Drosophila Notch and the related Caenorhabditis elegans proteins lin-12 and glp-1 function as mediators of local cell-cell interactions required for cell-fate decisions during invertebrate development. To investigate the possibility that similar proteins play determinative roles during mammalian development, we isolated cDNA clones encoding rat Notch. The deduced amino acid sequence of this protein contains 36 epidermal growth factor (EGF)-like repeats, and is remarkably similar in both its extracellular and cytoplasmic domains to the sequence of Xenopus Xotch and Drosophila Notch. In the developing central nervous system, in situ hybridisation analyses revealed that Notch transcripts were dramatically restricted to the ventricular proliferative zones of embryonic neuroepithelia. Notch was also strongly expressed during development of non-neural tissues, such as hair follicles and tooth buds, whose correct differentiation requires epithelial-mesenchymal interactions. These data support the hypothesis that Notch plays an essential role in mammalian development and pattern formation that closely parallels its role in the development of invertebrates.

Cell-cell interactions often underly decisions that direct cells toward specific differentiation pathways during development. Drosophila Notch and the C. elegans proteins lin-12 and glp-1 comprise a family of molecules that direct local cell-cell interactions required for celltype decisions during invertebrate development (Artavanis-Tsakonas, 1988; Campos-Ortega, 1988; Yochem et al. 1988; Yochem and Greenwald, 1989; Austin and Kimble, 1989). The structural features of these cell-fate determining proteins support the idea that they mediate interactions between neighbouring cells (Artavanis-Tsakonas, 1988; Campos-Ortega, 1988; Yochem and Greenwald, 1989; Greenspan, 1990). Although Notch was first described as a gene involved in determining cell-fate choices in the neurogenic region of the developing Drosophila embryo (Poulson, 1937), subsequent studies have demonstrated that this gene is expressed in a variety of cell types in Drosophila (Hartley et al. 1987; Markopoulou and Artavanis-Tsakonas, 1989; Johansen et al. 1989; Kidd et al. 1989; Fehon et al. 1991). Consistent with this expression profile, Notch is required for the determination of both neural and non-neural cells (Hoppe and Greenspan, 1986; Cagan and Ready, 1989; Hoppe and Greenspan, 1990; Hartenstein and Posakony, 1990).

The recent cloning of the Xenopus Notch-homo\og Xotch (Coffman et al. 1990) suggests that molecules analogous to Notch also function during vertebrate development. Both the structure and the expression pattern of Xotch (Coffman et al. 1990) are similar to those reported for Drosophila Notch, supporting the hypothesis that Notch functions in equivalent processes during invertebrate and vertebrate development. Since we are interested in identifying proteins that direct differentiation signals between dissimilar mammalian cell types, we asked whether a Notch gene was expressed during mammalian development. We report here the isolation of cDNA clones encoding rat Notch, the deduced structure of this protein, and an in situ hybridisation survey of Notch gene expression during mouse embryogenesis.

Cloning and sequencing

A rat Schwann cell cDNA, library (Monuki et aL 1,989) was probed with a 32P-radiolabilled ECOKI fragment from the EGF-like-repeat region of the Xotch DNA X-2 (Coffman et al. 1990). The screen was conducted under low stringency conditions (IM NaCl, 50mM Tris-HCL (pH7.5), 1% SDS, 5x Denhart’s solution and 0.2 mg ml’” salmon sperm DNA at 50°C) as described by Boulter and Gardner (1990). Hybond-N membrane filters (Amersham) were sequentially washed in 5xSSC and 0.5 % SDS at 21°C followed by 50°C. Using these conditions both Notch and Afofc/i-related cDNA clones were isolated. Clones were sequenced using the dideoxy chain termination method (Sequenase, US Biochemicals).

