.5, E18.5 and E20.5, Krit1 transcripts were mostly detected within layers containing neuronal cells as compared to layers enriched in glial cells (Fig. 2M–O). In adult brains, the same pattern persisted with strong signal in cortex (Fig. 2P–R), hippocampal dentate gyrus (Fig. 2S–U), olfactory bulb, habenula, cerebellum (granular layers and Purkinje cells, Fig. 2V–X) whereas the white matter was not labelled. Within the spinal cord, Krit1 mRNA expression pattern also predominated in the grey matter in embryonic (Fig. 2J–L) as in adult stages. Meninges and choroid plexus exhibited a weak signal. In the peripheral nervous system, a strong signal was detected in dorsal root ganglia by E11.5 up to adult stages (Fig. 2G–I, J–L) and a weaker signal was observed in sympathetic chains ganglia (Fig. 2J–L). In the eye, Krit1 labelling was coincident with the appearance of the optic placods, located from E10.5 in both inner neural and outer retinal layers. Strong expression persisted in the different neural cells layers in all studied developmental stages and in adult mouse retina (Fig. 2Y–ZZ). 2.3. Differentiating and adult thoraco-abdominal organs Krit1 mRNA was detected in the lung bud. By E12.5– E14.5, signal predominated in the tracheal/bronchial epithelium. The hybridisation signal was weaker in lung parenchyma and alveolar walls. This pattern persisted in adult mouse. In liver, a homogeneous pattern of expression existed as early as the primitive epithelial hepatic bud was formed. Liver expression level decreased from E16.5, but a weak signal persisted in adult mouse. Pancreas and spleen parenchyma exhibited a similar expression pattern. Krit1 expression was observed all along the digestive tract (oesophagus, stomach, intestine) at all developmental stages. By E14.5–E16.5, it predominated in epithelia compared to subjacent mesenchyma, and persisted up to adulthood (Fig. 3A–C, D–F). Krit1 kidney expression was detected all along organogenesis. It was restricted to glomeruli and tubules once medullary and cortical regions differentiated at E14.5. It was also present in ureter and bladder epithelia. In adult, it persisted moderately in glomeruli and distal convoluted tubules, in collecting ducts and ureter epithelium. The adrenal gland was strongly labelled from the time of adrenal primordium formation at E12.5 until adult in both cortex and medulla.
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In the aorta, inter-somitic, head and neck vessels, a weak signal was detected from E9.5, increased up to E14.5 (carotid arteries, aorta; Fig. 2D–F, G–I) and then diminished to nearly disappear at E18.5–E20.5. In the adult, Krit1 was not clearly detected in large blood vessels. 2.4.2. Capillary networks From embryonic to adult stages, on the contrary to what was observed in large blood vessels, we did not detect a reproducible signal in the small capillary networks of the heart, lung, liver, kidney, skeletal muscle and CNS; no clear difference was detected when comparing capillaries in sections labelled with sense and antisense probes. CNS negative capillaries are shown in Fig. 2. 2.5. Other tissues In mouse skin, Krit1 mRNA expression was detected at all stages studied, particularly from E14.5 in germinal cell layers of the epidermis and in the hair follicles and vibrissiae. In skeletal muscles, only a weak signal was observed at embryonic stages. In bones, variable levels of Krit1 mRNA expression were observed: moderate in precartilage (E12.5), low during cartilage formation (E14.5), stronger in ossification centres (E16.5). Late expression (E18.5–E20.5) persisted mostly in peri-osteal ossification regions (vertebrae and craniofacial bones).
2.4. Embryonic and adult cardio-vascular system 2.4.1. Heart and large vessels Krit1 mRNA was detected in the myocardium at all studied embryonic stages (Fig. 2G–I), and persisted at a weak level in adult.
Fig. 3. Krit1 mRNA expression in various epithelia of embryo and adult mouse. (A–C) Adult intestine. (D–F) Adult oesophagus. (G–I) Adult seminiferous tubules and (J–L) ovarian follicules (fol). Abbreviations: gl, germinal layers, mu, mucosa; spz, spermatozoids; scy, spermatocytes; sgn, spermatogonias. Organisation of the pictures like Fig. 1. Scale bar, 120 mm.
