The EMBO Journal vol.10 no.12 pp.3693-3702, 1991
Transient and locally restricted expression of the protooncogene during mouse development
Eva Sonnenberg1, Axel Godecke1, Barbara Walter, Friedheim Bladt and Carmen Birchmeier2 Max-Delbruck-Laboratorium, in der Max-Planck-Gesellschaft, Carl-von-Linne-Weg 10, 5000 Koin 30, Germany 'Authors contributed equally to this work 2Corresponding author Communicated by M.Birnstiel
The rosi gene was detected originally by virtue of its transforming potential; the cDNA of the human protooncogene was isolated from a tumor cell line expressing the gene ectopically. It encodes a receptor-type tyrosine specific protein kinase which is closely related to sevenless in Drosophila. Here we report the novel and remarkable in vivo expression pattern of c-rosl, which was determined in the mouse. By a combination of RNase protection and in situ hybridization, we find transient c-rosl expression during development in the kidney, intestine and lung, coinciding with major morphogenetic and differentiation events in these organs. This temporally restricted nature of expression is unusual for tyrosine kinase receptors and suggests a role for rosl during development. Furthermore, in kidney development c-rosl transcripts are confined to subgroups of ureter cells known to be involved directly in inductive interactions between ureter epithelium and metanephric mesenchyme. Thus, this study implicates for the first time a tyrosine kinase receptor in mesenchymal epithelial interactions and suggests a molecular basis for these important inductive events in development. Key words: kidney development/mesenchymal -epithelial interactions/receptor/tyrosine kinase
Introduction Receptor-type tyrosine kinases and their specific ligands are important components of signalling pathways, by which cells communicate with each other. Several genes which encode such receptors have been discovered through their ability to induce malignant transformation when mutated, among them rosi (Yamamoto et al., 1983; Downward et al., 1984; Fasano et al., 1984; Hampe et al., 1984; Neckameyer and Wang, 1985; Bargmann et al., 1986; Besmer et al., 1986; Birchmeier et al., 1986; Martin-Zanca et al., 1986). Previously, an extensive expression analysis carried out on a variety of human cell lines did not lead to the identification of particular cell types of tissues expressing c-rosl; only tumor cell lines expressing the gene ectopically were found (Birchmeier et al., 1987). The structural analysis of the intact (C Oxford University Press
ros 1
rosl cDNA from one of these cell lines indicates, that it encodes a cell surface receptor with an unusually large extracellular domain closely related to sevenless in Drosophila. The protein products of both, c-rosl and sevenless, are similar in size and overall structure, and extensive sequence homology exists in the cytoplasmic and the extracellular domains (Birchmeier et al., 1990). Therefore, the two proteins define a distinct subclass of transmembrane, tyrosine specific protein kinases. Many receptors with tyrosine kinase activity, as well as their ligands, function in growth control and the maintenance of normal homeostasis (for review, see Yarden and Ullrich, 1988). Recent evidence also demonstrates a role during vertebrate and invertebrate development. Three Drosophila genes coding for such receptors, sevenless, torso and the Drosophila homologue for the epidermal growth factor (EGF) receptor (DER), have a proven developmental function: sevenless is required for the determination of cell fate in eye development, torso for establishment of terminal structures of the early embryo and the DER gene, depending on the exact mutation, in early embryogenesis and eye development (Schejter and Shilo, 1989; Price et al., 1989; Sprenger et al., 1989; Basler et al., 1991). In the mouse, the W locus (encoding the c-kit receptor tyrosine kinase) and steel (encoding the c-kit specific ligand) are required for proliferation and migration of various stem cells (Russell, 1979; Anderson et al., 1990; Martin et al., 1990). A variety of developmental processes depend on signal transduction events between distinct cell types. In particular, morphogenesis and differentiation of most parenchymal organs are controlled by interactions between epithelial and mesenchymal cells (Kratochwil, 1983). In some instances, the epithelium triggers the differentiation of the mesenchyme, but in most tissues the mesenchyme initiates the differentiation of the epithelium. The factors involved in these processes are either ubiquitous or produced in a restricted and even tissue-specific manner (Saxen, 1987). One particularly well studied example, the differentiation of the kidney, depends largely on the exchange of information between epithelium and mesenchyme. The epithelial ureter branches in response to signals received from the surrounding metanephric mesenchyme, and the ureter in turn induces the conversion of nephrogenic mesenchyme to new epithelia (Saxen, 1987; Ekblom, 1991). The molecular nature has not been determined for either the exchanged signals or the receptors receiving the signals. As a step towards the understanding of the function of the ros] protooncogene, we have determined its expression pattern in mice during embyrogenesis and in adult life. Here we demonstrate that c-rosl is expressed transiently during kidney, intestine and lung development. The time course and the spatial pattern of expression suggest a role for rosl in the epithelial mesenchymal interactions known to control morphogenesis and differentiation of these organs. 3693
E.Sonnenberg
et
al.
