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Keywords:

  • Notch1;
  • Jagged;
  • limb;
  • AER;
  • mouse conditional deletion

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The Notch family of receptors is involved in a wide variety of developmental processes, including cell fate specification, cell proliferation, and cell survival decisions during cell differentiation and tissue morphogenesis. Notch1 and Notch ligands are expressed in the developing limbs, and Notch signalling has been implicated in the formation of a variety of tissues that comprise the limb, such as the skeleton, musculature, and vasculature. Notch signalling has also been implicated in regulating overall limb size. We have used a conditional allele of Notch1 in combination with two different Cre transgenic lines to delete Notch1 function either in the limb mesenchyme or in the apical ectodermal ridge (AER) and limb ectoderm. We demonstrate that Notch signalling, involving Notch1 and Jagged2, is required to regulate the number of Fgf8-expressing cells that comprise the AER and that regulation of the levels of fibroblast growth factor signalling is important for the freeing of the digits during normal limb formation. Regulation of the extent of the AER is achieved by Notch signalling positively regulating apoptosis in the AER. We also demonstrate that Notch1 is not required for proper formation of all the derivatives of the limb mesenchyme. Developmental Dynamics 234:1006–1015, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The Notch signalling pathway is involved in a large number of developmental processes, including cell fate specification, cell proliferation, and cell survival decisions during development as well as in the adult. Disruption of Notch receptors, their ligands, and downstream signalling components of the Notch pathway have been implicated in numerous human developmental defects and adult pathological conditions (reviewed in Lai, 2004).

Notch1 belongs to a family of four transmembrane receptors, Notch1–4, that signal between cells that are in direct contact with one another. Upon binding to one of its ligands, Jagged1/2 or Delta1/3/4, the Notch receptor is cleaved through a series of proteolytic steps to release the intracellular domain of Notch (NICD), which then translocates to the nucleus and forms a complex with the CSL (CBF1/RBP-κ, suppressor of hairless, LAG-1) DNA-binding protein to act as a transcriptional activator. When CSL is not bound to NICD, it can recruit repressors, so that activation of Notch signalling can switch from active repression to activation of target genes. The CSL/NICD complex can bind to specific sequences in the promoter region of targets and activate their expression, such as the Hairy/Enhancer of Split (Hes) and Hes-related repressor proteins (Herp) genes (for review, see Lai, 2004; Schweisguth, 2004).

In Drosophila, Notch signalling creates a specialized margin of cells at the dorsoventral boundary of the wing imaginal disc. This wing margin produces molecular cues that organize wing outgrowth, such as its downstream targets, vestigial and wingless (Milan et al., 2002). In vertebrates, several components of the Notch signalling pathway are expressed in the developing limbs and have been implicated in the development of individual tissues of the limb, such as the bones, muscles, and vasculature (Williams et al., 1995; Myat et al., 1996; Vargesson et al., 1998; Delfini et al., 2000; Iso et al., 2003; Watanabe et al., 2003; Nobta et al., 2005) and in regulating the size of the limb (Vasiliauskas et al., 2003). The vertebrate structure analogous to the wing margin in the Drosophila wing imaginal disc is the apical ectodermal ridge (AER). The AER is a ridge of epithelial cells running along the anterior-to-posterior axis at the distal tip of the limb bud and controls the pattering of the proximodistal axis (Martin, 1998). Mutations in mouse Jagged2 cause the AER to expand in size, which ultimately leads to digit fusions (syndactyly) and provides evidence that Notch signalling is required for proper functioning of the AER (Sidow et al., 1997; Jiang et al., 1998).

However, because the deletion of Notch1 is lethal before limb bud stages, its role in development remains poorly understood (Swiatek et al., 1994; Huppert et al., 2000). The Cre/Lox system allows specific temporal and spatial recombination of appropriately targeted mouse alleles allowing the disruption of genes in specific tissues at distinct times so that tissue-specific gene function can be assessed (Sauer, 1998). To address the role of Notch1 in the developing limb, we have used a conditional allele of Notch1, Notch1lox/lox (Radtke et al., 1999), in combination with two distinct Cre deleter transgenic lines that enable us to separately delete Notch1 function in either the limb mesenchyme or the limb ectoderm during early stages of embryonic limb development.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Components of Notch Signalling Are Expressed in the Limbs

