Expression of Flt3 tyrosine kinase receptor gene in mouse hematopoietic and nervous tissues

Differentiation (1995) 58:351-359

Expression of 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 I

2 3

Laboratoire d’Oncologie MolCculaire, U.119 Inserm, 27 Blvd. Leii Roure, F-13009 Marseille, France Centre I’lmmunologie Inserm-CNRS de Marseille-Luminy, Marseille. France 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 receptor 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 lymphohematopoietic 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 trophoblasts 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 protein is present in the tissues positive for Flr3 RNA expression. 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 protein, 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 lo: D. Birnbaum

closely linked to the FLTl gene, which encodes a receptor 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 hybridization [21, 221, amplification by polymerase chain reaction of reverse transcribed RNAs (RT-PCR) [ 1 I] and assays of kinase activity [lo], and in humans using RT-PCR [19, 231 or Northern-blot hybridization [2]. Flt3 is predominantly 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 lymphoid 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 hematopoietic stem cells. Even though one primary function of FLT3 appears to be within the hematopoietic system, the above-mentionned preliminary analyses of the expression of the gene have suggested that this receptor could mediate signalling 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 potential developmental roles for these receptors in nonhematopoietic 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 using this technique, with special emphasis on hematopoietic 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 m M MgCI,; 1 mM EGTA; 10 mM NaPyrophosphate; 100 mM NaF 10 mM dithiothreitol (DTT), 10 pg.ml-1 aprotinin, 10 pg.ml-1 leupep-

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Fig. 1A-F. FIr3 expression during thymus development. A, C, E Bright-field; B, D, E Dark-field views of sections through mouse embryonic thymus hybridized with a Flf3 probe, E16.5 (A, B), E18.5 (C, D).Bright-field (E) and dark-field (F) photographs of a section through a thymus from a new-born mouse were hybrid-

ized with the same probe. In all sections including the corticomedullary junction, FIr3 transcripts were localized in the medulla (m), whereas the cortex (c) was negative; bars, 100 pm (A, B); 50 pm (C-F)

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Fig. 2 A-G

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field. B, D Dark-field. Coronal sections through an adult mouse brain were hybridized with an Flr3 probe. Flr3 transcripts were detected in nuclei of the mesencephalon (A, B): red nucleus (N.

ru, arrow) and another nucleus in the gray matter (arrowhead). Some cells scattered in the substantia nigra (S. n i ) were positive for Flt3, K [It, third ventricle; Hip, hippocampus; bar, 300 pm (A, B); 100 pm (C, D)

Fig. 2A-C. Flr3 expression in adult lymph nodes, embryonic liver and adult spleen; bright-field (A, C, F) and dark-field (B, D, G ) views of sections were hybridized with an Flt3 antisense probe. A, B Transverse section through an axillary lymph node; Flr3 RNAs were localized in the paracortical area @) surrounding the germinal centers (gc), which were negative. C, D, E Sagittal section of a day-14.5 embryo; Flr3 expression exhibited a spotlike pattern (arrowhead) in the liver (li); diaphragm (d) was negative. Control sense probe (E) was negative. F, C Section of an adult spleen; Flr3 was expressed in the red pulp (rp);white pulp (wp) was negative; bars, 100 pm

tin, 1 mM phenylrnethylsulfonylfluoride). Tissue lysates were clarified by centrifugation at 13,000 rprn for 15 min, heated in sodium dodecyl sulfate (SDS) sample buffer, separated by polyacrylamide gel electrophoresis (PAGE), transferred to lmmobilon (Millipore, St-Quentin-Yvelines) and probed with FLT3 antibodies. Detection was performed with '2Wabelled Protein A (Amersham, Les Ullis, France).