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By E14.5, dentition primordia with first incisor tooth buds were labelled and Krit1 mRNA expression persisted through tooth differentiation, particularly in odotonblasts layers, up to E20.5. In gonads, Krit1 expression was detected from the primitive sexually undifferentiated stages (E11.5) and the primitive seminiferous tubules (E12.5–E14.5) up to adult stages with strong labelling in premeiotic spermatocytes (Fig. 3G– I) and ovarian follicles (Fig. 3J–L). 3. KRIT1 mRNA expression in adult human tissues KRIT1 mRNA expression pattern in human adult tissues was similar to mouse adult Krit1 expression pattern. Within the adult human CNS, mRNA expression also predominated in neuronal layers, in the brainstem as in cerebral frontal cortex (Fig. 4A–C). Human retina also exhibited the neuronal pattern observed in mouse retina (Fig. 4D–F). In the cardio-vascular system, human expression pattern was similar to the murine one: myocardium weakly expressed KRIT1, and human large vessels did not show labelling (aorta, renal and carotid arteries and vena cava). At last, we did not detect any signal in capillaries of the adult heart, liver, lung, kidney, skeletal muscle and CNS, contrasting with the intense signal observed in all types of vessels including capillaries, when using the JAGGED1 control probe (Section 4/data not shown). Concerning other organs, adult human KRIT1 expression
predominated also in epithelia. In skin, hybridisation signal was detected mostly in basal layers of the epidermis (Fig. 4G–I), a weaker signal was observed in superficial layers and we did not detect any labelling in derma and hypodermis, including in blood vessels. In lung, KRIT1 labelling was observed in the epithelium of main bronchi (Fig. 4J– L), whereas only a very weak signal was detected in lung parenchyma and alveolar walls. In liver and kidney, expression patterns were similar to those observed in mice. In conclusion, our analysis has shown a first phase of ubiquitous Krit1 mRNA expression during early embryogenesis, followed by a second phase of restricted expression which persisted up to adult life. During this second phase, Krit1 was predominating in neuronal cells of central and peripheral nervous system and in various epithelia in mouse. KRIT1 mRNA expression pattern in human adult tissues was similar to the mouse. Quite unexpectedly, in vascular tissues, Krit1 mRNA expression appeared to be weak and limited to large vessels of the embryo, with no signal above background in capillary networks. 4. Material and methods Techniques used in this study have been previously described (Sibony et al., 1995). Only specific features will be mentioned. 4.1. Mouse and human tissues Mouse embryos (C57BL/6) were collected from pregnant mothers at E7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 14.5, 16.5, 18.5 and 20.5 days post-coitum. Murine adult tissues (5-monthold) were fixed overnight in 4% paraformaldehyde and embedded in paraffin after stepwise dehydratation in ethanol. Autopsic paraffin-embedded normal adult human tissues, including brain (frontal cerebral cortex and brainstem), eye, skin, lung, heart, skeletal muscle, liver, kidney, aorta, renal and carotid arteries, vena cava from different donors (provided by C. Godfraind and J.-J. Hauw) had been previously tested positive using in situ hybridisation with NOTCH3 and JAGGED1 probes (Joutel et al., 2000 and unpublished data of the authors). 4.2. Mouse and human probes
Fig. 4. KRIT1 expression in adult human tissues. (A–C) Frontal cerebral cortex, (D–F) retina, (G–I) skin, and (J–L) lung. Abbreviations: ch, choroid; bro, bronchus; art, artery. Choroid and blood red cells exhibited spontaneous auto-fluorescence. Organisation of the pictures like Fig. 1. Scale bar, 120 mm.
