A new role for Notch and Delta in cell fate decisions: patterning the feather array

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1 Development 125, (1998) Printed in Great Britain The Company of Biologists Limited 1998 DEV A new role for Notch and Delta in cell fate decisions: patterning the feather array Rebecca Crowe 1, Domingos Henrique 3, *, David Ish-Horowicz 3 and Lee Niswander 2, 1 Cell Biology and 2 Molecular Biology Programs, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA 3 Developmental Genetics Laboratory, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln s Inn Fields, London WC2A 3PX, UK *Present address: Instituto Histologia e Embriologia, Faculdade de Medicina de Lisboa, Av. Prof. Egas Moniz, 1699 Lisboa Codex, Portugal Author for correspondence ( L-niswander@ski.mskcc.org) Accepted 29 November 1997: published on WWW 22 January 1998 SUMMARY Chick embryonic feather buds arise in a distinct spatial and temporal pattern. Although many genes are implicated in the growth and differentiation of the feather buds, little is known about how the discrete pattern of the feather array is formed and which gene products may be involved. Possible candidates include Notch and its ligands, Delta and Serrate, as they play a role in numerous cell fate decisions in many organisms. Here we show that Notch-1 and Notch-2 mrnas are expressed in the skin in a localized pattern prior to feather bud initiation. In the early stages of feather bud development, Delta-1 and Notch-1 are localized to the forming buds while Notch-2 expression is excluded from the bud. Thus, Notch and Delta-1 are expressed at the correct time and place to be players in the formation of the feather pattern. Once the initial buds form, expression of Notch and its ligands is observed within each bud. Notch-1 and -2 and Serrate-1 and -2 are expressed throughout the growth and differentiation of the feathers whereas Delta-1 transcripts are downregulated. We have also misexpressed chick Delta-1 using a replication competent retrovirus. This results in induction of Notch-1 and-2 and a loss of feather buds from the embryo in either large or small patches. In large regions of Delta-1 misexpression, feathers are lost throughout the infected area. In contrast, in small regions of misexpression, Delta-1 expressing cells differentiate into feather buds more quickly than normal and inhibit their neighbors from accepting a feather fate. We propose a dual role for Delta-1 in promoting feather bud development and in lateral inhibition. These results implicate the Notch/Delta receptor ligand pair in the formation of the feather array. Key words: Delta, Feather bud, Lateral inhibition, Notch, Pattern formation, Chick INTRODUCTION The Notch/Delta receptor/ligand pair is part of an evolutionarily conserved signaling system used in numerous cell fate decisions (Artavanis-Tsakonas et al., 1995; Chitnis et al., 1995; Henrique et al., 1995; Myat et al., 1996; Shawber et al., 1996). In C. elegans, lin12 and lag2 are involved in the cell fate decision between the ventral uterine precursor cell and the anchor cell. In Drosophila, Notch and Delta help to single out a sensory organ precursor from a set of equivalent cells (reviewed by Greenwald and Rubin, 1992). In the retina of Xenopus and chick, Notch signaling restricts the number of cells which can progress along their differentiation pathway at any given time (Austin et al., 1995; Dorsky et al., 1995, 1997; Henrique et al., 1997). This conserved signaling system is also present in mammals with homologues found in mouse, rat and human (Artavanis-Tsakonas et al., 1995). In the mouse developing immune system, Notch has been implicated in the CD8/CD4 T cell decision (Robey et al., 1996). Notch and Delta homologues are expressed in dynamic temporal and spatial patterns in many organisms suggesting that they may play numerous and diverse roles throughout development (Weinmaster et al., 1991; Reaume et al., 1992; Kopan and Weintraub, 1993; Lardelli and Lendahl, 1993; Austin et al., 1995; Chitnis et al., 1995; Conlon et al., 1995; Henrique et al., 1995; Mitsiadis et al., 1995; Williams et al., 1995; Myat et al., 1996; Bao and Cepko, 1997; Dorsky et al., 1997). Gene targeting of Delta-1 and Notch-1 have illustrated their importance for early mouse development and somitogenesis (Swiatek et al., 1994; Conlon et al., 1995; de Angelis et al., 1997). Although Notch and Delta are expressed in many different cell types, in each circumstance where their function has been determined, they act by restricting cell fates or by allowing only some cells to respond to fate-determining signals. Another ligand for Notch is Serrate (Artavanis-Tsakonas et al., 1995; Lindsell et al., 1995; Nye and Kopan, 1995; Myat et al., 1996; Shawber et al., 1996). It is not known whether Serrate