Northern analysis

RNA was isolated, processed and hybridised for northern analysis as described previously (Monuki et al. 1989; Weinmaster and Lemke, 1990). Methylene blue staining was used to verify that equal amounts of RNA were present on membranes following transfer. Random-primed 32 P radiolabelled probes were prepared from either a 4.5 kb Xhol fragment of cDNA clone SN6 encoding rat Notch or a genomic fragment encoding histone H2A (Heintz et al. 1983), according to the manufacturers instractions (BRL).

In situ hybridisation

Mouse embryo sections were prepared and hybridised as described previously (Bettier et al. 1990). The specificity of the rat Notch, probes was verified by determining that 35S-radrolaoelled riboprooes synthesised from DNA antisense inserts encoding amino acid residues 82-213 in the EGF-like repeat domain (see Fig. 1) subcloned into Bluescript SK+ (SN6-15), or amino acid residues 1755-1903 in the intracellular portion of rat Notch subcloned into Bluescript SK+ (SN6-7) gave identical hybridisation patterns on both mouse and rat tissues under high stringency conditions. Since more information is available on the anatomical development of the mouse embryo (Rugh, 1968), we performed a detailed in situ hybridisation analysis using mouse tissues. The hybridised sections were exposed to Hyperfilm Betamax (Amersham) for 48 h. For higher resolution analysis, slides were dipped in Kodak NTB-2 nuclear emulsion, developed in D19 (Kodak) after 2 weeks exposure and stained with Giemsa. Identification of the labelled tissues and organs was aided by reference to Rugh (1968).

Fig. 1.

Alignment of the deduced amino acid sequence for rat, Xenopus and Drosophila Notch. The amino acid sequence of rat Notch (3) (R) was deduced from the nucleotide sequence of cDNA clones and aligned with the published sequences of Xotch (Coffman et al. 1990) (X) and Drosophila Notch (Wharton et al. 1985a; Kidd et al. 1986) (D) using the Intelligenetics Genalign sequence analysis program. Identical amino acids are highlighted. Characteristic structural motifs following the signal peptide are boxed and indicated as: (1) EGF-repeats for the 36 cysteine-rich repeats related to epidermal growth factor; (2) LNR for the 3 repeats related to lin-Yl and Notch sequences; (3) TM for the hydrophobic transmembrane domain sequences; (4) cdclO/SWI6 for the 6 repeats related to the genes indicated in the text; (5) OPA for sequences related to the M-repeats of Drosophila (Wharton et al. 1985b), and (6) C-TERM for the carboxy-terminal region. The University of Wisconsin GAP program was used to determine the following amino acid identity scores for the different structural motifs, as well as the entire protein sequences, between R and X, R and D, and X and D respectively: (1) EGF-repeats; 76%, 51 % and 51%, (2) LNR; 81%, 50% and 50%, (3) cdclO/SW16; 91%, 70% and 70%. (4) OPA; 52%, 36% and 42%, (5) C-TERM; 91 %, 54%, and 54%, and (6) the entire sequence; 74%, 47% and 47%. Asterisks identify the conserved cysteines discussed in the text. The single-letter abbreviations for the amino acids are A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, He: K, Lys; L. Leu; M, Met: N, Asn: P, Pro; Q, Gin; R. Arg; S, Ser: T. Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 1.