Previous studies have shown that Notch1 and several of its ligands are expressed in the vertebrate limbs (Williams et al., 1995; Myat et al., 1996; Jiang et al., 1998; Vargesson et al., 1998) and that a downstream component of Notch signalling, Hes1, is expressed in the limb mesenchyme and regulates the size of the limb (Vasiliauskas et al., 2003). At embryonic day (E) 10.5, Notch1 transcripts are detectable in the AER (data not shown), and by E11.5, expression is clearly observable in the AER, while lower levels of expression are apparent in the ectoderm (Fig. 1A). At E11.5, three Notch ligands, Jagged1, Jagged2, and Delta-like3, are expressed in the limbs in nonoverlapping domains. Jagged1 is expressed in the distal mesenchyme and is absent from the AER (Fig. 1B), while Jagged2 is restricted to the AER (Fig. 1C), in a similar pattern to Notch1. Delta-like3 is expressed at very low levels in the deep, medial limb mesenchyme (data not shown). We were unable to detect the expression of the other Notch receptors (Notch2–4) or the Notch ligands Delta-like1 and Delta-like4 by in situ hybridization in the developing mouse limb.

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Figure 1. Notch1 and Jagged2 are expressed in the apical ectodermal ridge (AER). A:Notch1 is expressed in the AER in the limb (arrow in the hindlimb). B,C: The Notch ligand, Jagged1 (B) is expressed in the distal mesenchyme of the limb (arrow in the hindlimb), whereas Jagged2 (C) is expressed in the AER in a similar pattern to Notch1 (arrow in the hindlimb). Left lateral views of E11.5 embryos are shown in all cases.

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Notch1 Is Not Required in the Limb Mesenchyme

To study the role of Notch1 in the limb mesenchyme, we used a conditional allele of Notch1 (Notch1lox/lox; Radtke et al., 1999) and a Cre-driver that is capable of expressing Cre recombinase throughout the developing limb mesenchyme, Prx1-Cre (Logan et al., 2002). The Prx1-Cre has been used successfully with other conditional alleles to efficiently delete genes in the mesenchyme of the developing limb (Akiyama et al., 2002; Compagni et al., 2003; Rallis et al., 2003; Selever et al., 2004). In this line, Cre activity is first detectable by E9.5 and is found throughout the limb mesenchyme of the forelimb and hindlimb by E10.5. Because Cre activity is detectable at relatively early stages, all the decendents of these cells, which will give rise to the muscle, tendon, bone/cartilage, and vasculature, will have had conditional alleles recombined.

E16.5 embryos mutant for Notch1 in the limb mesenchyme (Notch1lox/lox; Prx1-Cre) are indistinguishable from control littermates (Notch1wt/lox; Prx1-Cre; Fig. 2). As described in the introduction, roles for components of the Notch signalling pathway have been implicated in the development of bone and muscle. However, skeletal preparations of these mutant embryos demonstrate that all of the skeletal elements of the limb appear normal (Fig. 2A–H). To study the muscle patterning in the conditional deletion embryos, anti-muscle myosin antibody staining was carried out on control (Notch1wt/lox; Prx1-Cre) and mutant (Notch1lox/lox; Prx1-Cre) E14.5 embryos (Fig. 2I–L). Patterning of the limb musculature is unaltered in embryos where Notch1 has been deleted from the limb mesenchyme. The Notch signalling pathway has also been implicated in angiogenesis, where Notch1 carries out a cell-autonomous role in the endothelium to form the mature vascular network (Vargesson et al., 1998; Iso et al., 2003; Limbourg et al., 2005). To assess vasculature development in mutant limbs in which Notch1 has been deleted in all the mesenchymal cells, we performed immunohistochemistry on sectioned E12.5 limb buds. We observed no abnormalities in limb bud vasculature of these mutant embryos, as assessed using platelet endothelial cell adhesion molecule-1 (PECAM-1), a marker for endothelial vasculature (Fig. 2M,N).