Mouse and human cDNA fragments encoding full length Krit1 coding sequence were generated by reverse transcriptase polymerase chain reaction (RT-PCR) using brain total RNA. cDNA clones were entirely sequenced and sequences were shown to be identical to previously reported mouse and human Krit1 coding sequences (mouse Krit1 cDNA clone: nt 298-2508//Accession No. AF310134; human KRIT1 cDNA clone: nt 790-3000//Accession No. AF296765). Antisense and sense riboprobes were generated as follows: 1 mg of linearised cDNA was transcribed in vitro using 50 mCi 35S-UTP and 50 mCi 35S-CTP. At the end of
C. Denier et al. / Mechanisms of Development 117 (2002) 363–367
the reaction, 80% of radioactive nucleotides were recovered in the precipitable material. 4.3. In situ hybridisation Adjacent paraffin-embedded sections (7 mm) were hybridised with antisense and sense Krit1 riboprobes using 4 £ 10 5 cpm/section. For each tissue and embryonic stage, two different embryos were studied. Human JAGGED1 (Joutel et al., 2000) and murine ACE riboprobes (Sibony et al., 1994) were used as positive controls of the quality of our tissues. For both probes, we observed patterns of expression similar to those previously reported; in particular, a strong labelling was observed with the JAGGED1 probe in small capillary vessels. Acknowledgements We thank J.-J. Hauw (Raymond Escourolle Neuropathology Laboratory, Pitie´ -Salpeˆ trie`re Hospital, Paris) and C. Godfraind (Neuropathology Laboratory, Clinique Saint Luc, Bruxelles, Belgium) for providing human tissues and A. Eichmann for critical review of the paper (INSERM U36, Paris). C.D. is a recipient of a fellowship from Poste Accueil INSERM. This work was supported by INSERM, Association Franc¸ aise contre les Myopathies, Fondation de France, Ministe`re Franc¸ ais de la Recherche. References Craig, H.D., Gunel, M., Cepeda, O., Johnson, E.W., Ptacek, L., Steinberg, G.K., Ogilvy, C.S., Berg, M.J., Crawford, S.C., Scott, R.M., SteichenGersdorf, E., Sabroe, R., Kennedy, C.T., Mettler, G., Beis, M.J., Fryer, A., Awad, I.A., Lifton, R.P., 1998. Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous malformation, at 7p15-13 and 3q25.2-27. Hum. Mol. Genet. 7, 1851–1858. Dubovsky, J., Zabramski, J.M., Kurth, J., Spetzler, R.F., Rich, S.S., Orr, H.T., Weber, J.L., 1995. A gene responsible for cavernous malformations of the brain maps to chromosome 7q. Hum. Mol. Genet. 4, 453–458. Eerola, I., Plate, K.H., Spiegel, R., Boon, L.M., Mulliken, J.B., Vikkula, M., 2000. KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation. Hum. Mol. Genet. 9, 1351–1355.
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Faisst, A.M., Gruss, P., 1998. Bodenin: a novel murine gene expressed in restricted areas of the brain. Dev. Dyn. 212, 293–303. Joutel, A., Andreux, F., Gaulis, S., Domenga, V., Cecillon, M., Battail, N., Piga, N., Chapon, F., Godfrain, C., Tournier-Lasserve, E., 2000. The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients J. Clin. Invest. 105, 597–605. Labauge, P., Laberge, S., Brunereau, L., Levy, C., Maciazek, J., TournierLasserve, E., 1998. Hereditary cerebral cavernous angiomas: clinical and genetic features in 57 French families. Socie´ te´ Franc¸ aise de Neurochirurgie. Lancet 352, 1892–1897. Labauge, P., Enjolras, O., Bonerandi, J.-J., Laberge, S., Dandurand, M., Joujoux, J.-M., Tournier-Lasserve, E., 1999. An association between autosomal dominant cerebral cavernomas and a distinctive hyperkeratotic cutaneous vascular malformation in 4 families. Ann. Neurol. 45, 250–254. Laberge-le Couteulx, S., Jung, H.H., Labauge, P., Houtteville, J.P., Lescoat, C., Cecillon, M., Marechal, E., Joutel, A., Bach, J.F., TournierLasserve, E., 1999. Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas. Nat. Genet. 23, 189–193. Otten, P., Pizzolato, G.P., Rilliet, B., Berney, J., 1989. 131 cases of cavernous angioma (cavernomas) of the CNS, discovered by retrospective analysis of 24,535 autopsies. Neurochirurgie 35, 82-3, 128–131. Russel, D.S., Rubinstein, L.J., 1989. Pathology of Tumors of the Nervous system, . 