Alignment of the deduced amino acid sequence for rat, Xenopus and Drosophila Notch. The amino acid sequence of rat Notch (3) (R) was deduced from the nucleotide sequence of cDNA clones and aligned with the published sequences of Xotch (Coffman et al. 1990) (X) and Drosophila Notch (Wharton et al. 1985a; Kidd et al. 1986) (D) using the Intelligenetics Genalign sequence analysis program. Identical amino acids are highlighted. Characteristic structural motifs following the signal peptide are boxed and indicated as: (1) EGF-repeats for the 36 cysteine-rich repeats related to epidermal growth factor; (2) LNR for the 3 repeats related to lin-Yl and Notch sequences; (3) TM for the hydrophobic transmembrane domain sequences; (4) cdclO/SWI6 for the 6 repeats related to the genes indicated in the text; (5) OPA for sequences related to the M-repeats of Drosophila (Wharton et al. 1985b), and (6) C-TERM for the carboxy-terminal region. The University of Wisconsin GAP program was used to determine the following amino acid identity scores for the different structural motifs, as well as the entire protein sequences, between R and X, R and D, and X and D respectively: (1) EGF-repeats; 76%, 51 % and 51%, (2) LNR; 81%, 50% and 50%, (3) cdclO/SW16; 91%, 70% and 70%. (4) OPA; 52%, 36% and 42%, (5) C-TERM; 91 %, 54%, and 54%, and (6) the entire sequence; 74%, 47% and 47%. Asterisks identify the conserved cysteines discussed in the text. The single-letter abbreviations for the amino acids are A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, He: K, Lys; L. Leu; M, Met: N, Asn: P, Pro; Q, Gin; R. Arg; S, Ser: T. Thr; V, Val; W, Trp; and Y, Tyr.

Cloning the rat Notch gene

We used a Xotch cDNA probe (Coffman et al. 1990) to screen a rat cDNA library under low stringency conditions (see Materials and Methods). One cDNA clone identified in this screen (SN6) contains a 7.6 kb cDNA insert with an open reading frame that encoded the entire Notch amino acid sequence except for the first two EGF-like repeats and the signal peptide sequence. The missing N-terminal sequence was found in a second Notch cDNA clone (SN32), which was isolated by rescreening,the raXJSchwann .cell cDNA library with a 32P-radiolabelled SN65-terminal restriction fragment under high stringency conditions. Clone SN32 contains a 3.1 kb insert that overlaps the cDNA insert of SN6 by 2.6 kb and extends approximately 0.5 kb further in the 5’-direction. The DNA sequence of the overlapping cDNA clones (8221 nucleotides) carries a large open reading frame of 2531 amino acids (Fig. 1), starting with an initiator methionine at nucleotide 241 and terminating with a stop codon at nucleotide 7830. The nucleotide and deduced amino acid sequences of rat Notch have been deposited with GenBank under the accession number X57405.

Notch structural domains are highly conserved

The deduced amino acid sequence of rat Notch carries several structural motifs that have been strongly conserved throughout evolution (see Fig. 1 for amino acid identity scores). One of these, located in the intracellular domain of the protein, consists of six cdclO/SWI6 repeats, first identified in the yeast cell cycle genes cdclO, SWI4 and SWI6 (Breeden and Nasmyth, 1987; Andrews and Herskowitz, 1989). These repeats are also present in the cell-fate determining proteins /m-12 and glp-l (Yochem et al. 1988; Yochem and Greenwald, 1989; Austin and Kimble, 1989), the human proto-oncogene bcl-3 (Ohno et al. 1990), the C. elegans sex-determining gene fem-l (Spence et al. 1990), and human erythrocyte ankyrin (Lux et al. 1990). The carboxy-terminal 77 amino acids of rat Notch constitute a second highly conserved intracellular domain. This region of the protein contains several sets of serine and threonine residues surrounded by prolines, which are potential sites for protein phosphorylation by either the proline-directed serine/threonine protein kinase (Vulliet et al. 1989) or glycogen synthase kinase-3 (Woodgett, 1990). In this regard, phosphoserine has been detected in the cytoplasmic portion of the Drosophila Notch protein (Kidd et al. 1989). The carboxy-terminal region of rat Notch also contains a PEST sequence implicated in the control of protein degradation (Rogers et al. 1986).

