Expression of the tyrosine kinase receptor gene trkB is ... - Development

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Development 109, 845-850 (1990) Printed in Great Britain © The Company of Biologists Limited 1990

Expression of the tyrosine kinase receptor gene trkB is confined to the murine embryonic and adult nervous system RUDIGER KLEIN2, DIONISIO MARTIN-ZANCA1, MARIANO BARB ACID2 and LUIS F. PARADA 1 * ' Molecular Embryology Croup, BRl-Basic Research Program, NCI-Frederick Cancer Research Facility, PO Box B, Frederick, Maryland 21701, USA 2 Squibb Institute for Medical Research, Department of Molecular Biology, PO Box 4000, Princeton, New Jersey 08543-4000, USA

• Author for correspondence

Summary We have examined the expression of the trkB gene, which encodes a member of the family of protein tyrosine kinase (TK) transmembrane receptors, during mouse embryogenesis using in situ hybridization and Northern analysis. Transcripts were first detected in the neuroepithelium and in the neural crest of 9.5 day embryos with regions of high expression in the neural folds and at the lateral neuroepithelium. However, during the process of cephalization and development of the peripheral nervous system, transcripts were detected in most neural tissues, including the brain, spinal cord,

cranial and spinal ganglia, and along the pathways of axonal tracts extending peripherally. In the adult brain, expression continues in a complex pattern that is confined to specific regions or neuron types. The expression of trkB, a TK receptor, in early embryogenesis, and specifically in neural tissues, is consistent with the notion that this gene plays a role in the events that regulate the development of the nervous system.

Introduction

as evidence for a gene having a role in gene regulation (Herr et al. 1988). Krox-20 and mKrl are examples of genes encoding zinc finger proteins that are expressed in the embryonic nervous system. KroxlO is transcribed in the second and fourth neuromeres during early neurogenesis (Wilkinson et al. 1988), and mKrl is expressed in various tissues of the developing nervous system (Chowdhury et al. 1988). Similarly, a series of genes that encode POU sequences have been shown to have neural specific patterns of expression during development and in the adult mouse (He et al. 1989). It has been suggested that these genes may be participants in specifying cellular phenotypes during development of the nervous system. Cell surface receptors containing tyrosine kinase (TK) catalytic domains have been extensively studied for their role in cell growth regulation and oncogenic transformation (Hunter and Cooper, 1985; Carpenter, 1987; Yarden and Ullrich, 1988). However, evidence for their involvement in embryonic development has only recently been obtained. The mouse locus W, which affects the proliferative or migratory properties of primordial germ cells, melanoblasts and hematopoietic stem cells, encodes a TK receptor (Silvers, 1979; Geissler et al. 1988; Chabot et al. 1988). In Drosophila, the genes sevenless and torso encode putative TK receptors. By mutation analysis, it is known that the

An important objective in the field of vertebrate developmental biology is the identification of genes whose products mediate the regulatory signals required for the intricate process of embryogenesis. Various approaches have been undertaken in efforts to identify such genes. For example, the search for mammalian homologues of invertebrate embryonic regulatory genes has led to the isolation of a number of genes that are related by sequence to the homeodomain-containing genes of Drosophila (McGinnis et al. 1984). The discovery of murine homeobox (//ar)-containing genes has provoked investigation into their potential role in embryogenesis, which gained considerable support once restricted embryonic patterns of Hox expression were observed. For example, distinct Hox genes are transcribed at specific rostro-caudal levels of the neural tube and spinal cord as development proceeds, suggesting a role for Hox genes in the spatial alignment of the nervous system (reviewed by Holland and Hogan, 1988; Gaunt et al. 1989; Wilkinson et al. 1989). Another approach toward searching for developmentally important regulatory genes stems from the existence of conserved sequence motifs among transcriptional factors (ie., zinc fingers, POU-domains, etc.). The presence of such sequences within a gene is widely accepted

Key words: tyrosine kinase, in situ hybridization, embryogenesis, proto-oncogene.