mEx2, 310bp
H
N
H
S
mKi2,170 bp
HH
S
_~~
N
q
mrosEx
~~~mrosKi
_
1kb
a 1kb
*
L TM
S
I
PK
mc3-1
I
mc4
Fig. 1. Schematic representation of human and murine c-rosl DNA clones. The structure of the human c-rosl cDNA is shown translated sequences are indicated by a box; black boxes represent the coding sequences for the putative signal peptide (S), the schematically. The transmembrane domain (TM) and the tyrosine specific protein kinase domain (PK). The restriction map of the two genomnic murine c-rosl clones is shown above. Hatched boxes show the location of c-rosl exon sequences; the corresponding sequences in the cDNA are indicated. Two genomic used as probes in RNase protection experiments (mEx2 and mKi2) are shown above. Two murine cDNA fragments (mc4 and mc3-1) used fragments for in situ hybridization analysis are indicated below the human cDNA.
Results In order to obtain homologous probes for the analysis of c-rosl expression in the mouse, parts of the murine gene and cDNA were isolated and characterized (see Materials and methods). First, two X clones containing genomic c-rosl DNA were isolated (mrosEx and mrosKi, Figure 1). Exon sequences present in this DNA were used as probes for an initial expression analysis by RNase protection, which led to the identification of embyronal tissues transcribing c-rosl. Subsequently, a cDNA library was synthesized from poly(A)+ RNA isolated from embryonal intestine, which is the tissue with the highest levels of c-rosl specific transcripts. Murine c-rosl cDNA clones were isolated and sequenced, and two cDNA fragments (mc4 and mc3-1, indicated in Figure 1) were chosen as probes for further expression analysis by in situ hybridization. The entire tyrosine-specific protein kinase domain is encoded by the cDNA fragment mc3-1. The predicted amino acid sequences of this murine cDNA clone and human c-rosl as well as various other kinases were compared (Table I). The similarity between murine and human isolates in the kinase domain is 93 % indicating that these sequences are indeed derived from identical genes in mouse and man. In the kinase domain, the similarities between murine or human ros 1 and Drosophila sevenless are 72 % and 69 %, respectively. This is comparable to the similarities observed between the kinase domains of the human insulin and EGF receptor and their Drosophila analogues (Table I). The initial analysis of c-rosl expression by RNase protection, a technique by which less than one copy of mRNA per cell can be detected (Melton et al., 1984), was performed with 32P-labeled RNA probes transcribed from two different genomic subclones (mEx2 and mKi2, as indicated in Figure 1). Both probes revealed the same pattern of expression as summarized in Table II. In adult tissues, low levels of c-rosl specific transcripts were detected in lung, somewhat higher amounts in testis, but all other adult organs were negative. In contrast, we found c-rosl specific transcripts in kidney, intestine and lung from mice shortly after birth (Table II). 3694
Table I. Sequence comparison of tyrosine murine rosl and several other receptors
Hros sev
HIR DIR HER DER
Mros
Hros
sev
93 72 67 62 61 59
69 66 59 60 58
62 61 58 57
(89) (53) (49) (43) (37) (35)
(54) (48) (42) (37) (36)
(46) (44) (37) (37)
specific kinase domains of
HIR
DIR
HER
73 (63) 56 (34) 56 (28)
56 (34) 55 (30)
75 (56)
The predicted amino acid sequences of murine rosl (Mros), human rosl (Hros), sevenless from Drosophila (sev), human insulin receptor (HIR) as well as its Drosophila analogue (DIR), and human EGF receptor (HER) and its Drosophila analogue (DER) were compared by the Bestfit program (University of Wisconsin, Genetics Computer Group, UWGCG, Devereux et al., 1984). Only the sequence of the protein kinase domains as defined by Hanks et al. (1988) were used for the analysis. Indicated in the matrix is the degree of similarity between the sequences; the percentage identity is shown in brackets. T'he sequences were from the following sources: Hros (Birchmeier et al., 1990), sev (Basler and Hafen, 1988), HIR (Ebina et al., 1985; Ullrich et al., 1985), DIR (Nishida et al., 1986), HER (Ullrich et al., 1984), DER (Livneh et al., 1985).