Fig. 3. F1r3 expression in mesencephalon peduncles. A, C Bright-

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Fig. 4A-D. F/t3 expression in brainstem and cerebellum. Brightfield (A, C) and dark-field (B,D) views of horizontal sections through adult mouse brainstem (A,B) and cerebellum (C, D)

were hybridized with an F/13 probe; Ce, cerebellum; Ci, inferior colliculus; Purkinje cells; mof, molecular layer; g r m , granular layer; burs, 100 pm

In situ hybridization. The F/t3 probe was a 1.1-kb fragment from the 5' part of the MP14 cDNA clone 1211 subcloned into a Bluescript vector and encompassing a portion encoding the last three immunoglobulin-like domains and the transmembrane region. Radiolabeled sense and antisense probes were generated after linearization of plasmid DNA by Pstl and EcoRl digestion and in vitro transcription, in the presence of %-labeled UTP, using T3 and T7 polymerases. Embryos and tissues were dissected from C3H/He inbred mice, fixed in 4% buffered paraformaldehyde at 4"C and embedded in paraffin wax. Noon of the day on which the vaginal plug was detected was considered to be 0.5 day of gestation (the corresponding stage is designated E0.5). Serial microtome sections (6 pm) were deparaffinized and hybridized with 50,000-75,000 cpm/pl probe, according to the protocol described in [25]. Slides were then treated as previously described [3].

Results Expression of Flt3 during thymus development

To determine the expression pattern of Flt3 in the mouse, a Flr3-specific probe was used for in situ hybridization of selected sections from mouse embryo organs. Since preliminary studies have shown that the hematopoietic and nervous systems represent the two major sites of expression of Flt3, we first hybridized sections of mouse thymus at various stages of development. Flt3 RNA was detected in the thymus at day 16.5 in a spotlike pattern (Fig. I A , B). After day 16.5 of embryonic life, cortical and medullary differentiation is readily visible in the fetal thymus. At day 18.5 a high level of Flt3

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Fig. 5A, B. Localization of Flr3 RNA in placenta. Bright-field (A) and dark-field (B) views of a section through an E14.5 placenta were hybridized with an Flr3 probe. Labyrinthine trophoblasts

(la) were positive whereas spongiotrophoblasts ( s p ) were negative; bar, 100 pm

expression was found in the medulla (Fig. lC, D). The same pattern of expression was observed in the newborn mouse (Fig. lE, F) and in the adult mouse (not shown). There was little or no expression in the cortex. No Flt3 expression was seen in sections of E14.5 and E15.5 (not shown). Earlier stages were not analyzed. The sense probe did not give a specific signal above background in any of the sections examined (not shown).

gene was found in different brain structures. A weak signal was present in the deeper layers of the cerebral cortex, in some, thalamic nuclei (paraventricular, medial geniculate body) and in the globus pallidus (data not shown). In the mesencephalon, Flt3 transcripts were found in the superior colliculus (data not shown), red nucleus (Fig. 3A, B arrow), some deep nuclei located in the gray matter (Fig. 3A, B arrowhead), in the substantia nigra (Fig. 3C, D) and in the inferior colliculus (Fig. 4A, B). In the cerebellum, Purkinje cells expressed high levels of Flt3 RNA whereas molecular and granular layers were negative (Fig. 4C, D).

Expression of Flt3 in lymph nodes, fetal liver m d spleen

We examined the expression of Flt3 in three other hematopoietic organs, adult lymph node, fetal liver and adult spleen. Adult lymph node is the site of B cell lymphopoiesis. Fetal liver represents a major site of hematopoiesis during development; interestingly, the Flk2/Flt3 gene was initially isolated from populations of enriched hematopoietic progenitors from fetal liver [ 1 13. In lymph note, Flt3 RNA was localized to the paracortical T cell areas, whereas germinal centers (Fig. 2A, B) and hilum (not shown) were negative. Spotlike expression of Flt3 was detected in E14.5 fetal liver in regions which probably represented areas of hematopoiesis (Fig. 2C, D). In spleen, Flr3 was detected, also in a spotlike pattern, in the red pulp only (Fig. 2F, G). The sense probe did not give a signal above background (Fig. 2E).