5th ed.Williams and Wilkins, Baltimore, MD pp. 730–736. Sahoo, T., Johnson, E.W., Thomas, J.W., Kuehl, P.M., Jones, T.L., Dokken, C.G., Touchman, J.W., Gallione, C.J., Lee-Lin, S.Q., Kosofsky, B., Kurth, J.H., Louis, D.N., Mettler, G., Morrison, L., Gil-Nagel, A., Rich, S.S., Zabramski, J.M., Boguski, M.S., Green, E.D., Marchuk, D.A., 1999. Mutations in the gene encoding Krit1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1). Hum. Mol. Genet. 8, 2325–2333. Serebriiskii, I., Estojak, J., Sonoda, G., Testa, J.R., Golemis, E.A., 1997. Association of Krev-1/rap1a with Krit1, a novel ankyrin repeat-containing protein encoded by a gene mapping to 7q21-22. Oncogene 15, 1043–1049. Sibony, M., Segretain, D., Gasc, J.-M., 1994. Angiotensin-converting enzyme in murine testis: step-specific expression of the germinal isoform during spermiogenesis. Biol. Reprod. 50, 1015–1026. Sibony, M., Commo, F., Callard, P., Gasc, J.-M., 1995. Enhancement of mRNA in situ hybridization signal by microwave heating. Lab. Invest. 73, 586–591. Zawistowski, J.S., Serebriiski, I.G., Lee, M.F., Golemis, E.A., Marchuk, D.A., 2002. Krit1 association with the integrin-binding protein icap1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis. Hum. Mol. Genet. 11, 389–396. Zhang, J., Clatterbuck, R.E., Rigamonti, D., Chang, D.D., Dietz, H.C., 2001. Interaction between krit1 and icap1alpha infers perturbation of integrin beta1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation. Hum. Mol. Genet. 10, 2953–2960.
Gene expression pattern
Krit1/cerebral cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in embryo and adult C. Denier a,b, J.-M. Gasc c, F. Chapon d, V. Domenga a, C. Lescoat a,b, A. Joutel a,b, E. Tournier-Lasserve a,b,* a
INSERM EMI 99-21, Faculte´ de Me´decine Lariboisie`re, 10, avenue de Verdun, 75010 Paris, France b Laboratoire de Cytoge´ne´tique, AP-HP, Hoˆpital Lariboisie`re, Paris, France c INSERM U36, Colle`ge de France, Paris, France d Service d’Anatomopathologie, CHU de Caen, Caen, France Received 24 May 2002; received in revised form 17 June 2002; accepted 18 June 2002
Abstract Cavernous malformations are capillaro-venous lesions mostly located within the central nervous system (CCM/OMIM#116860) and occasionally within the skin and/or retina. They occur as a sporadic or hereditary condition. Three CCM loci have been mapped, and the sole gene identified so far, CCM1, has been shown to encode KRIT1, a protein of unknown function. In an attempt to get some insight on the relationship between KRIT1 mutations and CCM lesions, we investigated Krit1 mRNA expression during mouse development from E7.5 to E20.5 and in adult tissues, of both mouse and human origin. A ubiquitous Krit1 mRNA expression was detected from E7.5 up to E9.5. Then, it became progressively restricted from E10.5 to E12.5, to become detectable later essentially in the nervous system and various epithelia. Strong labelling was observed in neurons in the brain, cerebellum, spinal cord, retina and dorsal root ganglia. In epithelia, Krit1 mRNA expression was detected in differentiating epidermal, digestive, respiratory, uterine and urinary epithelia. A similar pattern of expression persisted in mouse and man adult nervous system and epithelia. Unexpectedly, in vascular tissues, expression of Krit1 was detected only in large blood vessels of the embryo. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Krit1; Cerebral cavernous malformations; Cavernous angiomas; In situ hybridisation
1. Introduction Cerebral cavernous malformations (CCM/ OMIM#116860) are vascular anomalies mostly located within the central nervous system (CNS) and characterised by abnormally enlarged, endothelial lined, vascular sinusoid cavities, without intervening brain parenchyma (Russel and Rubinstein, 1989). Clinical features include seizures and/or focal neurological deficits mostly due to cerebral haemorrhages. Mean age of clinical onset is around 20–30 years old. CCM prevalence has been estimated to 0.1–0.5% (Otten et al., 1989). They can occur as a sporadic or familial, autosomal dominant, condition. Familial CCM are characterised by the presence of multiple cerebral lesions whose number is strongly correlated to patient age, suggesting a dynamic nature of this disease (Labauge et al., 1998).