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sevenless TK receptor is required for the determination of the R7 photoreceptor cell in the retina during embryogenesis (Basler and Hafen, 1987; Banerjee etal. 1987; Hafen et al. 1987). Similarly, the torso TK receptor is a maternally encoded member of the terminal class of genes required for the determination of the terminal antero-posterior structures of the embryo (Sprenger et al. 1989). We report here the neural specific embryonic expression pattern of another gene that codes for a TK receptor, trkB. The trk family of proto-oncogenes consists of at least two loci in the mouse: trk and trkB (Klein et al. 1989, and MartinZanca et al. 1990). Trk was first identified as an oncogene following transfection of NIH-3T3 cells with DNA obtained from a human colon carcinoma (MartinZanca et al. 1986). Northern analysis using a human Trk cDNA probe to hybridize RNA from mouse tissues revealed a weakly hybridizing, complex pattern of bands in brain. Subsequent studies have shown that these brain transcripts originate from a locus distinct from that of the trk gene, which we have designated trkB (Klein et al. 1989). Organization of the predicted trkB transmembrane, extracellular and catalytic TK domains is colinear with that of the trk protein and is described in detail elsewhere (Klein et al. 1989). Furthermore, we have shown, in a preliminary study, that trkB transcripts can be found in the mouse embryo nervous system. In this study, we present a more detailed RNA in situ analysis for the trkB gene in the embryo and in the adult brain. Our data demonstrate exclusive expression for this gene in tissues of neuroepithelial and neural crest origin, which are destined to constitute the central and peripheral nervous systems (CNS and PNS) from the time of morphogenesis, through development, and into maturity.

Materials and methods

Mice and embryos Mouse embryos were derived from C57BL/6 NCRXC3H/ HeN MTV" F2 litters and were staged under the dissecting microscope by counting somites through midgestation. In older embryos, external markers such as state of limb development were used (Theiler, 1972). The morning of vaginal plug was considered day 0.5 (E0.5).

RNA isolation and Northern analysis Embryos were dissected under the microscope, frozen in liquid nitrogen and stored at —70°C. RNA was extracted using RNAzol™ (Cinna/Biotecx) following the manufacturer's recommendations. For Northern analysis, 20ng of total RNA were electrophoresed through 1.2% agarose gels containing 0.37 M formaldehyde as described (Maniatis et al. 1982), transferred to ZETABIND membranes (CUNO) and hybridized at 65°C according to Church and Gilbert (1984). Washes were carried out at 65°C in 0.5% BSA, 40 mM phosphate buffer pH7.2, 1% SDS.

In situ hybridization In situ hybridization protocols were as described in detail elsewhere (Klein et al. 1989; Martin-Zanca et al. 1990).

Construction of probes RNA (Krieg and Melton, 1987) and DNA probes were prepared from the 482 bp insert in pFRKl6 (see Fig. 1A). This insert encompasses sequences encoding a portion of the putative extracellular domain (nucleotides 1181-1663 in Klein et al. 1989). This region was chosen to avoid possible recognition of related TK sequences. Results

Our previous analysis had revealed the existence of at least six independent transcripts in adult brain whereas only a single abundant 2.5 kb transcript was detected in E14 and E18 mouse embryos (Klein et al. 1989). We wished to extend this study and to determine whether additional trkB transcripts were present in the embryo that had been below our sensitivity of detection in prior analyses (Klein et al. 1989). We therefore performed Northern analysis of total RNA isolated from mouse embryos of stages E6.5 to E17.5. A 0.5 kbp DNA fragment from the trkB cDNA that corresponds to a portion of the region encoding the putative ligandbinding extracellular domain was used for preparing DNA and RNA probes (Fig. 1A; see Materials and methods). As shown in Fig. IB, a 2.5 kb mRNA species is first observed in E8.5 embryos, albeit at extremely low levels. This 2.5 kb transcript and a smaller 2.0 kb species that appears at stage E10.5 are the most abundant transcripts throughout gestation, while additional transcripts that may exist below our level of detection early, are evident only at later stages of development. For example, at stages E12.5 to E13.5, two RNA bands of approximately 8.0 and 9.0 kb appear preferentially in RNA from heads of embryos, although trace amounts can be detected in RNA from trunks. By stage E15.5, the larger transcript is no longer visible in heads while the 8.0 kb transcript remains present at low levels in trunks. In E16.5 embryos, the complexity of trkB transcripts increases, approaching that observed in the adult (Klein et al. 1989). The existence of multiple size trkB encoded transcripts ranging from 2.0kb to 9.0 kb has important implications for the interpretation of the results discussed below. trk5 is differentially expressed in the early neural tissues To determine whether temporal appearance of trkB expression coincided spatially with particular developmental events, in situ hybridization analysis was performed on embryo sections corresponding to all stages tested by Northern analysis. trkB expression was first seen in E9.5 embryos. At this stage, the embryo body plan is well established, about 25 somites are formed, the rudiments of several organs are appearing and the CNS and PNS are in the process of differentiating into anatomically recognizable structures. These include the primary divisions of the brain into forebrain, midbrain and hindbrain, and the formation of the cranial and spinal ganglia. We detect trkB transcripts in forebrain, caudal midbrain, hindbrain, spinal cord and differentiating neural crest cells, which form dorsal root ganglia

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