Expression of Flt3 in placenta

In addition to their role in hematopoiesis, RTKs of class I11 appear to be involved in placental development or function. Flt3 expression has been observed by Northern-blot hybridization of placenta at various stages of development. By day 15.5 of gestation, the three different cell types of the mature placenta can be distinguished. An outer layer of spongiotrophoblasts surrounds the inner labyrinthine trophoblast. By in situ hybridization, Flt3 RNA was found in the labyrinthine part of the placenta, whereas spongiotrophoblast cells did not express the gene (Fig. 5).

Expression cgFlt3 in the adult nervous system

Expression of the FLT3 protein in adult tissues

As Northern-blot hybridizations show Flt3 transcripts in the nervous system [21], we performed RNA in situ hybridization of sections from day-14.5 embryo and adult central nervous system. We were not able to detect Flt3 expression in E14.5. In the adult, the expression of this

To determine whether the expression pattern of the FLT3 protein correlated with that of Flt3 RNA determined previously by Northern-blot hybridization [21] and in this work by in situ hybridization, we attempted to identify the mouse Flt3 gene product in adult tissues by using a

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FLT3 145kDa

I

FLT3 155 kDa 132 kDa

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Fig.6. lmmunoblot analysis of the expression of the FLT3 proteins in adult mouse tissues. Tested tissues are indicated above each Imc. Arrows point to the bands corresponding to FLT3 proteins (molecular m m s is indicated in kDa)

FLT3 rabbit polyclonal antibody [lo] in a Western-blot analysis. Results are shown in Fig. 6. FLT3 protein was found mainly in brain, cerebellum, placenta, and lympho-hematopoietic organs. A low level of protein was also detected in large intestine, kidney and heart. It may correspond to the presence of FLT3-positive blood cells or to a true low level of expression. However, in situ hybridization of these tissues remained negative. This would support the former hypothesis or mean that the expression level was below the sensitivity of the in situ hybridization method. No FLT3 protein was detected in skeletal muscle, liver, lung, uterus or gonads. The protein was represented by two bands in placenta and lympho-hematopoietic organs (lower panel) whereas only one band was visible (at various times of exposure) in tissues of nervous origin (upper panel). The levels of the signals observed with thymus, spleen and lymph node protein extracts correlated well with the corresponding RNA signals detected by in situ hybridization.

Discussion Growth and differentiation of hematopoietic cells are strictly regulated phenomena, which rely on a complex network of growth factors. The FLT3 tyrosine kinase receptor appears to play an important role in these processes. In this work, we determined the expression pattern of the Flr3 gene in adult and embryonic mouse tis-