Hereditary CCM are occasionally associated with retinal and/or cutaneous vascular malformations (Labauge et al., 1999; Eerola et al., 2000). Three CCM loci have been mapped (Dubovsky et al., 1995; Craig et al., 1998). CCM1 was identified as coding KRIT1 (Laberge-le Couteulx et al., 1999; Sahoo et al., 1999), a protein of unknown function, previously shown to interact in vitro with Rap1A, a small Ras like GTPase (Serebriiskii et al., 1997). KRIT1 also interacts with ICAP1, a modulator of b1 integrin signal transduction, whose murine ortholog gene is Bodenin (Zhang et al., 2001; Zawistowski et al., 2002; Faisst and Gruss, 1998). Pathophysiological mechanisms underlying CCM are unknown. As a first step towards the investigation of CCM mechanisms, we studied Krit1 expression by in situ hybridisation during C57BL/6 mouse development (E7.5– E20.5) and in normal adult mouse and human tissues.
* Corresponding author. Tel.: 133-1-44-89-77-50; fax: 133-1-44-89-7755. E-mail address: [email protected] (E. TournierLasserve). 0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00209-5
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networks, including those of the developing brain and eye (Fig. 2A–C). 2.2. Developing and adult nervous system and eye Neural tube labelling was homogeneous from E7.5 to E11.5 (Figs. 1 and 2A–C). By E12.5, Krit1 predominated in proliferating neural cells: maximal expression was
Fig. 1. Krit1 mRNA expression in early mouse embryos. Krit1 is ubiquitously detected in embryos at E7.5 (A–C), E8.5 (D–F) and E9.5 (G–I). First column, brightfield views for histology; second column, darkfield views of the same sections with antisense probe; third column, darkfield views with Krit1 sense probe. Scale bar, 120 mm.
2. mRNA Krit1 expression in embryo and adult mouse 2.1. Early embryonic development From E7.5 to E9.5, a ubiquitous embryonic Krit1 expression was detected in primordial layers, contrasting with the weak signal observed in the adjacent uterus (Fig. 1). At E10.5, E11.5 and E12.5, Krit1 transcripts were widely expressed in the neural tube (Fig. 2A–B) and in various epithelia including coelomic and intestinal ones. Comparatively, only moderate labelling was observed in adjacent mesenchyma, limb buds, dermomyotome and scleromyotome. Weak Krit1 labelling was observed in vascular structures such as heart and large vessels (aorta, lateral vascular network, cardinal and head veins, and umbilical vessels), whereas there was no difference between sense and antisense labelled sections when examining small capillary
Fig. 2. Krit1 mRNA expression in central and peripheral nervous system of embryo and adult mouse. (A–C) E10.5 axial sections. (D–F) E14.5 brainstem sagittal section with adjacent trigeminal ganglia (tg) and internal carotid (ic). (G–I) E14.5 sagittal thoracic section. (J–L) E20.5 axial thoracic section with the spinal cord and adjacent dorsal root (drg) and sympathetic ganglia (syg). (M–O) E20.5 and (P–R) adult brain cortical sections. (S–U) Adult sagittal brain sections showing hippocampus (h) and subjacent corpus callosum (cc). (V–X) Adult cerebellum sections. (Y–ZZ) adult retinal section. Arrow-heads point to capillaries; no labelling was detected in small blood vessels. Choroid and red blood cells exhibited spontaneous auto-fluorescence in darkfield, also seen with the sense probe. Abbreviations: nt, neural tube; ao, aorta; cv, cardinal vein; tg, trigeminal ganglia; bst, brainstem; ic, internal carotid; drg, dorsal root ganglia; sc, spinal cord; ve, vertebrae; hea, heart; thy, thymus; syg, sympathetic ganglia; v, meningeal vessel; hip, hippocampus; cc, corpus callosum; pkj, Purkinje cells; gr, granular cells; ch, choroid. Organisation of the pictures like Fig. 1. Scale bar, 120 mm.