2 768 R. Crowe and others activation of Notch results in the same downstream sequence of events as Delta activation of Notch; however, Serrate can at least partially substitute for Delta during Drosophila neurogenesis (Gu et al., 1995). A distinct role for Serrate and Notch in the establishment of the wing margin in Drosophila has been demonstrated (Speicher et al., 1994; Couso et al., 1995; Diaz-Benjumea and Cohen, 1995). Serrate is conserved through evolution with homologues identified in chick, mouse, and rat (Artavanis-Tsakonas et al., 1995; Lindsell et al., 1995; Myat et al., 1996). The Serrate genes, like Notch and Delta, are expressed in a wide range of tissues during vertebrate embryogenesis (Lindsell et al., 1995; Myat et al., 1996; Shawber et al., 1996; Bao and Cepko, 1997; Mitsiadis et al., 1997). Although its roles in vertebrate development have not yet been determined, it has been demonstrated that Jagged (rat Serrate) can activate Notch in a cell culture assay to inhibit myogenesis (Lindsell et al., 1995). Due to the involvement of Notch, Delta, and possibly Serrate, in many cell fate decisions during embryogenesis, we sought to determine if Notch signaling may also play a role in the establishment or refinement of the feather array in chick embryos; perhaps in the choice between a feather or smooth skin fate. The embryonic chick feather buds are hexagonally arranged and arise progressively in tracts. This pattern is thought to be set up from signals in the dermis which then influences the epidermis (Cairns and Saunders, 1954; Saunders and Gasseling, 1957; Saunders, 1958; Linsenmayer, 1972; Sengel, 1976). Further feather development is dependent on reciprocal interactions between the epidermis and dermis (Saunders and Gasseling, 1957; Sengel and Abbott, 1963; Wessells, 1965). Prior to the morphological formation of the feather buds, the mesenchyme begins to condense uniformly under the ectoderm to form a dense dermis starting about Hamburger-Hamilton (HH) stage 28 (5.5-6 days of incubation); (Wessells, 1965; Sengel, 1976; Mayerson and Fallon, 1985; Hamburger and Hamilton, 1951). By HH stage (6.5-7 days of incubation) the first feather buds arise in tracts along the dorsal midline and around the shoulders and pelvis. At this stage, the dermis induces a morphological change in localized regions of the ectoderm resulting in a thickened, columnar epithelial placode. Shortly thereafter, the dermal cells migrate and condense specifically beneath each epidermal placode thus resulting in a small raised feather bud (placode stage). After initial bud formation, the feather continues to grow out and points posteriorly. Later as the feather differentiates, the epithelium invaginates and folds around the feather pulp, forming the marginal and barb plates. The marginal plates are destined to die by programmed cell death while the barb ridges will later keratinize and form the fully differentiated feather barbs (Saunders and Gasseling, 1957; reviewed by Chuong, 1993). A number of signaling molecules are expressed during the various stages of feather development. One well characterized marker of the feather bud is Sonic hedgehog (Shh). Shh, a secreted signaling molecule implicated in numerous morphogenetic processes, is first detected at the placode stage when it localizes to the epithelium. During feather growth, Shh transcripts are localized to the leading edge of the feather bud and expression is later restricted to the marginal plate showing a striped pattern (Nohno et al., 1995; Ting-Berreth and Chuong, 1996b). Shh has been implicated in feather bud differentiation as ectopic expression of Shh results in feathers of aberrant morphology (Ting-Berreth and Chuong, 1996b). Other signaling molecules, such as members of the FGF family and TGFβ superfamily, are also expressed in the epithelium during development of the feather bud (Noji et al., 1993; Jakowlew et al., 1994; Nohno et al., 1995; Chuong et al., 1996; Widelitz et al., 1996). Application of beads soaked in either FGF or TGFβ protein can substitute for the epithelial placodes in the induction of dermal condensations and can alter feather polarity and morphology (Ting-Berreth and Chuong, 1996a; Widelitz et al., 1996). FGFs were also shown to be important for feather bud development, as beads soaked in FGF protein can induce ectopic feather buds (Widelitz et al., 1996) and can rescue the phenotype of a naturally occurring chick mutant, scaleless (Song et al., 1996). scaleless chick embryos normally grow only a few feathers and FGF application causes additional feather growth (Abbott and Asmundson, 1957; McAleese and Sawyer, 1981; Song et al., 1996; Song and Sawyer, 1996). Thus, many signaling molecules are implicated in the regulation of various aspects of feather morphogenesis. However, a role for these or other molecules in establishment or refinement of the spatial array of feathers has not been demonstrated. Moreover, it is not known how the spacing of feather buds is achieved. To investigate a possible role for Notch and its ligands during the formation of the chick embryonic feather bud array, the expression patterns of the chick Notch, Delta, and Serrate genes were analyzed by RNA in situ hybridization. In this paper, we show that Notch-1 and -2 mrnas are detected prior to feather bud initiation, with expression of Delta observed in each bud as it forms. Overexpression of chick Delta-1 using a replication competent retrovirus results in the loss of feather buds from the normal fields. We also demonstrate both a cell autonomous and cell non-autonomous effect of Delta-1 misexpression. Large patches of viral misexpression correlate with widespread feather loss. However, in small patches of misexpression, Delta-1 misexpressing cells differentiate into buds and inhibit neighboring cells from doing the same. Also, feathers infected early with the Delta-1 virus appear to be more mature as compared to their uninfected neighbors. These results suggest that Delta-1 may play a role in the cell choice between a feather or smooth skin fate. We propose a model in which Delta-1 may act both to promote feather differentiation and to inhibit neighboring cells from accepting a feather fate. MATERIALS AND METHODS RNA probes Antisense digoxigenin probes were prepared as described: (Notch-1, Serrate-1, Myat et al., 1996]; Serrate-2, Laufer et al., 1997). The c- Notch-2 cdna was provided by Gerry Weinmaster. The c-delta-1 cdna was constructed by subcloning a 600 base pair ClaI/ClaI fragment from RCASBP(A)/c-Delta-1 into Bluescript SK +. The RCAS probe was constructed by subcloning a 1.6 kb KpnI/XbaI fragment corresponding to bases of the pol region, from RCASBP(A) into Bluescript SK +. Shh was provided by C. Tabin (Riddle et al., 1993) and Bmp7 by B. Houston (Francis-West et al., 1995). In situ hybridization White Leghorn chicken eggs were obtained from SPAFAS (Norwich,