To determine the temporal expression pattern of c-rosl in kidney, lung, and intestine during development, we dissected these organs from mice at various embryonic and postnatal stages. RNA prepared from the tissues was then tested for the presence of c-rosl transcripts by RNase protection using the RNA probe transcribed from clone mKi2 (Figure 2). In the kidney, we detected c-rosl specific transcripts already at the earliest stage tested, i.e. day 14 of embryonal development. Transcripts continue to be present throughout further embyrogenesis, are still detectable in the kidneys of newborn animals [3 days post natal (p.n.)], but are absent in kidneys of 21-day old and adult animals (Figure 2). In the intestine, c-rosl specific transcription commences on day 16 of development, reaches a high level on day 18 and declines from thereon. Small amounts of c-rosl message are still present 21 days postnatally, but none was found in the adult intestine (Figure 2). In the lung, c-rosl expression was generally low. Specific transcripts were first
:
Kidney
ros 1 protooncogene functions in development
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Transient and locally restricted expression of the protooncogene during mouse development
Eva Sonnenberg1, Axel Godecke1, Barbara Walter, Friedheim Bladt and Carmen Birchmeier2 Max-Delbruck-Laboratorium, in der Max-Planck-Gesellschaft, Carl-von-Linne-Weg 10, 5000 Koin 30, Germany 'Authors contributed equally to this work 2Corresponding author Communicated by M.Birnstiel
The rosi gene was detected originally by virtue of its transforming potential; the cDNA of the human protooncogene was isolated from a tumor cell line expressing the gene ectopically. It encodes a receptor-type tyrosine specific protein kinase which is closely related to sevenless in Drosophila. Here we report the novel and remarkable in vivo expression pattern of c-rosl, which was determined in the mouse. By a combination of RNase protection and in situ hybridization, we find transient c-rosl expression during development in the kidney, intestine and lung, coinciding with major morphogenetic and differentiation events in these organs. This temporally restricted nature of expression is unusual for tyrosine kinase receptors and suggests a role for rosl during development. Furthermore, in kidney development c-rosl transcripts are confined to subgroups of ureter cells known to be involved directly in inductive interactions between ureter epithelium and metanephric mesenchyme. Thus, this study implicates for the first time a tyrosine kinase receptor in mesenchymal epithelial interactions and suggests a molecular basis for these important inductive events in development. Key words: kidney development/mesenchymal -epithelial interactions/receptor/tyrosine kinase
Introduction Receptor-type tyrosine kinases and their specific ligands are important components of signalling pathways, by which cells communicate with each other. Several genes which encode such receptors have been discovered through their ability to induce malignant transformation when mutated, among them rosi (Yamamoto et al., 1983; Downward et al., 1984; Fasano et al., 1984; Hampe et al., 1984; Neckameyer and Wang, 1985; Bargmann et al., 1986; Besmer et al., 1986; Birchmeier et al., 1986; Martin-Zanca et al., 1986). Previously, an extensive expression analysis carried out on a variety of human cell lines did not lead to the identification of particular cell types of tissues expressing c-rosl; only tumor cell lines expressing the gene ectopically were found (Birchmeier et al., 1987). The structural analysis of the intact (C Oxford University Press
ros 1