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observed at E14.5, notably in subventricular cell layers, in the ganglionic eminence projecting into the third ventricle, in the thalamus, brainstem and trigeminal ganglia (Fig. 2D– F). During late embryogenesis, at E16.5, E18.5 and E20.5, Krit1 transcripts were mostly detected within layers containing neuronal cells as compared to layers enriched in glial cells (Fig. 2M–O). In adult brains, the same pattern persisted with strong signal in cortex (Fig. 2P–R), hippocampal dentate gyrus (Fig. 2S–U), olfactory bulb, habenula, cerebellum (granular layers and Purkinje cells, Fig. 2V–X) whereas the white matter was not labelled. Within the spinal cord, Krit1 mRNA expression pattern also predominated in the grey matter in embryonic (Fig. 2J–L) as in adult stages. Meninges and choroid plexus exhibited a weak signal. In the peripheral nervous system, a strong signal was detected in dorsal root ganglia by E11.5 up to adult stages (Fig. 2G–I, J–L) and a weaker signal was observed in sympathetic chains ganglia (Fig. 2J–L). In the eye, Krit1 labelling was coincident with the appearance of the optic placods, located from E10.5 in both inner neural and outer retinal layers. Strong expression persisted in the different neural cells layers in all studied developmental stages and in adult mouse retina (Fig. 2Y–ZZ). 2.3. Differentiating and adult thoraco-abdominal organs Krit1 mRNA was detected in the lung bud. By E12.5– E14.5, signal predominated in the tracheal/bronchial epithelium. The hybridisation signal was weaker in lung parenchyma and alveolar walls. This pattern persisted in adult mouse. In liver, a homogeneous pattern of expression existed as early as the primitive epithelial hepatic bud was formed. Liver expression level decreased from E16.5, but a weak signal persisted in adult mouse. Pancreas and spleen parenchyma exhibited a similar expression pattern. Krit1 expression was observed all along the digestive tract (oesophagus, stomach, intestine) at all developmental stages. By E14.5–E16.5, it predominated in epithelia compared to subjacent mesenchyma, and persisted up to adulthood (Fig. 3A–C, D–F). Krit1 kidney expression was detected all along organogenesis. It was restricted to glomeruli and tubules once medullary and cortical regions differentiated at E14.5. It was also present in ureter and bladder epithelia. In adult, it persisted moderately in glomeruli and distal convoluted tubules, in collecting ducts and ureter epithelium. The adrenal gland was strongly labelled from the time of adrenal primordium formation at E12.5 until adult in both cortex and medulla.