3 Notch signaling in feather bud formation 769 CT) and incubated at 39 C for noted lengths of time. Embryos were fixed and processed as described for either RNA whole mount in situ hybridization (Henrique et al., 1995) or for paraffin sectioning and RNA section in situ hybridization (Neubuser et al., 1995 and Neubuser personal communication). Modifications to the whole mount procedure included a proteinase K concentration of 5, 7.5, or 10 µg/ml for all probes except Dl-1 which was 30 µg/ml, and use of Boehringer- Mannheim BM purple substrate in place of BCIP/NBT. Viral infection RCASBP(A)/c-Delta-1 was constructed as described by Henrique et al. (1997). Transfection and growth of RCAS viruses were performed as described by Morgan and Fekete (1996). Concentrated virus was injected either into the presumptive limb region of Hamburger and Hamilton (1951) stage embryos to achieve widespread infection or into the amniotic cavity of stage embryos for infection predominantly localized to the skin. FGF application and skin culture scaleless eggs were kindly provided by Jacqueline Pisenti (UC Davis) for use as controls. Embryos were injected at day 2 of incubation into the presumptive limb region with RCAS/Delta-1 and then incubated until day 8. The skin from the back of a day 8 chick embryo was removed in 1 Tyrode s solution and placed on the chorioallantoic membrane (CAM) of a day 10 chick embryo host µm acrylic heparin beads (Sigma) were soaked in 2.5 µl of 1 mg/ml FGF4 protein (provided by Genetic Institute, Cambridge, MA) for over 1 hour before application to the skins. Hosts were returned to the incubator to allow the skins to develop (Song et al., 1996). RESULTS Notch, Delta and Serrate genes are expressed in dynamic patterns during feather development To study a possible role for Notch signaling in formation of the chick embryonic feather pattern, we examined the expression patterns of Notch and its ligands during various stages of feather development. Stage 29 embryos are at the stage just prior to initial epithelial placode formation, whereas at stages 30-31, epidermal placodes and dermal condensations are beginning to form along the midline and around the shoulders and pelvis (Mayerson and Fallon, 1985; Hamburger and Hamilton, 1951). Thus, for a molecule to be involved in the establishment of the feather pattern, expression should be seen prior to bud initiation. This is indeed the case for Notch-1 and 2. Notch-1 transcripts are detected at stage 29 in two epithelial stripes on the back which converge into one stripe more caudally, prefiguring the region where the initial feather tracts will form at stage 30 (Fig. 1A). Notch-2 transcripts are also detected at stage 29 in two broader stripes which overlap the Notch-1 stripes (Fig. 1D). As the placodes form (stage 30+), Notch-1 gradually becomes restricted to each epithelial placode (Fig. 1B,C). Interestingly, at the same stage (stage 30), Notch-2 transcripts are excluded from the early bud and instead are detected in the surrounding dermis (Fig. 1E). However, as the buds start to mature (stage 31), Notch-2, like Notch-1 is detected in the epithelial layer of the bud itself (Fig. 1F). Delta-1 transcripts are not detected at stage 29, but are observed in the mesenchyme underlying the forming epithelial placodes at stage 30 (Fig. 1G,H). Thus, when the first feather buds are observed at stage 30, Notch-1, Notch-2 and Delta-1 transcripts are expressed in complementary patterns. But as the buds begin to mature (stage 31), all three mrnas are localized to the feather bud. As Notch-1 and -2 are both expressed prior to epidermal placode formation, and Delta-1 is expressed during early bud initiation, it is possible that these genes may play a role in establishing or refining the pattern and spacing of the placodes within the feather fields or in the epithelialmesenchymal interactions necessary for placode induction. As the feather buds continue to grow and develop, Notch and Delta-1 continue to be expressed in dynamic patterns. By stages (day 8 of incubation), many small buds are seen along the main body axis as well as along the thighs and shoulders. At this time, Delta-1 expression is detected in a posterior crescent within the bud mesenchyme (Fig. 2E,J and Chen et al., 1997) while Notch-1 and Notch-2 switch from the epithelium to the mesenchymal layer (Fig. 2A,B,F,G). As the buds elongate, Delta-1 expression is downregulated and is no longer detected in the large midline feathers at stage 36 (day 10 of incubation; data not shown; and Chen et al., 1997). Notch-1 and -2 mrnas are detected in the mesenchyme until about stage 36, when the expression in the more mature buds along the midline changes. Here, Notch-1 mrna is localized once again to the epithelium in a striped pattern corresponding to the barb plates, the region destined to become the mature feather barbs (Fig. 2K,O). Conversely, Notch-2 is