The Drosophila slit protein is a secreted ligand produced in a number of tissues, including midline glial cells and body wall muscle attachment sites (Rothberg et al., 1988; Rothberg et al., 1990). At the midline it is a repellant signal critical for regulating the location and crossing over of neuronal axons, which express receptors for slit, the robo proteins (Kidd et al., 1999; Rajagopalan et al., 2000; Simpson et al., 2000). The secretion of slit by midline glial cells is also necessary for the normal migration of muscle precursor cells away from the midline (Kidd et al., 1999). Three slit gene homologues (-1, -2, and -3) have been isolated in the human (Holmes et al., 1998; Itoh et al., 1998), mouse (Holmes et al., 1998; Yuan et al., 1999), and rat (Brose et al., 1999; Nakayama et al., 1998). slit-2 and slit-3 have been isolated in the zebrafish (Yeo et al., 2001), and slit-2 in Xenopus and chicken (Li et al., 1999). Each is widely expressed, including in the central nervous system (CNS), where their functions have begun to be elucidated. slit-2 is a repulsive signal received by robo receptors for the guidance of the axons of spinal motor (Brose et al., 1999), olfactory bulb (Li et al., 1999; Nguyen Ba-Charvet et al., 1999), hippocampal (Nguyen Ba-Charvet et al., 1999), and retinal ganglion cell neurons (Erskine et al., 2000; Niclou et al., 2000; Ringstedt et al., 2000), and for the migration of neuronal cells themselves (Hu, 1999; Wu et al., 1999; Zhu et al., 1999).
The slit proteins are modular, containing four types of protein-protein interaction domains (Rothberg et al., 1988; Rothberg et al., 1990), and may interact with a wide variety of as-yet unidentified proteins. Netrin, laminin (Brose et al., 1999), and glypican-1 (Liang et al., 1999) for example, bind slit protein, but the functional significance of these interactions has not been determined. Potentially, slit proteins may interact with other proteins to affect cell movement and adhesion, growth factor activity, or to modulate slit activities themselves. As the chick provides a versatile system for in vivo studies of a variety of tissues, we have sought to isolate chick slit genes and characterize their expression as the basis for further functional studies, with particular emphasis on their expression during limb development.
RESULTS AND DISCUSSION
Isolation of Chicken slit-2 and slit-3
A previously identified fragment of chick slit-2 (kindly provided by Y. Rao (Li et al., 1999)) was used to screen a Hamburger and Hamilton (1951) Stage (St) 12–15 chick embryo library (generously provided by D. Wilkinson) at low stringency. Clones homologous to human slit-2 and slit-3 genes were obtained. The largest slit-2 clone (pcslit-2(26a)) was approximately 5,000 bp long and encompassed the entire coding region. A section of approximately 1,250 bp, within the leucine-rich repeat (LRR) 1-2 region (human Gly125-Asn542) could not be sequenced (GenBank Accession numbers are AF387319 for the 5′ region and AF387320 for the 3′ region). The nucleotide and amino acid homologies between the sequenced coding regions of chick slit-2 and human slit-2 were approximately 81% and 92% identity, respectively. The largest slit-3 clone obtained (pcslit-3(3)) was 3,542 bp long and extended from Gln430 of human slit-3 to beyond the C-terminus (GenBank Accession number AF387318). The nucleotide and amino acid homologies to human slit-3 within the known coding region were 75% and 81% identity, respectively. A second, shorter clone of 2,576 bp (pcslit-3(2)) extended from human Pro751 to beyond the C-terminus. These chick slit genes have the same predicted modular protein structure as their vertebrate counterparts, in particular having the nine EGF repeats common to all vertebrate slit homologues so far identified, compared to seven in Drosophila and C. elegans slit proteins. For whole-mount in situ hybridization (WISH), riboprobes were transcribed from the shorter slit-2 probe (Li et al., 1999) and from pcslit-3(2) (fragmented to approximately 300 bp). For section in situ hybridization (ISH) the full-length riboprobes transcribed from pcslit-2(26a) and pcslit-3(2) were used. For ISH, the pcslit-2(26a) riboprobe and shorter slit-2 riboprobe revealed identical expression patterns when tested, but the former gave a stronger signal, and was used subsequently.