The hallmark of the extracellular region of Drosophila Notch and related proteins is the presence of multiple, cysteine-rich EGF-like repeats (Wharton et al. 1985a; Kidd et al. 1986; Yochem et al. 1988; Davis, 1990). The extracellular region of rat Notch contains 36 of these repeats, which are arrayed in exactly the same order as the 36 repeats of Notch (Wharton et al. 1985a; Kidd et al. 1986) and Xotch (Coffman et al. 1990). This strong conservation in number and order suggests that these repeats serve distinct functions. Consistent with this idea is the observation that single amino acid changes in different EGF-like repeats of Drosophila Notch produce different mutant phenotypes (Hartley et al. 1987; Kelley et al. 1987; Markapoulou et al. 1989; Xu et al. 1990). The high degree of conservation of these sequences further supports previous genetic (Hartley et al. 1987; Kelleyet al. 1987; Markapoulou et al. 1989; Xu et al. 1990) and biochemical data (Fehon et al. 1990) that implicate them in ligand binding. Also highly conserved in number and relative position is a second set of 3 cysteine-rich domains, designated the lin-12/Notch repeats (LNR) (Yochem et al. 1988; Yochem and Greenwald, 1989; Austin and Kimble, 1989) (see Fig. 1). Two additional cysteine residues, located between the LNR and the transmembrane domain (see Fig. 1) and proposed to promote dimerization following ligand binding (Greenwald and Seydoux, 1990), are conserved in all the Notch proteins and in lin-Yl and glp-l.

Notch expression during mammalian development

The requirement of Notch for cell-fate decisions in Drosophila led us to investigate the expression of Notch during mammalian development, first by northern blot analysis in the developing rat brain and then by in situ hybridisation during mouse embryogenesis. Northern analysis of RNA isolated from embryonic, postnatal and adult rat whole brains indicated that high Notch expression occurs between embryonic day 12 and 14, and decreases rapidly thereafter to much lower levels in the adult (Fig. 2A). This pattern of expression is consistent with a role for Notch in proliferation and/or differentiation of developing neuroepithelia (Rakic, 1988; McConnell, 1988; Price and Thurlow, 1988). We observed a nearly identical expression pattern when the same RNA samples were hybridised with a probe to histone 2A (H2A), a gene whose transcription is tightly coupled to DNA synthesis (Heintz et al. 1983) (Fig. 2B). This observation is reminiscent of the close association of Notch expression with mitotically active cells in Drosophila (Markopoulou and Artavanis-Tsakonas, 1989; Kidd et al. 1989; Fehon et al. 1991).

Fig. 2.

Northern blot hybridisation analyses of Notch and Histone 2A mRNA isolated from embryonic, postnatal and adult rat brains. RNA blots were prepared using 2jig of poly(A)+ RNA isolated from the brains of rat embryos (E12-E19), postnatal (P1-P34) and adult (2 and 7 months) rats, and hybridised with a rat Notch probe (A) and a human H2A probe (B). A Notch transcript of ∼10kb in A and the locations of the 28S and 18S RNAs are indicated.

Fig. 2.

Northern blot hybridisation analyses of Notch and Histone 2A mRNA isolated from embryonic, postnatal and adult rat brains. RNA blots were prepared using 2jig of poly(A)+ RNA isolated from the brains of rat embryos (E12-E19), postnatal (P1-P34) and adult (2 and 7 months) rats, and hybridised with a rat Notch probe (A) and a human H2A probe (B). A Notch transcript of ∼10kb in A and the locations of the 28S and 18S RNAs are indicated.