expressed in the peripheral epithelium and also along the edges of each barb ridge (Fig. 2L,P). These dynamic expression patterns are suggestive of a role for Notch/Delta signaling in early patterning and growth of the feather. Alternate ligands for Notch, Serrate-1 and -2, are also expressed during development of the feather buds. Prior to bud initiation and in the early placode stages, Serrate-1 and -2 transcripts are not detected (data not shown and Chen et al., 1997). However, once small buds have formed, both mrnas are expressed. Serrate-1 is expressed in the mesenchyme (Fig. 2C,H and Chen et al., 1997) while Serrate-2 transcripts are localized to the epithelium (Fig. 2D,I). This expression pattern is maintained until stage 36. At this stage, in the midline feathers, Serrate-1 transcripts are now detected in a ring of the epithelial layer (Fig. 2M,Q) whereas Serrate-2 is localized to a striped pattern in the epithelium (Fig.2N,R). The epidermal stripes of Serrate-2 are in the opposing cell types to the Notch- 1 stripes, and they correspond to the marginal plates, the region destined to undergo programmed cell death later in development. The expression patterns of Serrate-1 and -2 suggest that the Notch/Serrate signaling system may play a role during growth and differentiation of the feather. Misexpression of Delta-1 results in a loss of feathers Notch-1 and -2 are expressed prior to the appearance of feather buds, and Delta-1 expression is observed in each bud as it forms, suggesting that these genes may play a role in patterning the feather array. To explore this possibility, we examined the effects on feather production of misexpressing Delta-1. This was accomplished by injection of a replication competent retrovirus (RCAS) expressing the c-delta-1 gene into chick embryos at various stages of their development followed by further incubation (see Materials and Methods). Injection into the presumptive limb region at stage 14 results in a widespread infection whereas injection into the amniotic cavity of stage 20 embryos results in both large and small regions of infection that usually remain superficially localized to the epidermal and

4 770 R. Crowe and others loss often appear in a less ordered pattern than feathers further from the regions of loss (Fig. 3B and data not shown). To determine if the loss of feather buds by Delta-1 was due to a block of bud initiation, a failure of bud maintenance, or a delay in bud formation, we performed histological and molecular marker analyses. Our results indicate that feather bud formation is never initiated in the regions of ectopic smooth skin following Delta misexpression. The ectoderm does not thicken into a placode and the underlying dermis shows no sign of localized condensation. Although the morphological signs of feather formation are absent, the smooth skin regions display normal histology with a uniformly dense dermis and unaltered epidermis (n=8) indicating that the virus does not affect the overall structure of the skin (Fig. 3C). Markers of early feather bud development such as Shh and Bmp 2, 4, and 7 (Nohno et al., 1995; Chuong et al., 1996; Ting- Berreth and Chuong, 1996b; and this report) are not detected in the regions of smooth skin (Fig. 3B and data not shown; n>30). Analyses of the phenotype at various stages indicates that Delta-1 overexpression blocks feather formation rather than delays it, as the smooth areas remain throughout embryonic development (analyzed through day 14 of incubation). Thus, it appears that Delta-1 misexpression results in an inhibition of bud formation as no signs of feather bud initiation were observed in the regions of ectopic smooth skin. Fig. 1. Notch and Delta expression during early feather bud development. Whole mount RNA in situ hybridizations with digoxigenin-labeled probes were carried out (rostral is at the top); some embryos were then processed for paraffin sectioning (stages 29 (A, bottom) and 30 (E,G, bottom) transverse sections, 31 (C,F, bottom) sagittal sections with rostral at the left). (A,D) At stage 29, prior to bud initiation, Notch-1 and Notch-2 are expressed in two stripes on the back (arrow). Notch-1 stripes are in the epidermis and prefigure the initial feather tracts. Notch-2 expression is in the dermis and is broader, encompassing the Notch-1 stripes. (B,E,G) At stage 30, the initial feather tracts have formed. (B) Notch-1 is now expressed in the epithelial placode of the newly forming buds (arrow). (E) In contrast, Notch-2 expression persists in the dermis but is excluded from the forming bud (arrow points to bud). (G) Delta-1 expression is observed at this time in the mesenchyme underlying the epithelial placode (arrow). (C,F,H) At stage 31 expression of Notch-1 (C) and Delta-1 (H) persists in the developing bud epithelium and dermis, respectively, while Notch-2 (F) now localizes to the epithelium of each bud. N-1, Notch-1; N-2, Notch-2; Dl-1, Delta-1. dermal layers (data not shown). By either infection protocol, Delta-1 overexpression results in a loss of feather buds (>90% of embryos). Infection at stage 14 results in a widespread feather loss involving large portions of the body (Fig. 3A). Injections into the amniotic cavity at stage 20 results in both large and small patches of smooth skin within a region of feathers (Fig. 3B). Also, feathers that do form near areas of Cells exposed to Delta-1 signaling are inhibited from adopting a feather fate To begin to understand how misexpression of Delta-1 inhibits feather formation, RCAS/Delta-1 injected embryos