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Figure 2. Deletion of Notch1 in the limb mesenchyme has no effect on limb development. Alcian blue/Alizarin red staining of the forming skeletal elements in control (Notch1wt/lox; Prx1-Cre; A,B,E,F) and mutant (Notch1lox/lox; Prx1-Cre: C,D,G,H) embryos at embryonic day (E) 16.5. A,B,E,F: Forelimb (A) and detail of the forefoot (B), hindlimb (E), and detail of the hindfoot (F) from a Notch1wt/lox; Prx1-Cre embryo. C,D,G,H: Forelimb (C) and detail of the forefoot (D), hindlimb (G), and hindfoot (H) from a Notch1lox/lox; Prx1-Cre embryo. I,J: Immunohistochemical staining of the limb muscles in forelimb (I) and hindlimb (J) from a Notch1wt/lox; Prx1-Cre embryo at E14.5. K,L: Identical staining of forelimb (K) and hindlimb (L) from a Notch1lox/lox; Prx1-Cre embryo. M,N: No difference in platelet endothelial cell adhesion molecule-1 (PECAM-1) staining, a marker of vascular endothelial cells, on comparable transverse sections of E12.5 control (M) and mutant (N) forelimbs (arrows mark assembled endothelial cells). U, ulna; R, radius; FL, forelimb; HL, hindlimb

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Newborn pups also have no evident phenotype compared with control littermates, and mutant animals develop to adulthood apparently normally (data not shown). Two secreted molecules expressed at early limb bud stages in two key signalling centers, Fgf8 in the AER (Martin, 1998) and Shh in the zone of polarizing activity (ZPA; Riddle et al., 1993), are expressed normally (data not shown). In contrast to previous data suggesting a role for Notch signalling during muscle and bone development (Vargesson et al., 1998; Delfini et al., 2000; Pereira et al., 2002; Watanabe et al., 2003; Nobta et al., 2005), these results demonstrate that Notch1 is not required for the development of the limb mesenchyme in vivo.

Notch1 Is Required in the Limb Ectoderm/AER

To delete Notch1 in the AER and ectoderm of mouse limbs, we used the conditional Notcth1lox/lox mouse in combination with a Brn4-Cre transgenic mouse line that expresses the Cre recombinase in ectoderm and AER but not the mesenchyme of the developing limb (Ahn et al., 2001). In this line, Cre-catalyzed recombination occurs within the hindlimb at relatively earlier times and more completely than in the forelimb (Ahn et al., 2001). Cre activity is detected in the hindlimb at E10, particularly robustly in pre-AER region at the distal tip of the limb. By E10.5, activity is detected throughout the AER and ventral ectoderm, with more limited activity in the dorsal ectoderm. Only very mild phenotypes were observed in forelimbs of mutant animals (data not shown). The phenotypes observed in the hindlimb were highly penetrant and reproducible. We, therefore, concentrated our analysis on hindlimbs.

E16.5 conditional knockout embryos (Notch1lox/lox; Brn4-Cre) have a “clenched foot” phenotype, with a fibular deviation of the first digit and a tibial deviation of the fifth digit of the hindlimbs (Fig. 3B,D). The three central digits of the hindfoot exhibit complete, simple syndactyly, the digits being joined along their entire length but without fusions between bones of adjacent digits (Fig. 3B,D,G). Syndactyly of the three central digits was evident as early as E14.5 (Fig. 3F).

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Figure 3. Deletion of Notch1 in the limb ectoderm leads to syndactyly. Hindlimb defects of Notch1lox/lox; Brn4-Cre embryos. A,C: Dorsal (A) and ventral (C) views of Notch1wt/lox; Brn4-Cre (control) postnatal day (P) 0 pups. B,D: Dorsal (B) and ventral (D) views of Notch1lox/lox; Brn4-Cre mutant embryos. As a result of a failure of interdigital cell death that normally frees the digits, the elements of the mutant hindfoot are brought together to form an arched footplate rather than the flattened footplate in the control. E,F: The footplate of a control (E) and mutant (F) embryonic day (E) 14.5 embryo. The failure in normal interdigital cell death is already evident and is most obvious between the three central digits. G: Alcian blue staining of the skeleton of a mutant embryo. Syndactyly between the three central digits is obvious, although there are no fusions between the phalangeal elements (arrows). Hindlimbs are shown in all cases, and the anterior digit 1 is uppermost in all panels.