General Expression of slit-2 and slit-3 in the Developing Chick Embryo
A brief description of slit-2 expression in the early chick embryo (St4–21) has previously been published (Li et al., 1999). For slit-2 and slit-3 we have focused on St19-32, with particular attention given to sites of expression within the developing limb. slit gene expression patterns were determined by WISH and ISH.
The high level of sequence homology between the chick slit genes and their mammalian counterparts is paralleled to a reasonable degree by the conservation of their respective tissue distributions, but qualitative differences in expression between chicks and other vertebrates do exist. A summary of their respective expression patterns in major tissues of the developing chick (this paper, and data not shown), in comparison with their expression reported for the mouse (Holmes et al., 1998; Yuan et al., 1999), is presented in Table 1.
Table 1. Comparison of slit Expression Patterns Between Mouse and Chick Embryos
Expression is indicated as present (+) or absent (−) during the development of the tissue; “Low” indicates expression levels compared to slit-2 in the same tissues; “Limited” indicates a discontinuous expression pattern within a structure.
In the neural tube slit-2 expression is present between St19–27 in the dorsal part of the floor plate, the roof plate, motor neurons and in the notochord (Fig. 1A; St22, and data not shown). Definitive expression in the dorsal root ganglia is seen from St26, and by St27 expression fades rostrally in the notochord (data not shown). Between St19-27 slit-3 expression is present in the notochord and motor neurons (Fig. 1F; St22, and data not shown). Very low levels of expression are seen in the roof plate and floor plate by St27 (data not shown).
The slit-2 expression seen in the midline of the neural tube (Fig. 1A) is continuous along the dorsal and ventral midlines of the brain (Fig. 1B; D), but in the ventral midline it is restricted to the dorsal part of the floor plate (Fig. 1C). By St22 ventral midline expression of slit-2 has broadened in the anterior brain to include the medial walls of the telencephalic vesicles and the floor of the diencephalon, and expression in the epithelia of the optic nerve, especially ventrally, continues along its length into the diencephalon (not shown). At St22 slit-3 is expressed selectively in the midline of the brain. Very low expression is seen in the anterior-most dorsal midline (Fig. 1G) fading out near the floor of the median telocoele, but by St25 expression expands to include the medial telencephalic walls (not shown). At St22 intense slit-3 expression is seen in restricted domains flanking the midline slit-2 expression in the ventral cephalic fold (Fig. 1G). A short region of the floor plate in the caudal hindbrain expresses slit-3 in its ventral most part (Fig. 1H), and is complementary to the more dorsal slit-2 floor plate expression through this region (Fig. 1C).
In the eye slit-2 is expressed in the anterior epithelium of the lens and in the retina (Fig. 1D). At St25 expression extends into the lens body, and is present in the corneal epithelium (not shown). Expression in the anterior retina occurs in those cells of the sensory layer most closely opposed to the pigment layer, more readily evident at the level of the choroid fissure (Fig. 1E). No slit-3 expression is seen in the eye before St22 (Fig. 1I), but at St25 it is present in the anterior lens epithelium (not shown) and in the pigment layer of the anterior retina (Fig. 1J).
Between St17–24 slit-2 expression occurs in the distal branchial arches (particularly I and II), the clefts (II and III), the epibranchial placodes of cranial ganglia VII/VIII and IX, in the rim of the otic vesicle (Fig. 1D), and in cranial nerve V (not shown). Low levels of slit-2 expression are present in the head mesenchyme at St22, including around the optic cup and stalk (not shown). slit-3 expression occurs in the proximal aspect of branchial arches II and III, in clefts (II and III), in the epibranchial placodes of cranial ganglia VII/VIII and IX, in the medial walls of the otic vesicles (Fig.1I), and surrounding cranial nerve III (not shown). From St22 there are low levels of slit-3 expression in head mesenchyme (not shown). The expression of both genes is seen in the epithelia of Sesel's and Rathke's pockets (not shown).