were subjected to RNA in situ hybridization to detect viral transcripts. The embryos from such experiments show viral expression patterns which fall into two classes corresponding to the extent of feather loss. In embryos lacking feathers from large regions of the body, RCAS transcripts are detected within the areas of ectopic smooth skin (examined at stage 31-36; Fig. 4A,B; n=13). Localization of the virus to the large regions of smooth skin suggests that widespread misexpression does not allow cells to adopt a feather fate. In striking contrast, in embryos missing small patches of feathers, viral transcripts are detected at high levels in the feathers and interbud regions adjacent to the smooth patch but not in the smooth skin itself (examined at stage 31-36; Fig. 4C-F; n=11). Feather loss is most often observed lateral to infected feathers, but we also observe the converse in which feather loss is medial to the Delta-1-infected feathers (Fig. 4E-F). In addition, patches of feathers are frequently lost both medial and lateral to the small regions of virally infected feathers. In these cases, the data suggest a cell nonautonomous effect of Delta-1 in which it inhibits feather formation in neighboring nonexpressing cells. In embryos examined at stage 36 or later, viral expression was sometimes noted to a lesser degree and more variable extent in feathers away from the altered region, most likely due to later spread of the virus into the feathers after their initial formation. There was no apparent phenotypic effect on these later-infected feathers or surrounding skin suggesting that Delta inhibits feather formation at an early stage. To examine the possibility that Delta may be acting through secondarily induced genes to produce its inhibitory effect, we performed whole mount and section RNA in situ hybridization experiments with a number of feather-specific genes. In large

5 Notch signaling in feather bud formation 771 Fig. 2. Dynamic expression of Notch and its ligands during feather differentiation. Whole mount (A-E, K-N; rostral is at the top) and transverse section (F-J; O-R) RNA in situ hybridizations of stages (day 8) and stage 36 (day 10) feather buds. By day 8, Notch-1 and Notch-2 become restricted to the bud mesenchyme (A,B,F,G). Serrate-1 and Serrate-2 expression are first detected in the bud mesenchyme and epithelium respectively (C,D,H,I). Delta-1 RNA remains localized to the mesenchyme (E,J). By day 10, Notch-1 is expressed in stripes corresponding to the barb ridges (K,O) while Notch-2 is expressed more uniformly in the epidermis with some localization to the inner edges of the barb ridges (L,P). Serrate-1 is expressed in a ring of the epidermis (M,Q) while Serrate-2 localizes to the marginal plates (N,R). Delta-1 expression is not detected by this stage. Ser-1, Serrate-1; Ser-2, Serrate-2. patches of smooth skin (Fig. 5A,B) we observed induction of endogenous Notch-1 and Notch-2 transcripts and this induction correlates with regions of Delta-1 misexpression (alternate sections in Fig. 5C-E). We note that Notch induction is variable in that it is not detected in all regions of viral misexpression and that the induction seems to partially correlate with the stage of the embryos. Embryos with young feather buds (stages 31-34) often show Notch induction while older embryos (stage 35+) rarely show this effect (Fig. 5A,B compared to Fig. 3A). Other genes such as Serrate-1, Serrate-2, Shh, Bmp2, Bmp-4 and Bmp-7 are not detected in the smooth skin and are instead restricted to their normal pattern in the feather bud (Figs 3B, 4A and data not shown) Another effect of the Delta-1 misexpression that was often noted is that the infected feathers appear more mature than corresponding uninfected feathers (n=6 embryos). The infected feather buds show slightly accelerated development and are larger in size as compared to the corresponding contralateral uninfected feather buds (Fig. 6). These more mature feathers are often, but not always, adjacent to smooth patches of uninfected skin. Thus, small groups of cells expressing Delta-1 appear to differentiate into feather buds more quickly than nonexpressing cells. Feather loss cannot be rescued by fibroblast growth factor application scaleless is a naturally occurring chick mutant which lacks feathers over most of its body (Abbott and Asmundson, 1957). The defect is thought to lie in the epidermal layer, as recombination of wild-type epidermis with mutant dermis can rescue the feather defect (Sengel and Abbott, 1963; McAleese and Sawyer, 1981; Song and Sawyer, 1996). It was also shown that application of beads soaked in fibroblast growth factor to scaleless skin can rescue the formation of feathers near the beads (Song et al., 1996). As widespread misexpression of Fig. 3. Delta-1 misexpression results in feather loss. (A) Widespread feather loss is observed after injection of RCASBP(A)/Delta-1 at stage 14. Example shown is stage 36 embryo where remaining feather buds are visualized by RNA in situ hybridization of Notch-2 RNA (rostral is at the top). (B) Small patches of feather loss (some marked by arrows) result from RCASBP(A)/Delta-1 injection into the amniotic cavity at stage 20. Example shown is stage 32 embryo where remaining feather buds are visualized by Bmp7 RNA (rostral is at the top). (C) Histological section indicates that skin structure is not affected in regions of feather loss as shown by a uniformly dense dermis (transverse section through a stage 35 embryo; two feather buds can be observed in this section).

