Differentiation (1995) 58:351-359 zyxwvutsrq Expression of zyxwvu FZt3 tyrosine kinase receptor gene in mouse hematopoietic and nervous tissues Odile delapeyrierel, Philippe Naquet2, Jacqueline Planchel, Sylvie Marchettol, Robert RottapeP, Daniele Gambarelli4,Olivier Rosnetl, Daniel Birnbauml zyxwvu I Laboratoire d’Oncologie MolCculaire, U.119 Inserm, 27 Blvd. Leii Roure, F-13009 Marseille, France 2 Centre I’lmmunologie Inserm-CNRS de Marseille-Luminy, Marseille. France 3 Wellesley Hospital, Toronto, Ontario, Canada HBpital de la Timone, Marseille, France Accepted in revised form: 2 December 1994 Abstract. The Flr3 gene encodes a tyrosine kinase re- ceptor highly related to the Kit and Fms gene products. We have studied the expression of Flr3 by using in situ hybridization of mouse tissue sections. The results show that Flr3 RNAs are present in certain regions of lympho- hematopoietic organs, placenta and nervous system. Flr3 is expressed in the medullary area of fetal and newborn thymus, in the paracortical regions of lymph nodes and in the red pulp of spleen. In placenta, labyrinthine tro- phoblasts express Flr3. Finally, Flr3 RNAs are found in several regions of the brain and in cerebellar Purkinje cells. Western-blot analysis showed that the FLT3 pro- tein is present in the tissues positive for Flr3 RNA ex- pression. Our observations allow for a comparison with the distribution of the Kit gene and analysis of a possible redundancy between KIT and FLT3 receptors. Introduction The receptors for colony-stimulating factor 1 (CSFI ) and Steel factor, KIT ligand (SLF), respectively encoded by the FMS and KIT proto-oncogenes, and a third pro- tein, the FLT3 gene product ([17], for a review) are three hematopoeitic receptors endowed with tyrosine kinase activity. These receptor-type tyrosine kinases (RTKs) are characterized by an extracellular ligand-binding region divided into five immunoglobulin-like domains, and by an intracellular catalytic domain split into two parts by a stretch of hydrophobic amino acids known as the kinase insert [24]. The Flr3 gene (also named Flk2) was first identified in the mouse after a search for either Fms-related genes [ 181 or RTK genes (associated with hematopoietic stem cells) [ 1 I]. The mouse gene encodes a transmembrane receptor of one thousand amino acid residues, with an apparent molecular weight of 135-155 kDa [8, 101. The human gene is located in chromosomal region 13q12, Correspondence zyxwvutsrqp lo: D. Birnbaum closely linked to the FLTl gene, which encodes a recep- tor for vascular endothelial growth factor [4, 201, and its product is a protein of 993 amino acid residues [21]. The overall pattern of expression of Flr3 has been roughly established in the mouse, using Northern blot hybridiza- tion [21, 221, amplification by polymerase chain reaction of reverse transcribed RNAs (RT-PCR) [ zy 1 I] and assays of kinase activity [lo], and in humans using RT-PCR [19, 231 or Northern-blot hybridization [2]. Flt3 is pre- dominantly expressed in lympho-hematopoietic cells and in the nervous system. Within the hematopoietic tissues, and in contrast to C S F I W M S and SLFWKIT, FLT3 seems restricted to stem cells, early progenitor cells and immature lymphocytes [2, 11, 231. Accordingly, FLT3 RNA is found in leukemic blast cells of myeloid or lym- phoid origin [l]. The recently identified ligand of FLT3 is synthesized by stromal cells [4, 6, 71 and could prove to be of major importance for the physiology of hemato- poietic stem cells. Even though one primary function of FLT3 appears to be within the hematopoietic system, the above-mention- ned preliminary analyses of the expression of the gene have suggested that this receptor could mediate signal- ling in other types of tissues. The pattern of expression of the related genes Fms and Kit determined by in situ hybridization on mouse tissue sections has suggested po- tential developmental roles for these receptors in nonhe- matopoietic areas during embryogenesis and adult life [5, 9, 13, 14, 161. Therefore, we have studied the pattern of expression of Flr3 in mouse fetal and adult tissues us- ing this technique, with special emphasis on hematopoi- etic and nervous tissues. Methods Western immunoblot analysis. A polyclonal antibody to FLT3 was obtained by immunization of rabbits with a TrpE-FLT3 fusion protein [lo]. Tissues were lysed in lysis buffer (50 mM Hepes, pH 7.0; 150mM NaCI; 10% glycerol; 1% Triton x100; 1.5 mM MgCI,; 1 mM EGTA; 10 mM NaPyrophosphate; 100 mM NaF 10 mM dithiothreitol (DTT), 10 pg.ml-1 aprotinin, 10 pg.ml-1 leupep-