Both slit genes are expressed widely in the internal organs during chick embryonic development (not shown). Prominent sites of slit-2 expression are the mesenchyme surrounding the dorsal aorta and cardinal veins, the dorsal mesentery, the peritoneal epithelia, the mesenchyme and epithelia of the lung buds, the mesenchyme surrounding the oesophagous, in the proventriculus and gizzard (particularly concentrated in scattered clusters of cells), and in the lumenal epithelia of the intestine as it exits the gizzard. In the heart slit-2 is expressed in the truncus arteriosus and the atrio-ventricular cushion. It is also expressed in the mesonephric tubules and in the genital tubercle. slit-3 is expressed in mesenchyme around the dorsal aorta, in the dorsal mesentery, and in the peritoneal epithelium. Sites of gut tube expression include the proventriculus and the gizzard, in a similar clustered pattern to slit-2, and in the mesenchyme of the intestine. In the heart it is expressed in the pericardium and in the ventricles (not shown).
At St19, slit-2 somitic expression at the hindlimb level is restricted to the ventrolateral lip of the dermamyotome (Fig. 2A), and occurs subsequently in the myotome (not shown). slit-3 expression is found in the epidermis overlying the somites (Fig. 2D and not shown) and in the sclerotome by St22 (Fig 1F), and is down-regulated in mesenchyme immediately surrounding the notochord by St27 (not shown).
Expression in the Limbs
slit-2 expression in the limbs is complex, following similar patterns in both the fore and hind-limbs. In the hindlimb slit-2 is expressed at St19 in scattered cells extending from the ventrolateral dermamyotomal lip into the proximal limb (Fig. 2A). These are presumably muscle precursor cells, and at St22 slit-2 is expressed in dorsal and ventral muscle masses (Fig. 2B) and is associated with limb muscles until very late stages in limb development (Fig. 2C, K). Expression is also evident in the mesenchyme subjacent to the apical ectodermal ridge (AER) between St20–22 (Fig. 2B), continuing proximally along the anterior and (more broadly) posterior borders (not shown). The distal-most sub-AER expression is down-regulated by St23, while the anterior and posterior domains contract proximally with the onset of footplate development (not shown). slit-2 is also expressed in mesenchyme at the base of the limb bud up to St23 (Fig. 2B). Footplate expression of slit-2 begins at St24 around the site of the posterior-most metatarsal elements and as development proceeds, progressively more anterior domains of interdigital expression are established, being completed by St28 (Fig. 2C, I). During cartilage formation low expression levels of slit-2 are seen by St30 in some carpals/tarsals (not shown), at the ends of some adjacent long cartilage elements (Fig. 2G), and is present in the rounded cells of the inner layer of the periosteum along the center of long cartilage elements (Fig. 2G, K). By St32 inner periosteal cell expression of slit-2 is more extensive and is also present in mesenchymal cells immediately radial to the flattened cells of the outer layer of the periosteum (Fig. 2L, O) (Pechak et al., 1986). slit-2 is also expressed under regions of the epidermis (Fig. 2G, L), and peripheral to phalangeal joints (Fig. 2G, I). slit-3 expression was not detected in the hindlimb at St19 (Fig. 2D). At St22 expression is highest in proximal mesoderm, fading distally through the core of the limb bud (Fig. 2E), and from at least St29 slit-3 is expressed in the interdigital mesenchyme (Fig. 2F, J). At St27 low levels of expression are seen in the chondrocytes of the stylopod (not shown). By St30 it is detectable throughout the carpals/tarsals and in the proliferating chondrocytes of long cartilage elements (Fig. 2H), and at St32 it is also expressed in the phalanges (Fig. 2J). By St30 slit-3 is expressed in both the inner layer of rounded periosteal cells and mesenchyme radial to the outer flattened periosteal cell layer of long cartilage elements (Fig. 2H, N, M, P). In some regions the mesenchymal layer of expression extends to and is continuous with a layer of expression under the epidermis (Fig. 2N), or forms a bridge between elements such as the radius and ulna (Fig. 2N) or metacarpals (Fig. 2M).