Notch is required for the correct differentiation of both neural and non-neural tissues in Drosophila (Hoppe and Greenspan, 1986; Cagan and Ready, 1989; Hoppe and Greenspan, 1990; Hartenstein and Posakony, 1990). In order to determine whether Notch might play a role in mammalian development, we used in situ hybridisation to survey the expression of Notch mRNA during mouse embryogenesis beginning at embryonic day 9 and continuing through to postnatal day 5 (Fig. 3). Notch transcripts were initially detected during early neurogenesis, in the neural tube of E9 embryos (Fig. 3A). Very high expression was detected in the developing brain, spinal cord and dorsal root ganglia of E10 mouse embryos (Fig. 3B) and in the brain (Fig. 3C,D,E), eye, and dorsal root and trigeminal ganglia of E12 embryos (Fig. 3C). As in Drosophila, this expression pattern suggests a function for Notch in the development of both the central and peripheral nervous systems (Shellenbarger and Mohler, 1978; Cagan and Ready, 1989; Johansen et al. 1989; Kidd et al. 1989; Hartenstein and Posakony, 1990; Fehon et al. 1991). The strong expression in the developing peripheral nervous system is consistent with our detection of Notch transcripts in neonatal rat sciatic nerves and cultured Schwann cells (data not shown). Within the developing central nervous system, intense hybridisation signals were dramatically restricted to the periventricular zone where the cells are rapidly proliferating (Rakic, 1988; McConnell, 1988; Price and Thurlow 1988) (Fig. 3D,E). Neuroepithelial cells external to the proliferative zone were mostly negative, although punctate hybridisation signals, in what may correspond to secondary zones of proliferation (Rakic, 1988), were also observed. In E16 (Fig. 3F,K) and E18 embryos (data not shown), Notch exhibited a complex pattern of expression; however, the highest transcript levels were detected in organs that contain rapidly dividing and differentiating cells such as the brain (Fig. 3D,E), eye (Fig. 31,J), ear (Fig. 3G,H) and thymus (Fig. 3K). In the eye, strong hybridisation signals were again strictly confined to the proliferative cells of the retina and the lens (Fig. 31, J), similar to Xotch expression in the retinal marginal zone (Coffman et al. 1990). It is important to note that although many different cell types are dividing during these periods of embryogenesis, Notch is not ubiquitously expressed. For example, signals are virtually absent from osteogenic and cartilagenous tissues (Fig. 3M,H), and the choroid plexus (data not shown), indicating that high Notch expression is confined to a subset of mitotically active cells. Perhaps Notch expression is required in cells that are still dividing while in the process of initiating cell-fate choices. In the brain and thymus of the adult, Notch expression is substantially reduced in fully differentiated cells (G.W. and J. Lesley, unpublished data). Finally, a strong Notch signal was detected in tissues that are undergoing epithelial-mesenchymal interactions required for their correct cellular differentiation (Cutler and Chaudhry, 1973; Slavkin and Bringas, 1976; Saxen et al. 1986; Snead et al. 1988; Kopan and Fuchs, 1989; Lyons et al. 1990; Pelton et al. 1990). These include whisker follicles, tooth buds, salivary glands and kidney (Fig. 3F,K), and notably the matrix cells of developing hair follicles (Fig. 3N,O). Detection of Notch transcripts in these tissues, which depend upon reciprocal instructive signals for morphogenesis, is in keeping with the importance of Notch in cell-fate decisions indicated by Drosophila mutant analysis.

Fig. 3.

Localisation of Notch gene transcripts during, mouse embryogenesis. All-in situ hybridisations were performed, with [3%]UTP-labelled antisense rar Notch riboprooes. Photomicrographs of autoradiograms are presented for sagittal sections through E9 (A), E10 (B), E12 (C) and E16 (F,K) mouse embryos. Tissues and organs with strong hybridization signals (dark areas) are labelled. Photoemulsion-dipped sections at higher magnification show morphology (bright-field) and signal (white grains in dark-field) and are presented for the following organs and tissues: E12 brain bright-field (D) and dark-field (E) showing signal in the proliferative zone lining the 4th ventricle; E16ear bright-field (G) and dark-field (H) showing signal in the trigeminal ganglion and sensory hair cells in the cochlea of the ear; E16 eye bright-field (I) and dark-field (J) showing signal in the proliferating cells of the retinal layer and the lens; E16 spinal cord bright-field (L) and dark-field (M) showing signal in the dorsal root ganglia; and P5 hair follicles bright-field (N) and dark-field (O) showing signal in the matrix cells of the hair bulb. Abbreviations: ca, cartilage; co, cochlea; drg, dorsal root ganglia; e, ear; ey, eye; hf, hair follicles; hm, hair matrix cells; k, kidney; le, lens; lu, lung; mes, mesencephalon; met, metencephalon; nt, neural tube; pi, proliferating cells of the lens; prl, proliferating cells of the retinal layer; pz, proliferative zone of the brain; rh, rhombencephalon; sc, spinal cord; sh, sensory hair cells; sg, submandibular gland; t, tongue; tb, tooth buds; tel, telencephalon; tg, trigeminal ganglion; th, thymus; v, ventricle; vt, vertebrae; u, uterus; wf, whisker follicles. Boxed areas in C, F and K are shown at higher magnification in D-E, G-H, and L-M respectively. The bars in A, B, C, F, and K represent 1.5mm; the bars in E, H, J, M, and O represent 250mm.