rosl cDNA from one of these cell lines indicates, that it encodes a cell surface receptor with an unusually large extracellular domain closely related to sevenless in Drosophila. The protein products of both, c-rosl and sevenless, are similar in size and overall structure, and extensive sequence homology exists in the cytoplasmic and the extracellular domains (Birchmeier et al., 1990). Therefore, the two proteins define a distinct subclass of transmembrane, tyrosine specific protein kinases. Many receptors with tyrosine kinase activity, as well as their ligands, function in growth control and the maintenance of normal homeostasis (for review, see Yarden and Ullrich, 1988). Recent evidence also demonstrates a role during vertebrate and invertebrate development. Three Drosophila genes coding for such receptors, sevenless, torso and the Drosophila homologue for the epidermal growth factor (EGF) receptor (DER), have a proven developmental function: sevenless is required for the determination of cell fate in eye development, torso for establishment of terminal structures of the early embryo and the DER gene, depending on the exact mutation, in early embryogenesis and eye development (Schejter and Shilo, 1989; Price et al., 1989; Sprenger et al., 1989; Basler et al., 1991). In the mouse, the W locus (encoding the c-kit receptor tyrosine kinase) and steel (encoding the c-kit specific ligand) are required for proliferation and migration of various stem cells (Russell, 1979; Anderson et al., 1990; Martin et al., 1990). A variety of developmental processes depend on signal transduction events between distinct cell types. In particular, morphogenesis and differentiation of most parenchymal organs are controlled by interactions between epithelial and mesenchymal cells (Kratochwil, 1983). In some instances, the epithelium triggers the differentiation of the mesenchyme, but in most tissues the mesenchyme initiates the differentiation of the epithelium. The factors involved in these processes are either ubiquitous or produced in a restricted and even tissue-specific manner (Saxen, 1987). One particularly well studied example, the differentiation of the kidney, depends largely on the exchange of information between epithelium and mesenchyme. The epithelial ureter branches in response to signals received from the surrounding metanephric mesenchyme, and the ureter in turn induces the conversion of nephrogenic mesenchyme to new epithelia (Saxen, 1987; Ekblom, 1991). The molecular nature has not been determined for either the exchanged signals or the receptors receiving the signals. As a step towards the understanding of the function of the ros] protooncogene, we have determined its expression pattern in mice during embyrogenesis and in adult life. Here we demonstrate that c-rosl is expressed transiently during kidney, intestine and lung development. The time course and the spatial pattern of expression suggest a role for rosl in the epithelial mesenchymal interactions known to control morphogenesis and differentiation of these organs. 3693
E.Sonnenberg
et
al.
mEx2, 310bp
H
N
H
S
mKi2,170 bp
HH
S
_~~
N
q
mrosEx
~~~mrosKi
_
1kb
a 1kb
*
L TM
S
I
PK
mc3-1
I
mc4
Fig. 1. Schematic representation of human and murine c-rosl DNA clones. The structure of the human c-rosl cDNA is shown translated sequences are indicated by a box; black boxes represent the coding sequences for the putative signal peptide (S), the schematically. The transmembrane domain (TM) and the tyrosine specific protein kinase domain (PK). The restriction map of the two genomnic murine c-rosl clones is shown above. Hatched boxes show the location of c-rosl exon sequences; the corresponding sequences in the cDNA are indicated. Two genomic used as probes in RNase protection experiments (mEx2 and mKi2) are shown above. Two murine cDNA fragments (mc4 and mc3-1) used fragments for in situ hybridization analysis are indicated below the human cDNA.