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In the aorta, inter-somitic, head and neck vessels, a weak signal was detected from E9.5, increased up to E14.5 (carotid arteries, aorta; Fig. 2D–F, G–I) and then diminished to nearly disappear at E18.5–E20.5. In the adult, Krit1 was not clearly detected in large blood vessels. 2.4.2. Capillary networks From embryonic to adult stages, on the contrary to what was observed in large blood vessels, we did not detect a reproducible signal in the small capillary networks of the heart, lung, liver, kidney, skeletal muscle and CNS; no clear difference was detected when comparing capillaries in sections labelled with sense and antisense probes. CNS negative capillaries are shown in Fig. 2. 2.5. Other tissues In mouse skin, Krit1 mRNA expression was detected at all stages studied, particularly from E14.5 in germinal cell layers of the epidermis and in the hair follicles and vibrissiae. In skeletal muscles, only a weak signal was observed at embryonic stages. In bones, variable levels of Krit1 mRNA expression were observed: moderate in precartilage (E12.5), low during cartilage formation (E14.5), stronger in ossification centres (E16.5). Late expression (E18.5–E20.5) persisted mostly in peri-osteal ossification regions (vertebrae and craniofacial bones).
2.4. Embryonic and adult cardio-vascular system 2.4.1. Heart and large vessels Krit1 mRNA was detected in the myocardium at all studied embryonic stages (Fig. 2G–I), and persisted at a weak level in adult.
Fig. 3. Krit1 mRNA expression in various epithelia of embryo and adult mouse. (A–C) Adult intestine. (D–F) Adult oesophagus. (G–I) Adult seminiferous tubules and (J–L) ovarian follicules (fol). Abbreviations: gl, germinal layers, mu, mucosa; spz, spermatozoids; scy, spermatocytes; sgn, spermatogonias. Organisation of the pictures like Fig. 1. Scale bar, 120 mm.
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By E14.5, dentition primordia with first incisor tooth buds were labelled and Krit1 mRNA expression persisted through tooth differentiation, particularly in odotonblasts layers, up to E20.5. In gonads, Krit1 expression was detected from the primitive sexually undifferentiated stages (E11.5) and the primitive seminiferous tubules (E12.5–E14.5) up to adult stages with strong labelling in premeiotic spermatocytes (Fig. 3G– I) and ovarian follicles (Fig. 3J–L). 3. KRIT1 mRNA expression in adult human tissues KRIT1 mRNA expression pattern in human adult tissues was similar to mouse adult Krit1 expression pattern. Within the adult human CNS, mRNA expression also predominated in neuronal layers, in the brainstem as in cerebral frontal cortex (Fig. 4A–C). Human retina also exhibited the neuronal pattern observed in mouse retina (Fig. 4D–F). In the cardio-vascular system, human expression pattern was similar to the murine one: myocardium weakly expressed KRIT1, and human large vessels did not show labelling (aorta, renal and carotid arteries and vena cava). At last, we did not detect any signal in capillaries of the adult heart, liver, lung, kidney, skeletal muscle and CNS, contrasting with the intense signal observed in all types of vessels including capillaries, when using the JAGGED1 control probe (Section 4/data not shown). Concerning other organs, adult human KRIT1 expression
predominated also in epithelia. In skin, hybridisation signal was detected mostly in basal layers of the epidermis (Fig. 4G–I), a weaker signal was observed in superficial layers and we did not detect any labelling in derma and hypodermis, including in blood vessels. In lung, KRIT1 labelling was observed in the epithelium of main bronchi (Fig. 4J– L), whereas only a very weak signal was detected in lung parenchyma and alveolar walls. In liver and kidney, expression patterns were similar to those observed in mice. In conclusion, our analysis has shown a first phase of ubiquitous Krit1 mRNA expression during early embryogenesis, followed by a second phase of restricted expression which persisted up to adult life. During this second phase, Krit1 was predominating in neuronal cells of central and peripheral nervous system and in various epithelia in mouse. KRIT1 mRNA expression pattern in human adult tissues was similar to the mouse. Quite unexpectedly, in vascular tissues, Krit1 mRNA expression appeared to be weak and limited to large vessels of the embryo, with no signal above background in capillary networks. 4. Material and methods Techniques used in this study have been previously described (Sibony et al., 1995). Only specific features will be mentioned. 4.1. Mouse and human tissues Mouse embryos (C57BL/6) were collected from pregnant mothers at E7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 14.5, 16.5, 18.5 and 20.5 days post-coitum. Murine adult tissues (5-monthold) were fixed overnight in 4% paraformaldehyde and embedded in paraffin after stepwise dehydratation in ethanol. Autopsic paraffin-embedded normal adult human tissues, including brain (frontal cerebral cortex and brainstem), eye, skin, lung, heart, skeletal muscle, liver, kidney, aorta, renal and carotid arteries, vena cava from different donors (provided by C. Godfraind and J.-J. Hauw) had been previously tested positive using in situ hybridisation with NOTCH3 and JAGGED1 probes (Joutel et al., 2000 and unpublished data of the authors). 4.2. Mouse and human probes
Fig. 4. KRIT1 expression in adult human tissues. (A–C) Frontal cerebral cortex, (D–F) retina, (G–I) skin, and (J–L) lung. Abbreviations: ch, choroid; bro, bronchus; art, artery. Choroid and blood red cells exhibited spontaneous auto-fluorescence. Organisation of the pictures like Fig. 1. Scale bar, 120 mm.