sues. Fft3 is expressed in lympho-hematopoietic organs such as thymus, fetal liver and lymph node. It is also expressed outside the hematopoietic system, primarily in brain, cerebellum and placenta. Accordingly, the FLT3 protein was found in those tissues. The exquisite localization of Ffr3 RNA in the medullary region of E18.5 thymus and the paracortical arex of lymph nodes suggests that the gene is highly expressed in T-lymphocyte zones. In addition, scattered cells were found in fetal liver and day- 16.5 thymus, suggesting that few immature precursor cells may express Flr3 RNA. This is concordant with previous work on Flk2/Flr3 [ 1 I ] showing expression of the gene in immature T cells. However, the high level of observed Fft3 message, also in adult thymus and spleen, is not compatible with an exclusive positivity of immature thymocytes. Hematopoietic precursor cells can give rise to both lymphoid and nonlymphoid populations. Since the appearance and the development of thymic medullary stromal cells (including dendritic cells and macrophages) seems to be dependent upon the presence of mature thymocytes, one cannot exclude the possibility that Flr3 is also expressed in a population of stromal cells. Furthermore, the signal observed in spleen suggests that a subpopulation of macrophages might express Flr3. The distribution of F f f 3 RNAs reported here will have to be compared with analysis of the FLT3 receptor on the surface of hematopoietic cells, once efficient antibodies are available. The FLT3 receptor is highly related to two other hematopoietic ,RTKs, the receptors for CSFl and for the Steel factor, respectively encoded by the Fms and Kit proto-oncogenes [17]. Both Frns and Kit genes are expressed outside hematopoietic cells. Fms expression is found in gonads, placenta and myoblasts. The Kit gene is expressed in placenta, gonads, melanoblasts, and the nervous system. The tissue distribution of Flr3 RNAs has some similarity to that of Kit. However, we failed to observe Flt3 expression in testes or ovaries, and no FLT3 protein was detected in the gonads, although a faint band was seen in testes. It is unlikely that this band corresponded to FLT3, since only short aberrant Ffr3 transcripts are found in testis by Northern-blot analysis [21]. Interestingly, both Kit and Flf3 are expressed in placenta, brain and cerebellum. In placenta, Kit signals are predominantly found in the decidua[ 131, whereas Fft3 is mainly expressed in labyrinthine trophoblasts. This is also in contrast to Frns expression, which is predominant in trophoblast giant cells and spongiotrophoblast of day- 15.5 placenta but weak in labyrinthine trophoblast [ 161. In cerebellum, Kif RNAs are found mainly in the molecular layer [ 12, 131, whereas Flt3 is expressed in the Purkinje cells, as is the Steel gene coding for the ligand of KIT. In adult brain, Kit and Flt3 RNAs are both present in the colliculi and brainstem nuclei [ 5 , 131. In thymus, KIT expression has been studied by immunofluorescence analysis. Although it cannot be compared directly with our in situ hybridization analysis of Ffr3 RNAs, the pattern appears different from that of Flt3. The number of cells positive for KIT is maximal at day 15 but decreases thereafter [15]. By day 17, only very few cells express

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KIT in thymus. In contrast, KIT and Flt3 expression seem to be coincident in fetal liver. Establishing the expression pattern of Fft3 outside hematopoiesis is important for several reasons. Kit and Flt3 RNAs are found in the same areas, in particular placenta and nervous system. W or Steel mouse mutants, in which the Steel factor/KIT interaction does not function normally, do not have apparent placental or brain defects. Functional redundancy with another RTK pathway, possibly FLT3, could explain these observations. However, the precise sites of expression of Kit and Flt3 do not overlap. In placenta and cerebellum, the two genes are not expressed in the same cell layers. In both cases, Ffr3 RNA localization overlaps with that of the Steel gene. Presently no functional model sustains this observation. It will be of interest to extend this analysis by determining the expression of the FLT3 ligand in those tissues. It is intriguing that no RNA transcript corresponding to the FLT3 ligand was detected in brain or cerebellum by Northern-blot analysis (Rosnet et al., unpublished observations). A low level or a variant form of FLT3 ligand may be present in this tissue. This may relate to the FLT3 protein, detected in the brain and cerebellum, which appears to have a different molecular mass, possibly as the result of different glycosylations. In any case, the expression pattern of the FLT3 receptor appears more restricted than that of its ligand; indeed, the gene encoding the FLT3 ligand appears to be expressed at relatively high levels in almost all tissues tested except the central nervous system [4,6, 71. Thus, restricted expression of the receptor appears to play a major role in the specific activity of the ligand. The expression pattern of the Fft3 gene should obviously be confirmed in humans. We previously reported a preliminary analysis of the expression pattern of the human FLT3 gene [ 191. A FLT3 transcript was clearly pesent in various hematopoietic cells and tissues, but, in contrast to the mouse gene, no expression could be evidenced by Northern-blot hybridization of placenta or brain RNAs. These differences in the expression patterns of the murine and human genes should be interpreted with caution until more refined analyses are performed. Acknowledgements. We thank F. Birg, F. Coulier, P. Dubreuil and C. Mawas for enthusiastic support and critical reading of the manuscript. This work was supported by Inserm and by grants from the “Association pour la Recherche contre le Cancer”.

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