6 772 R. Crowe and others Fig. 4. Viral localization in Delta-1 infected embryos. (A,B) In large patches of feather loss (arrow; rostral at top), as visualized by loss of Shh transcripts (A), viral transcripts are detected in the region of feather loss (B). Embryo in A and B was injected at stage 20 and analysed at stage 31 by RNA in situ hybridization with successive Shh (A) and RCAS (B) probes. (C) In contrast, in small regions of feather loss (marked by *; rostral at top), viral transcripts are detected in the adjacent feather buds and interbud (arrow). Embryo in C was injected at stage 20 and analysed at stage 36 by RNA in situ hybridization with RCAS probe. (D) Transverse section in situ hybridization through a small region of feather loss (demarcated by arrows), viral transcripts are detected in the dermis and epidermis of the adjacent feather buds and skin. Embryo was injected at stage 14 and analysed at stage 36 with RCAS probe. (E,F) Feather loss is often noted medial to the RCAS infected feathers (arrow indicates infection). Embryo was injected at stage 20 and analysed at stage 35 with sequential RCAS (E) and Shh (F) probes (rostral at top; midline to the right). Delta-1 also results in large patches of featherless skin, we wanted to test if local application of FGF to the back skin of an infected chicken could rescue the phenotype. Experiments were performed either with scaleless back skins as a control or with skins in which Delta-1 was widely misexpressed. In control scaleless skins, feather buds were noticed around the beads after 2-3 days of incubation in 52% of the cases (n=23). In contrast, in all but one case of the Delta-1 infected back skins, no feathers were observed near the FGF beads even following 7 days of incubation (n=19). In the one case, a feather did form near the bead but in situ hybridization analysis with the viral probe indicated that virus was not localized to this area and thus feather formation in this region could occur normally. These results indicate that Delta-1 misexpression results in an irreversible block to feather bud formation which cannot be overridden by application of a feather stimulator, FGF. DISCUSSION Fig. 5. Delta-1 misexpression results in Notch induction. (A,B) Following RCAS/Delta-1 injection into the amniotic cavity at stage 20, endogenous Notch-1 (A; examined at stage 34) and Notch-2 (B; examined at stage 32) mrnas are induced in large regions of feather loss (white line demarcates the boundary between the region of normal expression of Notch in the bud and the region of Notch induction). (C-E) Notch induction correlates with regions of Delta-1 misexpression. Embryo was injected at stage 14 and fixed at stage 35 for hybridization of alternate transverse sections with Delta-1 (C), Notch-1 (D), and Notch-2 (E), probes. Alternate sections from embryo in C-E were also hybridized with Serrate-1, Serrate-2 and Shh but no induction was noted (not shown). Chick embryonic feather buds arise in a distinct pattern both spatially and temporally. How such a complex array is established and maintained is a fundamental question in developmental biology. The genes Notch and Delta are involved in many cell fate decisions and therefore we explored their potential role in establishing or refining the feather pattern. Here we show that Notch-1 and Notch-2 are expressed in the skin prior to feather bud development. Once the epithelial placode is morphologically distinguishable, Delta-1 and Notch-1 mrnas are expressed in adjacent cell layers within the bud while Notch-2 expression is excluded from the early bud. These early expression patterns are consistent with the idea that Notch and Delta are players in the early events related to formation of the feather array. To test this hypothesis, misexpression of Delta-1 was carried out using a replication competent retrovirus encoding the chick Delta-1 gene. Misexpression of Delta-1 results in a loss of feather buds. Feather formation appears to never be initiated and to be irreversibly blocked in the regions of feather loss, as molecular markers of the feather bud are not expressed and application of FGF beads cannot rescue feather formation. Examination of viral transcripts in relation to the phenotype suggests that Delta-1 has the ability to act cell nonautonomously to inhibit feather initiation. Delta-1

7 Notch signaling in feather bud formation 773 Fig. 7. Model for Delta-1 action in feather bud patterning. Delta-1 expression in the mesenchyme of a newly forming feather bud will have two effects. One is a positive feather promoting signal through activation of Notch-1 in the epithelial placode. The second is a negative lateral inhibitory signal mediated through Notch-2 in the adjacent mesenchyme. The combination of both a positive and a negative signal may refine the pattern of the feather bud array. Fig. 6. Delta-1 overexpressing feathers appear more mature than uninfected feathers. Sequential RNA in situ hybridizations were performed with RCAS and then Shh probes (rostral at top). Infected feather buds (arrow in A and C) appear older and, in some cases form earlier, than corresponding uninfected feathers as visualized by Shh (B and D; embryos were injected at stage 20 and fixed at stages 31 and 34 respectively). (E,F) In an older embryo, the size difference is more apparent in infected feathers on the right side of the embryo (arrow in E) compared to