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To understand the molecular mechanisms that lead to syndactyly, which is caused by a failure to remove the interdigital limb mesenchyme cells, after deletion of Notch1 in the AER, we analyzed the expression of Fgf8, a molecular marker of the AER (Fig. 4A–F). The anterior to posterior extent of the AER, which divides the dorsal and ventral ectoderm, is normal in the mutant hindlimb (Fig. 4B). In a distal view, however, an expansion along the dorsal–ventral axis of the domain of Fgf8 expressing cells is obvious (Fig. 4D), compared with control littermates (Fig. 4C). This expansion can vary along the anterior to posterior extent of the AER. Transverse sections of mutant limbs show an expansion of the AER and that this hyperplastic AER (Fig. 4F) protrudes into the mesenchyme of the limb (Fig. 4E). The hyperplastic AER phenotype is strikingly similar to that reported for mice mutant for the Notch ligand, Jagged2 (Sidow et al., 1997; Jiang et al., 1998).

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Figure 4. Deletion of Notch1 in the ectoderm leads to the formation of a hyperplastic apical ectodermal ridge (AER). Hyperplasia of the AER is evident by the expanded expression of Fgf8 in mutant embryonic day (E) 11.5 hindlimbs. A,B: Comparable dorsal views of control (Notch1wt/lox; Brn4-Cre, A) and mutant (Notch1lox/lox; Brn4-Cre, B) limbs processed by whole-mount in situ hybridization with an Fgf8 probe. The anterior to posterior extent of Fgf8 expression is unaffected. C,D: Views of the apex of the limb show the expanded domain of Fgf8-expressing cells in the mutant limb (arrow in D) when compared with the same view of a control limb (C). In sections of the limb, the extent of AER hyperplasia is evident. E,F: The AER is expanded and protrudes into the mesenchyme (red arrow in F; original magnification of inset detail, ×40) compared with the normal AER morphology in the control limb (E; original magnification of inset detail, ×40). A, anterior; P, posterior.

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Hyperplastic AERs Form in Notch1 Mutants Due to a Decrease in Programmed Cell Death

To determine whether the hyperplastic AER in the Notcth1lox/lox; Brn4-Cre hindlimbs is due to an increase in proliferation or a decrease in apoptosis, E11.5 mutant and control embryos were processed for phosphorylated histone H3 (PH3) and terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining. Relatively high levels of programmed cell death are observed in the AER of control limbs when compared with the rest of the limb ectoderm (Fig. 5A). A similar section through a mutant limb indicates there is a marked decrease in the number of cells undergoing apoptosis in the AER (Fig. 5B). When several samples were analyzed and the mean number of cells calculated, on average, we detect almost threefold more TUNEL-positive cells in the control limbs than mutant limbs (Student's t-test P value of 5.74E-06; see the Experimental Procedures section). In contrast, in sections of mutant embryos, there is no difference in the number of cells stained for PH3 (Fig. 5D), compared with control littermates (Fig. 5C; mutant mean, 0.8 PH3+ cells/section vs. control mean, 1 PH3+ cells/section and a t-test gave a P value of 0.30). This finding demonstrates that there is no change in the number of proliferating cells in the AER after deletion of Notch1. Most of the proliferating cells in the developing limb detected with PH3 are found within the underlying mesenchyme. We also detect no difference in the number of PH3-positive cells in the mesenchyme of control and mutant limbs (11.8 and 11.2 PH3+ cells/section, respectively). Taken together, these results demonstrate that Notch1 plays a role in controlling the size of the AER by regulating the extent of programmed cell death in cells of the AER.

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Figure 5. Deletion of Notch1 in the ectoderm leads to reduced programmed cell death in the apical ectodermal ridge (AER). A,B: A higher number of terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) -positive (apoptotic) cells are observed in the control (Notch1wt/lox; Brn4-Cre) AER (A) than in the mutant (Notch1lox/lox; Brn4-Cre) AER (B). C,D: Using phosphohistone-3 (PH3) as a marker of cells committed to mitosis, no difference in the number of cells undergoing cell division is observed between the AER in control (C) and mutant (D) hindlimbs. Expression of the Notch ligands Jagged1 and Jagged2 are not affected after deletion of Notch1 in the ectoderm. E,G: Control hindlimbs are shown. F,H: Mutant hindlimbs are shown. E,F: Jagged2. G,H: Jagged1. A–D: The AER is outlined on sections with a dashed line. A–D: Transverse sections of E11.5 hindlimbs. E–H: Left lateral views of E11.5 hindlimbs.