Mouse and human cDNA fragments encoding full length Krit1 coding sequence were generated by reverse transcriptase polymerase chain reaction (RT-PCR) using brain total RNA. cDNA clones were entirely sequenced and sequences were shown to be identical to previously reported mouse and human Krit1 coding sequences (mouse Krit1 cDNA clone: nt 298-2508//Accession No. AF310134; human KRIT1 cDNA clone: nt 790-3000//Accession No. AF296765). Antisense and sense riboprobes were generated as follows: 1 mg of linearised cDNA was transcribed in vitro using 50 mCi 35S-UTP and 50 mCi 35S-CTP. At the end of
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the reaction, 80% of radioactive nucleotides were recovered in the precipitable material. 4.3. In situ hybridisation Adjacent paraffin-embedded sections (7 mm) were hybridised with antisense and sense Krit1 riboprobes using 4 £ 10 5 cpm/section. For each tissue and embryonic stage, two different embryos were studied. Human JAGGED1 (Joutel et al., 2000) and murine ACE riboprobes (Sibony et al., 1994) were used as positive controls of the quality of our tissues. For both probes, we observed patterns of expression similar to those previously reported; in particular, a strong labelling was observed with the JAGGED1 probe in small capillary vessels. Acknowledgements We thank J.-J. Hauw (Raymond Escourolle Neuropathology Laboratory, Pitie´ -Salpeˆ trie`re Hospital, Paris) and C. Godfraind (Neuropathology Laboratory, Clinique Saint Luc, Bruxelles, Belgium) for providing human tissues and A. Eichmann for critical review of the paper (INSERM U36, Paris). C.D. is a recipient of a fellowship from Poste Accueil INSERM. This work was supported by INSERM, Association Franc¸ aise contre les Myopathies, Fondation de France, Ministe`re Franc¸ ais de la Recherche. References Craig, H.D., Gunel, M., Cepeda, O., Johnson, E.W., Ptacek, L., Steinberg, G.K., Ogilvy, C.S., Berg, M.J., Crawford, S.C., Scott, R.M., SteichenGersdorf, E., Sabroe, R., Kennedy, C.T., Mettler, G., Beis, M.J., Fryer, A., Awad, I.A., Lifton, R.P., 1998. Multilocus linkage identifies two new loci for a mendelian form of stroke, cerebral cavernous malformation, at 7p15-13 and 3q25.2-27. Hum. Mol. Genet. 7, 1851–1858. Dubovsky, J., Zabramski, J.M., Kurth, J., Spetzler, R.F., Rich, S.S., Orr, H.T., Weber, J.L., 1995. A gene responsible for cavernous malformations of the brain maps to chromosome 7q. Hum. Mol. Genet. 4, 453–458. Eerola, I., Plate, K.H., Spiegel, R., Boon, L.M., Mulliken, J.B., Vikkula, M., 2000. KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation. Hum. Mol. Genet. 9, 1351–1355.
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