corresponding uninfected feathers on the left side (arrow in F). The embryo was injected at stage 14 and analysed at stage 35. misexpression also results in the induction of Notch-1 and Notch-2 transcripts. These results suggest a role for Notch and Delta in the formation of the chick embryonic feather pattern. Based on our results presented here, Delta-1 seems to act as both a positive and negative signal for feather development. In our model (Fig. 7), early expression of Delta-1 at stage 30 will have two effects. One, as a feather promoting signal, through activation of Notch-1 in the epithelial placode. Two, as an inhibitory signal, through activation of Notch-2 to inhibit feather formation in neighboring cells. These two effects could occur simultaneously or sequentially. Thus, by combining a positive feather promoting signal with a lateral inhibitory signal, Delta-1 may act to pattern and refine the feather array. The early expression pattern of Notch and Delta supports a role for this ligand/receptor system in early bud development. Notch-1 and -2 are both expressed in the skin prior to bud formation. Although ligands for Notch are not detected at this stage, we cannot rule out the possibility that uncloned members of the family are expressed or that Delta-1 may be expressed at levels below detection. However, this early Notch expression correlates with studies of Linsenmayer (1972) that indicated that the position of each feather bud is established approx. 1 day before the feather placode is morphologically distinguishable and that the information for this position lies within the skin itself. As the buds form, Delta-1 expression is observed in the dermis underlying the forming epithelial placodes and Notch - 1 expression is restricted to the epithelial placode. Reciprocal interactions between the epidermis and dermis are important for feather formation. Hence, the complementary expression of Delta in the dermis and Notch-1 in the epidermis at the earliest stages of bud formation may indicate that Notch-1 signaling promotes feather fate. In contrast, Notch-2 transcripts are observed in the dermis, adjacent to each bud. This suggests that Notch-2 activity inhibits feather fate. Our experimental data supports a role for Delta-1 in promoting feather development. Feather buds that ectopically express Delta-1 virus from an early stage appear more mature than uninfected buds. During normal feather development, expression of Delta-1 in the early bud may provide a positive signal necessary for bud development. By misexpressing Delta-1 at an earlier stage, cells may begin to follow the feather differentiation program earlier than normal and thus appear older than neighboring buds. We cannot exclude the possibility that the larger feather size is due in part to a faster proliferation rate, as this has not been tested. However, slightly later, after feather buds have been initiated, Delta-1 infection yields normal sized feathers. Thus it appears that early Delta-1 misexpression results in precocious feather development. The misexpression data also supports an inhibitory role for Delta. Misexpression of Delta-1 at a stage prior to bud initiation results in a loss of feather buds. In widespread regions of feather loss, there was widespread expression of viral transcripts. In contrast, when small patches of feather loss were observed, viral transcripts were observed in the feathers adjacent to the region of feather loss and not within the smooth patch itself. These results are quite similar to those seen following misexpression of Delta in the Xenopus and chick retina (Dorsky et al., 1997; Henrique et al., 1997). In large regions of Delta misexpression, cells in the center were inhibited from accepting a neuronal fate. In contrast, single cells and small groups of cells expressing Delta were able to accept neuronal fates prematurely while inhibiting the neighboring cells from doing the same. In these circumstances, lateral inhibition was the model implicated to explain the actions of Delta. Our results are also compatible with a lateral inhibition model. In small areas of Delta misexpression, in either the retina or the feather field, Delta may signal to the neighboring cells through Notch to inhibit them from accepting a neural or feather fate. Signaling through Notch may also result in large regions

8 774 R. Crowe and others of feather loss, as Notch is induced to high levels by Delta-1 misexpression, thus leading to global inhibition. Hence, the misexpression data presented here are in accordance with the results obtained in other systems where Delta appears to act via a lateral inhibitory model. However, we cannot rule out that widespread infection affects feather formation by creating cell confusion in which widespread misexpression of a feather bud marker itself can prevent organization into distinct buds. Based largely on studies in Drosophila, Delta and Notch are thought to interact in a cell contact dependent manner. Moreover in Drosophila, Delta acts at a single cell level to influence the immediately neighboring cells (reviewed by Greenwald and Rubin, 1992). It is not clear during normal feather development whether a single cell or a group of cells is selected to become the feather primordium. The first localized expression of Delta and Notch that we observe in the skin is in more than one cell. Alternatively, lateral inhibition of the