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Expression Patterns of Notch Signalling Components Are Unaffected After Deletion of Notch1 in the Ectoderm

Studies on the regulation of the Notch signalling components have shown that there are often feedback loops between the Notch receptors and their ligands that regulate and often refine their expression domains (de Celis and Bray, 1997; Panin et al., 1997; Jen et al., 1999; Ross and Kadesch, 2004). Jagged2 is expressed in cells of the AER (Fig. 5E,C), in a pattern that overlaps with that of Notch1, but is unaffected after deletion of Notch1 (Fig. 5F), indicating that Notch1 is not required to regulate expression of Jagged2 in the AER. Jagged1, which is expressed in the distal mesenchyme of the limb (Figs. 5G, 1B), is also unaffected after deletion of Notch1 (Fig. 5H).

Deletion of Notch1 in the Ectoderm Has Consequences on Gene Expression in the Limb Mesenchyme

Three bone morphogenetic proteins (BMPs), Bmp2, Bmp4, and Bmp7, expressed in the cells of the interdigital mesenchyme, are involved in controlling interdigital programmed cell death (IPCD) and normal digit formation (Buckland et al., 1998; Ganan et al., 1998). In E13.5 mutant hindlimbs (Notch1lox/lox; Brn4-Cre), Bmp2, Bmp7, and Bmp4 (Fig. 6B, 6D, 6F, respectively) are down-regulated in the interdigital limb mesenchyme. In all examples, the domains where expression is most obviously down-regulated are limited to the distal-most extremes of the interdigital mesenchyme, either directly underneath or close to the hyperplastic AER and in the interdigital space between digits 2 and 3 and digits 3 and 4. Hoxa13 is required for normal IPCD, digit outgrowth, and chondrogenesis (Fromental-Ramain et al., 1996; Stadler et al., 2001), and it carries out these functions by regulating Bmp2 and Bmp7 expression (Knosp et al., 2004). Notcth1lox/lox; Brn4-Cre mutant embryos show a decrease in the expression Hoxa13 expression at E12.5. Of interest, Hoxa13 is most obviously down-regulated in the interdigital space between digits 2 and 3 and digits 3 and 4, precisely the same location where both Bmp2 and Bmp7, and also Bmp4, are most profoundly down-regulated.

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Figure 6. Factors that positively regulate interdigital cell death are down-regulated after deletion of Notch1. A,B:Bmp2, which is expressed in the interdigital mesenchyme of control limbs (A), is down-regulated in the mutant limb (Notch1wt/lox; Brn4-Cre, B). This down-regulation is most apparent in the interdigital regions between digits 2 and 3 (white arrow) and 3 and 4. C,D: The forming digits are numbered 1–5; Bmp7, expressed in the interdigital mesenchyme (C), is down-regulated in the mutant (D). The interdigital space between digits 3 and 4 is indicated with an arrow. E,F: Similarly, the expression of Bmp4, expressed in the interdigital mesenchyme, (E), is down-regulated in the mutant (F, white arrow). F: Bmp4 is also expressed in the AER, and this expression domain is expanded in the mutant (blue arrow). G,H:Hoxa13, expressed in the interdigital space in a control limb (G), is down-regulated in the mutant limb (H) most profoundly in the interdigital space between digits 2 and 3 and 3 and 4 (denoted with an asterisk). Left lateral views of hindlimbs are shown in all cases.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Notch1 Is Not Required to Form the Limb Skeleton, Musculature, or Vasculature

The Prx1-Cre deleter line we have used is detectable by E9.5 in the limb mesenchyme and is active throughout all limb mesenchyme cells by E11.5 (Logan et al., 2002), and, therefore, would delete Notch1 function in all progenitors that ultimately give rise to the limb bone, cartilage, muscles, and vasculature. This line has been used in combination with a Sox9 conditional allele to delete this gene's function in the early condensing cartilage primordia and in such mice the bones of the limb fail to form (Akiyama et al., 2002).