In summary, we have isolated two chick slit gene homologues. As in other organisms they are widely expressed in a variety of cell types during embryogenesis. In the limb both slit genes are expressed in a spatio-temporal manner that suggests their involvement in fundamental aspects of limb development such as muscle development, AER function, chondrogenesis, and osteogenesis. The early and widespread expression of slit genes in the mouse may limit or complicate the information yielded from future “knockout” or transgenic approaches to determine slit gene function in mice. The chick embryo provides a versatile experimental system that should allow a variety of approaches to elucidate the roles of the slit genes in limb and other organ development.
A St12–15 oligo dT-primed chick cDNA library constructed in λZapII (Stratagene, La Jolla, CA; a gift from D. Wilkinson) was screened with a PCR-derived fragment of chick slit-2 (pcslit2Exlox18B) (Li et al., 1999), labeled with (32P)dCTP by random-primed synthesis (Random Primed DNA Labeling Kit, Boehringer Mannheim, Germany). Approximately 500,000 plaques were screened, and filters were hybridized and washed under conditions of low stringency. Numerous positive plaques were obtained and purified by two further rounds of hybridization. Plasmid clones (pBluescript SK−) were obtained from plaques by excision as described by the manufacturer. The sequence identity of isolated clones was determined by automated dye terminator sequencing (Perkin Elmer/Applied Biosystems) using plasmid-specific and custom primers (Sigma Genosys, TX).
Full-length digoxigenin-labelled probes were synthesized from linearized templates using Promega (WI) reagents. After DNase I digestion of the DNA templates, riboprobes were purified on Quick Spin Columns (Boehringer Mannheim, Germany) and quantified by spectrophotometry.
Whole-Mount In Situ Hybridization (WISH)
For WISH the shorter pcslit2Exlox18B riboprobe was used to detect slit-2 expression; the pcslit-3(2) riboprobe was fragmented to approximately 300 bp by alkaline hydrolysis. White Leghorn chick embryos (eggs from SPAFAS, CT) were staged according to Hamburger and Hamilton (1951). WISH was performed as described previously (Henrique et al., 1995), except that the slit-3 signal was detected with 4-nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indoyl phosphate (BCIP).
In Situ Hybridization (ISH)
For ISH full-length riboprobes from pcslit-2(26a) and pcslit-3(2) were used. Hybridization of 10μm alternate sections cut from samples embedded in frozen OCT blocks was as described (Neubuser et al., 1995), except for the following modification. On the first day sections were fixed in 4% PFA/1xPBS for 10 min, washed twice in PBS for 5 min, digested with 5μg/ml Proteinase K in PBS for 6 min, then washed once in PBS for 5 min. After acetylation for 10 min in 0.5% acetic anhydride/0.1 M TEA-HCl (pH 7.5), sections were washed in PBS for 5 min then incubated in 0.1 M Tris/ 0.1 M glycine buffer for 30 min. Sections were then hybridized overnight as described.
We thank David Wilkinson and Yi Rao for the gifts of the chick embryo library and the chick slit-2 probe, respectively. This work was supported by NIH HD32427 award to L.N and by the MSKCC Support Grant. G.H. and L.N. are, respectively, a Research Associate and an Assistant Investigator of the Howard Hughes Medical Institute. This paper is dedicated to the memory of Toshiya Yamada.