Fig. 3.

Localisation of Notch gene transcripts during, mouse embryogenesis. All-in situ hybridisations were performed, with [3%]UTP-labelled antisense rar Notch riboprooes. Photomicrographs of autoradiograms are presented for sagittal sections through E9 (A), E10 (B), E12 (C) and E16 (F,K) mouse embryos. Tissues and organs with strong hybridization signals (dark areas) are labelled. Photoemulsion-dipped sections at higher magnification show morphology (bright-field) and signal (white grains in dark-field) and are presented for the following organs and tissues: E12 brain bright-field (D) and dark-field (E) showing signal in the proliferative zone lining the 4th ventricle; E16ear bright-field (G) and dark-field (H) showing signal in the trigeminal ganglion and sensory hair cells in the cochlea of the ear; E16 eye bright-field (I) and dark-field (J) showing signal in the proliferating cells of the retinal layer and the lens; E16 spinal cord bright-field (L) and dark-field (M) showing signal in the dorsal root ganglia; and P5 hair follicles bright-field (N) and dark-field (O) showing signal in the matrix cells of the hair bulb. Abbreviations: ca, cartilage; co, cochlea; drg, dorsal root ganglia; e, ear; ey, eye; hf, hair follicles; hm, hair matrix cells; k, kidney; le, lens; lu, lung; mes, mesencephalon; met, metencephalon; nt, neural tube; pi, proliferating cells of the lens; prl, proliferating cells of the retinal layer; pz, proliferative zone of the brain; rh, rhombencephalon; sc, spinal cord; sh, sensory hair cells; sg, submandibular gland; t, tongue; tb, tooth buds; tel, telencephalon; tg, trigeminal ganglion; th, thymus; v, ventricle; vt, vertebrae; u, uterus; wf, whisker follicles. Boxed areas in C, F and K are shown at higher magnification in D-E, G-H, and L-M respectively. The bars in A, B, C, F, and K represent 1.5mm; the bars in E, H, J, M, and O represent 250mm.

Overall, the structure of mammalian Notch is strikingly similar to that reported for Drosophila and Xenopus. This strong conservation, together with the marked similarity in expression in the developing tissues of flies, frogs and rodents, most probably reflects an equivalent role for Notch in cell-cell interactions that mediate both vertebrate and invertebrate development.

We thank Jim Boulter for advice on low stringency cloning and characterization of cDNA clones, Clark Coffman, Chris Kintner and Anna Neuman for providing Xotch DNA and sequence information prior to publication, Irm Hermans-Borgmeyer for tissue sections, advice and help with in situ hybridisation, Cary Lai for sharing Northern blots, Steve Hanks for the H2A DNA, Lisa Caballero and Anne O’Shea-Greenfield for help with computer analysis, Jim Boulter, Bill Harris, Tony Hunter, Chris Kintner, Jim Posakony and John Thomas for helpful discussions and comments, and Sara Barth, Chris McGaugh and Danny Ortuno for technical assistance. Supported by grants from the NIH and the National Multiple Sclerosis Society (G.L.) and the National Neurofibromatosis Foundation, Inc. (G.W.).

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