Notch signalling has been implicated in bone development and both Notch1 and Notch2 are expressed in osteoblasts. Apparently conflicting data from work in cell lines has suggested a role for Notch intracellular domain (ICD) in either inhibiting differentiation of osteoblasts or promoting osteoblast differentiation. Constitutive expression of Notch1ICD has been shown to impair osteoblast differentiation (Sciaudone et al., 2003). However, it has been reported also that Notch1ICD positively regulates osteoblastic cell differentiation (Tezuka et al., 2002). Notch1 expression has been detected also in condensed mesenchymal cells that will form cartilage as well as in chondrocytes at the developing articulations in mouse limb buds. It has been detected also in chondrogenic cell lines. In these cell lines, Notch signalling has an inhibitory effect on both differentiation and proliferation (Hayes et al., 2003; Watanabe et al., 2003).

Our results using the Prx1-Cre line in combination with a conditional allele of Notch1 demonstrate that Notch1 is not required for bone and cartilage formation. However, experiments using the Notch1ICD may indicate that another Notch receptor, for example Notch2, may have a role in bone formation. After its deletion in the mesenchyme, Notch2, or another Notch receptor, may compensate for the absence of Notch1. However, in our expression analyses, we were unable to detect expression of the Notch receptors Notch2–4 by in situ hybridization in the limb mesenchyme between E10.5 and E15.5.

Evidence for the involvement of Notch in muscle development has been provided from work in mouse cell lines, where activated Notch signalling inhibits muscle differentiation (Kopan et al., 1994; Shawber et al., 1996; Kuroda et al., 1999; Nofziger et al., 1999). In agreement, activation of the Notch pathway in the chick limb by misexpression of the Notch ligand Delta1 also suppresses myogenesis in vivo (Delfini et al., 2000). Recent studies have demonstrated that the Notch pathway is also involved in vascular development, and in particular, that Notch1 has an essential cell-autonomous role in the endothelium to form the mature vascular network during angiogenesis (Vargesson et al., 1998; Iso et al., 2003; Limbourg et al., 2005). The lack of any effect on the limb musculature or vasculature in Notch1lox/lox; Prx1-Cre mice indicates that Notch1 function is dispensable for their formation. Again, functional redundancy with other Notch family members could explain the lack of phenotypes in these tissues in Notch1 mutant mice.

Jagged2 Signalling Through Notch1 Regulates the Size of the AER

Notch1 and Jagged2 are coexpressed within the Fgf8-expressing cells that constitute the molecular and morphological AER (Fig. 1). Previous results describing the disruption of Jagged2 (Sidow et al., 1997; Jiang et al., 1998) and our own results following the conditional deletion of Notch1 in the limb ectoderm produce almost identical phenotypes; expansion of the Fgf8-expressing cells that define the AER, resulting in syndactyly of the digits. The only differing feature in the phenotypes of the two mutants is that osseus fusions of the digits are occasionally observed in the Jagged2 mutants (Sidow et al., 1997; Jiang et al., 1998), whereas these are never seen in the Notch1 mutants. Together these results suggest that Jagged2 is signalling through Notch1 to control the size of the AER. Furthermore, this signalling is occurring in a population of cells that express both ligand and receptor components of the signalling circuit.

High rates of cell death, as evidenced by TUNEL staining, have been described in the AER (Sun et al., 2002). Our results implicate Notch1 in the regulation of apoptosis of the cells in the AER, because following disruption of Notch1 signalling, the number of cells undergoing apoptosis is reduced and, as a result, a hyperplastic AER forms. Notch signalling has been implicated frequently in protecting cells from undergoing apoptosis (for review, see Miele and Osborne, 1999). However, few examples have been reported in which Notch1 positively activates apoptosis. Overexpression of NICD selectively induces apoptosis in neural progenitors (Yang et al., 2004), B cells (Morimura et al., 2000), and erythroid cells (Ishiko et al., 2005). Similarly, in Drosophila, Notch signalling can trigger apoptosis in cells that are specified incorrectly in either dorsal or ventral compartment of the wing imaginal disc (Milan et al., 2002), although activation of the Notch pathway itself is not sufficient to kill cells. Our results indicate that Notch signalling is needed to control cell number in the AER. These results do not indicate that Notch signalling itself is sufficient to cause cell apoptosis in the AER leaving open the possibility that other factors may be required, in conjunction with Notch, for the clearing of cells by means of this apoptotic mechanism.