Results In order to obtain homologous probes for the analysis of c-rosl expression in the mouse, parts of the murine gene and cDNA were isolated and characterized (see Materials and methods). First, two X clones containing genomic c-rosl DNA were isolated (mrosEx and mrosKi, Figure 1). Exon sequences present in this DNA were used as probes for an initial expression analysis by RNase protection, which led to the identification of embyronal tissues transcribing c-rosl. Subsequently, a cDNA library was synthesized from poly(A)+ RNA isolated from embryonal intestine, which is the tissue with the highest levels of c-rosl specific transcripts. Murine c-rosl cDNA clones were isolated and sequenced, and two cDNA fragments (mc4 and mc3-1, indicated in Figure 1) were chosen as probes for further expression analysis by in situ hybridization. The entire tyrosine-specific protein kinase domain is encoded by the cDNA fragment mc3-1. The predicted amino acid sequences of this murine cDNA clone and human c-rosl as well as various other kinases were compared (Table I). The similarity between murine and human isolates in the kinase domain is 93 % indicating that these sequences are indeed derived from identical genes in mouse and man. In the kinase domain, the similarities between murine or human ros 1 and Drosophila sevenless are 72 % and 69 %, respectively. This is comparable to the similarities observed between the kinase domains of the human insulin and EGF receptor and their Drosophila analogues (Table I). The initial analysis of c-rosl expression by RNase protection, a technique by which less than one copy of mRNA per cell can be detected (Melton et al., 1984), was performed with 32P-labeled RNA probes transcribed from two different genomic subclones (mEx2 and mKi2, as indicated in Figure 1). Both probes revealed the same pattern of expression as summarized in Table II. In adult tissues, low levels of c-rosl specific transcripts were detected in lung, somewhat higher amounts in testis, but all other adult organs were negative. In contrast, we found c-rosl specific transcripts in kidney, intestine and lung from mice shortly after birth (Table II). 3694
Table I. Sequence comparison of tyrosine murine rosl and several other receptors
Hros sev
HIR DIR HER DER
Mros
Hros
sev
93 72 67 62 61 59
69 66 59 60 58
62 61 58 57
(89) (53) (49) (43) (37) (35)
(54) (48) (42) (37) (36)
(46) (44) (37) (37)
specific kinase domains of
HIR
DIR
HER
73 (63) 56 (34) 56 (28)
56 (34) 55 (30)
75 (56)
The predicted amino acid sequences of murine rosl (Mros), human rosl (Hros), sevenless from Drosophila (sev), human insulin receptor (HIR) as well as its Drosophila analogue (DIR), and human EGF receptor (HER) and its Drosophila analogue (DER) were compared by the Bestfit program (University of Wisconsin, Genetics Computer Group, UWGCG, Devereux et al., 1984). Only the sequence of the protein kinase domains as defined by Hanks et al. (1988) were used for the analysis. Indicated in the matrix is the degree of similarity between the sequences; the percentage identity is shown in brackets. T'he sequences were from the following sources: Hros (Birchmeier et al., 1990), sev (Basler and Hafen, 1988), HIR (Ebina et al., 1985; Ullrich et al., 1985), DIR (Nishida et al., 1986), HER (Ullrich et al., 1984), DER (Livneh et al., 1985).
To determine the temporal expression pattern of c-rosl in kidney, lung, and intestine during development, we dissected these organs from mice at various embryonic and postnatal stages. RNA prepared from the tissues was then tested for the presence of c-rosl transcripts by RNase protection using the RNA probe transcribed from clone mKi2 (Figure 2). In the kidney, we detected c-rosl specific transcripts already at the earliest stage tested, i.e. day 14 of embryonal development. Transcripts continue to be present throughout further embyrogenesis, are still detectable in the kidneys of newborn animals [3 days post natal (p.n.)], but are absent in kidneys of 21-day old and adult animals (Figure 2). In the intestine, c-rosl specific transcription commences on day 16 of development, reaches a high level on day 18 and declines from thereon. Small amounts of c-rosl message are still present 21 days postnatally, but none was found in the adult intestine (Figure 2). In the lung, c-rosl expression was generally low. Specific transcripts were first
:
Kidney
ros 1 protooncogene functions in development
.ung
I ntest ne
u
r-:
d 4-
9-
9-1
_ 13 :3
Z--
a
cos
n
D0 -
-
{.N D