cells that immediately border the Delta region may be sufficient to result in proper spacing, as each feather is made up of a group of cells and therefore the next feather may only be able to form from cells at a distance from these boundary cells. The exclusion of Notch- 2 transcripts from the early stage feather buds is suggestive of a role of Notch-2 in mediating such an inhibitory signal. However, we cannot rule out the possibility that the inhibition of neighbors by Delta may be indirect, possibly through regulation of a diffusable inhibitory signal. In contrast to early Notch-2 expression, the pattern of Delta-1 and Notch-1 in the early feather bud is complementary in the dermis and epidermis, respectively. This cell layer-specific expression may reflect a continuation of the earlier process of feather promotion by Delta and Notch as dermal-epidermal interactions are critical for feather development. Therefore, by combining lateral inhibition with positive signaling from Delta in the bud itself, it can be imagined how a complex feather array can be patterned. Whether both the promotion and inhibition processes are direct consequences of Delta activation of Notch, or a secondary effect of possible downstream genes is not clear. Of the genes tested, (Shh, Bmp2, 4 and 7, Serrate-1 and 2, Notch-1 and 2), only Notch-1 and Notch-2 are induced within large regions of Delta misexpression. This suggests that inhibition of feather formation by Delta is direct. However, Notch induction is not observed in all cases. This may indicate that Notch is induced for a relatively short window of time after which it is downregulated or, that after a certain point in development, cells become refractory to the effects of Delta-1 infection. It also suggests that although Delta is misexpressed in a particular region throughout its development, it may signal through Notch only for a short time. One possible means to test whether inhibition is direct or indirect could be to express an activated form of Notch and determine whether its expression is or is not correlated with patches of feather loss. It will also be interesting to compare misexpression results of activated form of Notch-1 versus Notch-2 as we predict that the first may mediate the feather promoting role of Delta while the second may mediate the inhibition. It may also be possible to study the later events of feather differentiation as Chen et al. (1997) have suggested that Notch signaling may play a role in the posterior growth of each bud. Although we have not observed a consistent effect of Delta misexpression on feather polarity, it remains possible that Serrate could play a unique role in later events of feather differentiation that cannot be mimicked by Delta. It has been proposed that the feather tracts arise progressively due to the presence of a prepatterning morphogenetic wave (Sengel, 1976). This wave begins at the midline, for the dorsal feather field, and moves laterally, allowing for the specification of successive rows of feather buds. Sengel (1976) postulated that disruption of the wave would be manifested later as a disruption in the pattern of more lateral feathers. In terms of this prediction, it is possible that feather loss following Delta-1 misexpression could be due to a disturbance of the morphogenetic wave. If this were the case, it would be expected that virally-infected feathers should only be observed medial to the regions of smooth skin. In fact this is not the case. Viral misexpression is observed within the regions of feather loss in the case of large patches or, in the case of small patches, in the feathers either medial and/or lateral to the region of ectopic smooth skin. This latter result suggests that regions of the embryo infected with the Delta-1 virus have the ability to inhibit their neighbors either medially or laterally and thus this inhibition can occur prior to bud initiation. This also illustrates that the morphogenetic wave, referred to by Davidson (1983) as a wave of competence is unaltered by misexpression of Delta-1, since feathers form normally on either side of the ectopic smooth skin. Therefore, Delta-1 seems to inhibit feather bud initiation in the neighboring cells by a more direct mechanism than by disruption of a presumptive morphogenetic wave. Thus, Delta-1 may be involved in formation of the feather pattern rather than progression of the morphogenetic wave. In summary, Delta-1 seems to have a dual role in the establishment of the chick embryonic feather pattern. Early expression of Delta-1 promotes bud development in regions where it is expressed while inhibiting bud formation in neighboring non-delta expressing cells (Fig. 7). This positive signal may be mediated through Notch-1 activation while the inhibitory signal may be through Notch-2 activation. The combination of both a positive and an inhibitory signal from Delta-1 could lead to the spatially and temporally patterned feather fields. We are grateful to K. Manova and K. Witty-Blease of MSKCC Molecular Cytology Facility for assistance, to A. Neubuser for the section in situ protocol, to G. Weinmaster, C. Tabin, P. Brickell and B. Houston for probes, and J. Pizenti for scaleless eggs. Also thanks to S. Jeffrey for construction of the RCAS probe and to J. Kuhlman and S. Pizette for helpful suggestions and critical review of the manuscript. 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