Regulation of Fgf8 Signalling Levels From the AER Is Critical for Normal Limb Formation

Our results highlight the need to control AER size, because syndactyly results if this process fails. As a result of less apoptosis in the AER, there are increased levels of signalling from the hyperplastic ridge and this increase leads to down-regulation of Hoxa13 in the interdigits. This down-regulation in turn leads to lower expression, or lack of expression, of BMPs in cells of the interdigital mesenchyme. Lower levels of BMPs, which normally positively regulate interdigital apoptosis, lead to decreased programmed cell death in this region and subsequent syndactyly.

Application of exogenous sources of fibroblast growth factor (FGF) or BMPs, as well as inhibitors of these signalling pathways to the interdigit of the chick limb, have indicated that FGFs and BMPs control interdigital apoptosis. These observations suggest that both signalling pathways have pro-apoptotic effects (Ganan et al., 1998; Montero et al., 2001). However, it has also been reported that administration of FGFs in the chick limb can antagonize the BMP-induced apoptosis in the limb mesenchyme, resulting in the formation of soft tissue syndactyly (Ganan et al., 1996; Macias et al., 1996; Buckland et al., 1998). These results suggest that, in our mutant hindlimbs, increased FGF signalling from the hyperplastic ridge decreases the level of Hoxa13 expression in the mesenchyme. Consequently, the level of Bmp expression is lowered, leading to a reduction in interdigit-programmed cell death and syndactyly. In the chick, levels of Hoxa13 expression decrease when the AER is removed, which can be restored with an FGF bead. However, when FGF4 is applied to a limb bud, it is unable to activate the expression of Hoxa13 early or expand its domain of expression (Vargesson et al., 2001). Together with our results, these findings suggest there is fine balance of FGF signalling required for the correct expression of Hoxa13. Significantly, we did not observe any difference in the timing of Fgf8 expression in the AER, and expression was down-regulated in the regressing AER at the appropriate time during development (data not shown). This finding indicates that the defects observed result from inappropriate levels of FGF signalling rather than FGF signalling being maintained over an extended period of limb development. Of interest, the phenotypes we observe after disruption of Notch1 signalling in the AER are distinct from those produced when BMP signalling from the ridge is impaired by expressing the BMP antagonist, Noggin, in cells of the AER (Wang et al., 2004) or by conditional deletion of a BMP receptor, BMPR-1A, in the AER (Ahn et al., 2001). Deletion of BMPR-1A early leads to a failure of AER formation and dorsal transformation of ventral limb structures (Ahn et al., 2001). Disruption of BMP signalling from the ridge by the BMP antagonist Noggin leads to severely malformed limbs that have syndactyly similar to the Notch1 mutants but also postaxial polydactyly and dorsal transformation of ventral structures. The domain of Fgf8-expressing cells in the AER in these mutants is expanded in a similar way to that seen in the Notch1 mutant limbs. However, in addition to expansion of the domain of Fgf8, expression of Fgf8 also persists longer in the mutant AER than normal. This finding suggests that the extended period of Fgf8 expression caused by Noggin expression in the AER, rather than perturbation of BMP signalling directly, leads to the postaxial polydactyly. Furthermore, prolonged FGF signalling in the overlying apical ridge produces additional phalanges (Sanz-Ezquerro and Tickle, 2003), which is distinct from the phenotype in the Notch1lox/lox; Brn4-Cre mutant in which the timing of FGF expression is normal. Together, these findings suggest that the defects we observe in our mutant hindlimbs are due to an increase in the level of FGF signalling from the AER rather than an extension of the period of FGF signalling. Our results demonstrate that Notch signalling is required to maintain a delicate balance in the levels of FGF signalling from the AER and that this balance is critical for the correct formation of the interdigital gaps that free the digits of the limbs.

In Drosophila, Notch signalling between the dorsal and ventral compartments of the developing wing specify the position of the wing margin, a line of cells that play a role analogous to that of the AER. Failure of Notch signalling in the wing margin leads to the formation of notches in the wing blade from which the mutant derives its name. We do not see Notch1 playing an analogous role to that of Notch in the Drosophila wing margin because notches do not form in the AER after deletion of the gene. Our results demonstrate that Notch1 signalling is not required to establish the AER but plays an important role in maintaining the correct extent of Fgf8-expressing cells in the AER, and it does so by regulating apoptosis of these cells.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Mouse Embryos

Mouse embryos were staged according to Kaufman (2001). Noon on the day a vaginal plug was observed was taken to be E0.5 days of development. Mice with the conditional Notch1 allele Notch1lox/lox (Radtke et al., 1999) were crossed with two Cre deleter lines: The limb mesenchyme-restricted Prx1-Cre deleter line (Logan et al., 2002) and the limb ectoderm-restricted Brn4-Cre line (Ahn et al., 2001). To produce mice null for Notch1, mice homozygous for the Notch1 allele (Notch1lox/lox) were crossed with mice heterozygous for the Notch1 allele and Cre transgene (Notch1lox/wt; Prx1-Cre, or Brn4-Cre).

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridizations were carried out essentially as previously described (Riddle et al., 1993). Antisense RNA probes were obtained as follows: mFgf8 (Crossley and Martin, 1995); mNotch1 is a 1.2-kb fragment digested with EcoRI and transcribed with T7 RNA polymerase; mJagged2 is full-length sequence digested with BamHI, transcribed with T7; mJagged1 (Mitsiadis et al., 1997); mHes1 (Sasai et al., 1992); mBmp2 (Lyons et al., 1989); mBmp4 (Jones et al., 1991); mBmp7 (Dudley and Robertson, 1997); mHoxa13 (Ma et al., 1998).

Histology, TUNEL Analysis, and Immunofluorescence Assays

The cartilage and bone elements of newborn mouse pups were stained with Alcian blue and Alizarin red, respectively, essentially as described in (Hogan, 1994). E14.5 embryos were fixed overnight in 4% paraformaldehyde, washed in PBT, and dehydrated through a methanol series. After removing the skin, the muscle pattern was analyzed using the monoclonal My32 antibody (Sigma) essentially as previously described (Kardon, 1998). Wax microtome (Leica) sections (10 μm) were made of E11.5 mouse embryos that had been processed previously by whole-mount in situ hybridization with Fgf8 probe. E12.5 mouse embryos were fixed for 1 hr in 4% paraformaldehyde at room temperature, washed in phosphate buffered saline (PBS), left in 30% sucrose overnight, and then embedded in OCT (BDH, Merck). Transverse sections (12 μm) were processed for PECAM-1 (Pharmigen; 1:1,000) and a horseradish peroxidase–conjugated anti-rat secondary antibody (Calbiochem), essentially as previously described (Zimmermann et al., 2001).

Programmed cell death was assayed by TUNEL (Q-BIOgene). E11.5 mouse embryos were fixed overnight in 4% paraformaldehyde, washed in PBS, left in 30% sucrose, and then embedded in OCT. Transverse sections (12 μm) were assayed by TUNEL according to the manufacturer's protocol. To detect cells in mitosis, a rabbit anti-phosphorylated histone H3 primary antibody (Upstate Biotechnology) and Cy3-conjugated goat anti-rabbit IgG secondary antibody (Jackson Lab) were used following the protocol described previously (Yamada et al., 1993).

The average number of cells positive for TUNEL staining was calculated over an equal area in 10 comparable sections of control and mutant limbs. The mean number of TUNEL-positive cells found per control section was 7.18, whereas in mutants, it was 2.7. A Student's t-test on the number of TUNEL-positive cells on sections of control and mutant limbs gave a P value of 5.74E-06, demonstrating there is a significant difference in the number of apoptotic cells. A similar analysis was done on six comparable sections of control and mutant limbs stained with PH3 antibody. The mean number of positive cells in the AER per control section was 1, whereas the mean number of positives cells in mutant AERs was 0.83. In five comparable sections of control and mutant limbs stained with PH3, the mean number of positive cells in the mesenchyme was 11.8 and 11.2, respectively. A t-test on the PH3-positive cells in the AER of control and mutant limbs gave a P value of 0.30, whereas a t-test on the PH3-positive cells in mesenchyme gave a P value of 0.37, demonstrating no significant difference in the number of cells undergoing mitosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

F.R. and M.P.O.L. are both members of EMBO Young Investigator Programme (YIP). This work was initiated after interactions that arose out of an EMBO YIP meeting. We thank Raphael Kopan for communicating unpublished results and members of the lab for their critical input. We thank B. Crenshaw for supplying the Brn4-Cre transgenic line. We are indebted to the staff of the Biological Services and Procedural Services sections, NIMR, for assistance with the animal work.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES