Complement in glomerular injury

In recent years, research into the role of complement in the immunopathogenesis of renal disease has broadened our understanding of the fragile balance between the protective and harmful functions of the complement system. Interventions into the complement system in various models of immune-mediated renal disease have resulted in both favourable and unfavourable effects and will allow us to precisely define the level of the complement cascade at which a therapeutic intervention will result in an optimal effect. The discovery of mutations of complement regulatory molecules has established a role of complement in the haemolytic uremic syndrome and membranoproliferative glomerulonephritis, and genotyping for mutations of the complement system are already leaving the research laboratory and have entered clinical practice. These clinical discoveries have resulted in the creation of relevant animal models which may provide crucial information for the development of highly specific therapeutic agents. Research into the role of complement in proteinuria has helped to understand pathways of inflammation which ultimately lead to renal failure irrespective of the underlying renal disease and is of major importance for the majority of renal patients. Complement science is a highly exciting area of translational research and hopefully will result in meaningful therapeutic advances in the near future.
Introduction
The complement system involves approximately 30 plasma- and membrane-bound proteins (reviewed in [1, 2]). These proteins play an important role in anti-microbial defence and the clearance of immune complexes and apoptotic and necrotic cells. The role of complement is not restricted to the innate immune system but includes important functions in the regulation of the adaptive immune response. Although complement undoubtedly contributes to tissue damage in numerous forms of glomerulonephritis, we will also see that complement protects against immune-mediated tissue damage in a number of settings.
In the following, we will first give an introduction to the pathways of complement activation and their regulation. This will be followed by a review of glomerular renal diseases in which complement plays a prominent role. The role of complement in non-glomerular renal disease entities such as transplantation and ischaemia/reperfusion damage falls outside the scope of this study and is reviewed in references [3] and [4].
Overview of the complement pathways
The complement system consists of three different pathways that all converge in the activation of the central complement molecule C3 (Fig. 1). Sufficient activation of C3 will then lead to the formation of the membrane attack complex.
Fig. 1Schematic overview of the three pathways of complement activation
The first component in the activation of the classical pathway is C1. Binding of at least two bindings sites of C1q to antigen-bound IgG or IgM, acute phase proteins such as CRP or dead cells leads to conformational changes that result in the activation of the associated serine proteases C1r and C1s. Activated C1s cleaves C4 into C4a and C4b. C4b then covalently binds to nearby structures. The bound C4b then binds C2 whereupon the smaller C2b fragment is cleaved off, resulting in the formation of the C4bC2a complex, which is the classical pathway C3 convertase.
The lectin pathway is related to the classical pathway and uses the same C3 convertase, C4bC2a. The initiation molecules of the lectin pathway, mannose-binding lectin (MBL) and the ficolins, recognise carbohydrate ligands present on a wide range of microorganisms in a pattern-like fashion. The interaction of MBL with its ligand leads to the activation of the MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3). MASP-2 then cleaves C4 and subsequently C2 leading to the formation of the C3 convertase which is identical to the classical route C3 convertase, C4bC2a.
MBL consists of up to six trimeric subunits which are arranged in a structure similar to C1q. Frequently occurring single nucleotide polymorphisms within exon 1 of the MBL-2 gene and polymorphisms of the promoter region explain the up to 1,000-fold inter-individual variation in MBL plasma levels.
The activation of the alternative route of complement depends on spontaneous activation of the C3 molecule by hydrolysis of the internal thioester bond of C3. This results in the formation of the C3b-like molecule C3(H2O). Hydrolysed C3 then binds to factor B. This interaction renders factor B susceptible to cleavage by factor D resulting in the release of the Ba fragment and the formation of the C3 convertase C3(H2O)Bb. This initial convertase constantly cleaves C3 at a low rate generating C3b. This constant low rate generation of C3b is referred to as the “tick over” of the alternative pathway. The generated C3b can interact with factor B to form the more active alternative pathway C3 convertase C3bBb.
The majority of the generated C3b is rapidly inactivated by circulating factor I together with its co-factors, factor H (fH) and membrane co-factor protein (MCP). However, if C3b binds to an activator surface (e.g. a bacterial wall or damaged tissue), the molecule is protected against inactivation and further amplification of the alternative route will occur. Properdin is an important positive regulator of the alternative route. Binding of properdin leads to stabilisation of the labile C3 convertase and promotes the assembly of a complement-activating lattice by further binding factor B and C3b molecules [5–7].
The early activation steps of the classical, lectin and alternative route of complement activation converge in a common terminal pathway. The addition of a further C3b molecule to the C3 convertase complex leads to the formation of C3bBbC3b in the case of the alternative pathway and to the formation of C4bC2aC3b in the case of both the classical and lectin pathways. These C5 convertases then initiate the assembly of the membrane attack complex by cleavage of C5 to C5a and C5b. C5a can then function as a potent anaphylotoxin. The newly formed C5b forms a tri-molecular complex by binding C6 and C7. After inserting into a cell membrane, this complex binds C8 and multiple C9 molecules. This results in the completion of the pore-forming membrane attack complex (C5b-9). This complex can lead to cell lysis and, in the absence of complete lysis, to cell activation.
Regulation of complement activation
The complement system consists of numerous regulatory molecules that protect the host from uncontrolled tissue destruction and activation by the complement system. Recently, defective complement regulation has been shown to play an important role in the pathogenesis of some forms of the haemolytic uremic syndrome (HUS) and membranoproliferative glomerulonephritis (MPGN). The role of complement in these diseases will be discussed in more detail below.
C1-inhibitor is a powerful inhibitor of the classical pathway of complement activation. It binds to activated C1r and C1s and causes dissociation of these inactivated enzymes from C1q. Recent data shows that C1 inhibitor also inhibits the activation of the lectin pathway by inactivation of MASP-2 and, at higher concentrations, fluid phase activation of the alternative pathway [8].
Factor I is a circulating serine protease that proteolytically degrades C3b and C4b in the presence of the co-factors fH and C4-binding protein (C4bp). Next to its function as a co-factor, fH also inhibits activation of the alternative pathway by binding to C3b and displacing Bb from the C3 convertase complex. Similarly, C4bp regulates activation of the classical and lectin pathway by displacing C2a from C4b. Both fH and C4bp promote the degradation of the C3 and C5 convertases of the respective pathways.
Cell-membrane-bound inhibitors of complement activation also contribute to the defence against inappropriate tissue damage by homologous complement. Decay-accelerating factor (CD55) exerts its effect early in the complement cascade by inhibiting the activation of C3 by preventing the formation and accelerating the decay of both the alternative and classical pathway C3 and C5 convertases. Membrane co-factor protein (MCP, CD46) serves as a co-factor for the cleavage of C3b and C4b by factor I. CD59 interacts with the final section of the complement activation pathway by inhibiting the formation of C5b-9.
Complement receptor 1 (CD35, CR1) also functions as a complement regulator by accelerating the decay of the C3 convertases. A functionally intact soluble form of CR1 can be detected in plasma [9]. Recently, a new complement receptor, the human complement receptor of the immunoglobulin superfamily (CRIg), has been described [10]. CRIg is present on macrophages in both humans and mice and plays a role in pathogen clearance. It binds to C3b and selectively inhibits the C3 and C5 convertases of the alternative pathway. A recombinant soluble form of CRIg suppressed inflammation in two murine models of arthritis [11].
Immune-complex-mediated glomerulonephritis
Immune complex glomerulonephritis is a good example for the dual role of the complement system. Immune complexes can either be deposited in the glomerulus by passive deposition from the circulation or by in situ formation via binding of antibody to local antigens. Alternatively, local formation of immune complexes may occur when a circulation antigen is recognised by antibodies after deposition in the glomerulus (planted antigen). Subepithelial complement deposition as found in membranous nephropathy leads to a non-inflammatory complement-mediated damage because the anaphylotoxins produced during the local activation do not reach circulating leucocytes. Subendothelial deposition of complement factors is associated with a brisk inflammatory response because the produced anaphylotoxins easily come into contact with circulating cells. Subendothelial immune complex deposition is typical of proliferative lupus nephritis.
Various studies have underscored the role of complement in immune-complex-mediated glomerulonephritis. Complement depletion by treating rats with aggregated human IgG resulted in a marked decrease of neutrophil influx and renal damage in a nephrotoxic serum model of acute glomerulonephritis [12]. Later studies demonstrating a beneficial effect of complement depletion in the non-inflammatory Heymann nephritis model of membranous nephropathy showed that complement-mediated damage is not dependent on the influx of inflammatory cells [13].
The contribution of terminal pathway of complement-mediated injury was established in various glomerulonephritis models in C6-depleted or C6-deficient rats. Renal damage is ameliorated in both the anti-Thy-1 and the passive Heymann nephritis models in the absence of C6 [14, 15]. As a follow-up to these findings, soluble CR1 was successfully used to treat the disease in both models [16]. A recent paper pointed towards an important role of the alternative pathway of complement activation in mouse models of type I and type II cryoglobulinemia [17]. The glomerular influx of neutrophils was significantly less in mice deficient for C3, factor B and C5, whereas C1q deficiency had no protective effect, suggesting involvement of the alternative pathway or lectin pathway.
It is interesting to note that many of the complement-deficient models of renal disease show spontaneous or worsened renal disease. This is compatible with the observation that the complement system plays an important role in the clearance of immune complexes from the circulation and in the solubilisation of deposited immune complexes. Immune complexes are rapidly opsonised with C4b and C3b. These complement components mediate the binding of the immune complexes to CR1 on erythrocytes. The complexes are then stripped off the erythrocytes when they pass through the liver or spleen. Thus, CR1-mediated clearance plays an important role in the handling of immune complexes and in keeping soluble immune complexes away from the endothelial surface thereby preventing vascular injury.
Numerous animal models of immune-complex-mediated renal disease demonstrate a protective role of complement. C3 deficiency did not protect mice from the formation of immune complexes and proteinuria in a planted antigen model of immune complex nephritis [18]. This can be explained by the role of Fc-receptor-mediated activation of either resident or infiltrating cells at the site of injury. In the complete absence of complement activation, deposition of immune complexes can still lead to renal damage via Fc-receptor-mediated cell activation [19, 20].
An interesting recent study demonstrated that factor-D-deficient mice spontaneously develop immune complex glomerulonephritis with mesangial deposition of IgM and C3 [21]. Apparently, amplification of C3 activation is necessary for the processing or dissociation of IgM-containing immune complexes in the kidney.
The important role of the complement system in immune complex clearance is underscored by the finding that humans with complement deficiency are prone to immune-complex-mediated disease. Systemic lupus erythematosus (SLE) is a highly relevant example for this dual role of the complement system as will be discussed in the next section.
Role of complement in lupus nephritis
The degree of peripheral complement consumption and the heavy glomerular deposition of complement in SLE nephritis point towards an important role of complement in lupus nephritis. Various approaches at inhibiting complement activation have been successful in treating experimental lupus nephritis. Wang et al. prevented the development of glomerulonephritis with an anti-C5 antibody in lupus-prone NZB/W mice. Both treatments with the soluble rodent complement inhibitor rCrry and transgenic expression of this CR1-like molecule limited renal damage in the MLR/lpr mouse model of SLE [22, 23].
However, in humans, deficiencies of the early complement proteins C1q, C2 and C4 are associated with an increased risk of developing SLE [24]. Similarly, mice with C1q or C4 deficiency develop an autoimmune disease which resembles SLE [25–27].
Next to the loss of clearance of immune complexes, complement deficiency may also lead to auto-immune phenomena due to defective clearance of apoptotic cells. C1q binds to apoptotic cells [28] and C1q-deficient mice are impaired in their capacity to clear these cells [29]. Defective clearance of this rich source of auto-antigens may contribute to the emergence of auto-immunity in patients with SLE.
Taken together, it seems that, in lupus, the early components of the classical pathway of complement activation are beneficial due to their role in the clearance of immune complexes and apoptotic cells. Probably, the damage caused by Fc-receptor-mediated mechanisms in the presence of an increased deposition of immune complexes overrides the benefit of complement inhibition in these models. However, the inhibition of complement activation downstream of C3 may be a promising therapeutic approach.
Lupus nephritis is strongly associated with the presence of anti-C1q antibodies. These antibodies are present in 30–40% of SLE patients [30] and correlate with active lupus nephritis with a sensitivity of 87 to 97% and a specificity of 92% [31, 32]. Antibodies against MBL are also present in SLE, but no association with disease activity was detected [33]. The strong association of anti-C1q antibodies with active lupus nephritis suggests a pathogenic role of these antibodies. To study this question, our group has generated homologous mouse anti-mouse C1q antibodies. The administration of these antibodies to healthy mice resulted in deposition of C1q in the glomeruli together with an influx of granulocytes. However, this was not accompanied by a reduction of renal function or significant proteinuria [34]. When mice were pre-treated with a sub-nephritogenic dose of rabbit anti-C1q antibodies, the subsequent administration of mouse anti-C1q antibodies resulted in an increased deposition of immunoglobulin. The use of mice deficient for C3, C4 or for all three Fc gamma receptors showed that both complement and Fc-gamma-receptor-mediated damage is involved in this model.
IgA nephropathy
Mesangial IgA deposition is the hallmark of IgA nephropathy. Early studies on complement deposition in IgA nephropathy reported co-deposition of C3 and properdin [35]. Because C1q and C4 are usually not detected in kidneys with IgA nephropathy, this complement deposition was thought to be caused by alternative pathway activation. Mesangial deposition of C5b-9 is also present and underscores the possible pathogenic importance of complement activation in this setting [36]. More recent studies suggest a role of the lectin pathway of complement activation in IgA nephropathy. Co-deposition of IgA and MBL has been described by several authors in both IgA nephropathy [37–39] and the related entity Henoch Schönlein purpura [40]. Hisano et al. found a relation between the presence of MBL deposition and the severity of the disease [41].
The discovery of MBL-binding properties of IgA has provided a mechanistic explanation for the link between IgA and MBL deposition [42]. MBL binds to polymeric IgA in a calcium-dependent fashion via its lectin domain, suggesting an interaction with carbohydrates exposed on the IgA molecule. The binding of MBL to IgA results in complement deposition and offers an explanation for the complement deposition found in IgA nephropathy.
A recent publication underscored the potential clinical importance of MBL deposition in IgA nephropathy [43]. About 75% of the 60 biopsies in this study were negative for MBL and C4d, indicating that the C3 and C5b-9 deposition in these patients is most probably caused by the alternative pathway. The 25% of biopsies in which glomerular MBL deposition was found were positive for C4d in the absence of Clq, indicating complement activation via the lectin pathway. MBL deposition in the glomerulus was associated with both clinical and histological markers of more severe renal damage including more severe proteinuria, renal failure, extracapillary proliferation, glomerular sclerosis and interstitial fibrosis. The MASP-associated lectin, L-ficolin, was also present in these biopsies and may contribute to the activation of the lectin pathway next to MBL.
Membranoproliferative glomerulonephritis
Complement research has greatly contributed to the understanding of the pathophysiology of type II membranoproliferative glomerulonephritis (dense deposit disease) [44]. MPGN type II is characterised by deposits within the glomerular basement membrane together with staining for C3 along the glomerular basement membrane. In contrast to MPGN type I, deposition of immunoglobulins is usually not detected. The glomerular complement deposition is usually accompanied by decreased circulating C3 levels and alternative route activity. More than 80% of patients with MPGN type II are positive for serum C3-nephritic factor (C3NeF) [45].
C3NeF is an antibody directed against the alternative pathway C3 convertase. The binding of C3NeF to C3bBb prolongs the half-life of the C3 convertase by slowing down the dissociation of Bb form C3b [46]. One of the mechanisms by which C3NeF increases the half-life of C3bBb is by inhibition of fH-mediated inactivation of the convertase [47]. Consistent with complement activation by stabilisation of alternative pathway convertase activity, serum complement profiles of patients with MPGN II show predominant depletion of C3 with no consumption of C1q and C4. The glomeruli of affected kidneys show marked deposition of C3 along the glomerular capillary walls without deposition of C1q, C4 or immunoglobulins. MPGN II is also associated with acquired partial lipodystrophy. This entity is also associated with the presence of C3NeF and marked C3 depletion. In vitro data shows that C3NeF is capable of inducing alternative-pathway-mediated damage on adipocytes [48]
The role of a deregulated alternative pathway in MPGN type II was highlighted by the discovery of FH mutations in both humans [49] and pigs with MPGN type II. Mice with a targeted deletion of FH have significantly reduced levels of C3 and consistently develop MPGN with deposition of C3 in the capillary walls [50]. If fH-deficient mice are also deficient for factor B, they cannot activate the alternative pathway and no renal disease develops.
A chronic serum sickness model of immune complex disease demonstrated increased deposition if IgG immune complexes with increased C3 deposition in fH-deficient mice compared to wild-type mice. The fH-deficient mice developed diffuse proliferative glomerulonephritis, while the wild-type mice were protected against glomerular pathology. These findings indicate a role of fH in processing immune complexes and protecting the glomerulus against immune-complex-mediated disease.
Patients with MPGN type II develop ocular lesions which are similar to the drusen that are found in patients with age-related macular degeneration (AMD). The finding of a close association of AMD with factor H mutations suggests that complement is also involved in the pathogenesis of this visually disabling disease [51–54].
A recent study into the role of the terminal complement pathway in MPGN has pointed towards the potential of therapeutic inhibition of the complement system in MPGN [55]. Mice deficient for both fH and C5 developed less severe glomerulonephritis with better renal function, lower mortality and reduced glomerular cellularity in comparison to fH-deficient mice with normal C5. It is interesting to note that C5-deficient mice were not protected against proteinuria, suggesting that the glomerular C3 activation is sufficient to disrupt the glomerular permselectivity. The induction of heterologous nephrotoxic serum nephritis in fH-deficient mice resulted in markedly increased renal damage when compared to wild-type mice. The renal damage was clearly reduced in fH-deficient mice lacking C5 whereas C6-deficient mice were not protected. These findings demonstrated that the complement-mediated renal damage depended on the formation of the anaphylotoxin C5a but not on the formation of C5b-9. The role of C5 was supported by the reduction of proteinuria and glomerular neutrophil accumulation after treatment with an antibody against C5.
These observations suggest that anti-C5 treatment could serve as a treatment option in MPGN type II.
Complement and the atypical haemolytic uremic syndrome
The haemolytic uremic syndrome is characterised by microangiopathic haemolytic anemia, consumptive thrombocytopaenia and the formation of microvascular thrombi. The vascular damage is particularly severe in the kidney and can lead to acute renal failure. Most cases of HUS are associated with diarrhoea and are caused by the verotoxin-producing Escherichia coli strain O157:H7. The less common form that is not associated with diarrhoea is referred to as atypical HUS (aHUS). Especially in children, the outcome and prognosis of diarrhoea-associated HUS is good, whereas atypical HUS is associated with substantial chronic renal failure and mortality.
The familial occurrence of aHUS [56] and the occasional finding of complement consumption [57] and deposition [58] in familial aHUS suggested a hereditary defect in alternative pathway activation or control. Importantly low levels of C3 persisted in patients with familial HUS after remission of the disease and low C3 levels were also detected in unaffected relatives. The discovery of fH mutations in families with aHUS confirmed this hypothesis [59, 60]. Until now, more than more than 100 fH mutations have been described [61]. These can be searched in an interactive HUS database (http://www.FH-HUS.org). The fH mutation frequency is 38% in familial forms of aHUS and 20% in sporadic forms [62]. In contrast to the fH mutations in patients with MPGN type II, patients with aHUS are usually heterozygous for the fH mutation. The penetrance is around 60%. Patients usually have normal levels of circulating fH protein, but reduced C3 levels are found in about 50% of the aHUS cases with a fH mutation [62]. Also in contrast with the mutations associated with MPGN, the fH mutations in patients with aHUS are located in the C-terminal region which is important for binding to cellular surfaces [63–65] via an interaction with C3b deposited in the surface of these cells [66]. Because mutated fH cannot bind to surface-bound C3b, circulating factor B can associate with the C3b and C3 convertase is formed, leading to unopposed complement activation on the endothelium.
The creation of a transgenic mouse that lacks the exons encoding for the C-terminal region of fH that is responsible for the binding to cellular surfaces has resulted in an aHUS model that is highly similar to the human disease [67]. These mice have a preserved capacity to regulate fluid phase complement activation and did not develop glomerulonephritis. The absence of systemic complement depletion in the presence of defective endothelial protection against complement attack led to a typical picture of HUS including the formation of glomerular microthrombi, fragmentocytes in peripheral blood and thrombocytopenia.
Next to fH mutations, other mutations in complement regulatory proteins have been discovered in patients with aHUS. MCP mutations are found in approximately 14% of the patients with aHUS [62] and until now 43 mutations have been reported [61]. The course of the disease is milder in patients with MCP mutations, and plasma therapy does not seem to contribute to the outcome.
Factor I mutations are quite rare and are found in about 4.5% of the patients with aHUS. More recently, two factor B mutations have been discovered [68]. One of these mutations increases the affinity of factor B for C3b, while the other increases the half-life of C3bBb. Both mutations result in an increased activity of the alternative pathway.
Taken together, the clinical and experimental findings clearly point towards an important role of complement regulation in the pathogenesis of aHUS. However, until now, mutations of complement-regulatory proteins are only found in about 50% of the affected patients and family members of affected patients can share the mutations without manifesting aHUS. It seems that both additional predisposing factors and triggering circumstances, e.g. infections, are necessary to initiate the full-blown microangiopathy of aHUS.
Nevertheless, it is clinically useful to screen patients with aHUS for the known mutations because the findings may influence the prognosis and therapeutic decisions. Although evidence is lacking, patients with fH deficiency are usually treated with plasma therapy [69]. On the other hand, MCP is a membrane-bound protein and there is no rationale for plasma substitution in patients with MCP mutations and aHUS. As mentioned above, retrospective data do not indicate that plasma therapy results in improved outcomes in these patients [62]. However, patients with MCP mutations do well after kidney transplantation [70] as functional MCP is present on the endothelium of the transplanted kidney whereas patients with fH mutations have a very high rate of disease recurrence and graft loss after kidney transplantation [71]. Preemptive plasma therapy may be an option in these patients, but data are lacking to support this approach. Others have attempted combined liver and kidney transplantation in children with fH-associated HUS, but liver transplantation is associated with greatly increased risks when compared with kidney transplantation alone, and this procedure has not been uniformly successful until now [72–74].
Complement and progressive renal damage
Independent of the underlying renal disease, proteinuria is associated with tubulointerstitial fibrosis and a progressive loss of renal function [75–77]. A number of mechanisms by which proteinuria may cause renal function loss have been suggested. These include oxidative damage induced by transferrin, lysosomal rupture caused by overload of the resorptive capacity for urinary proteins and pro-inflammatory effects of albumin-bound free fatty acids [78–81].
For many years, complement deposition along the brush border has been noted in proteinuric renal disease [82]. C5b-9 is found in urine from patients with various proteinuric renal diseases including diabetic nephropathy [83]. The C5b-9 in the urine is very probably generated within the tubulular lumen due to an intrinsic complement-activating property of the tubular cells [84, 85]. The exact mechanism of tubular complement activation is not understood, but tubular ammonia production [86] and a low expression of complement regulatory proteins on the apical cell surface [87] are thought to contribute.
The insertion of sublytic amounts of C5b-9 in the cell membrane of tubular cells leads to the production of pro-inflammatory cytokines [88, 89] and collagen. In vivo evidence for the role of complement in proteinuria-mediated renal damage has been derived from studies in C6-deficient rats. Rats with an inherited C6 deficiency were protected against tubulointerstitial damage in both the remnant kidney model [90] and the puromycin model of proteinuric renal disease [91]. A therapeutic intervention with either the murine complement inhibitor Crry or CD59 targeted to the renal tubulus resulted in improved renal function and less interstitial damage when compared with untreated animals [92].
Conclusions
Increasing knowledge about the complement system has taught us about both the protective and harmful roles of complement in renal disease. In the course of this review, it has repeatedly become clear that complement inhibition early on in both the classical and alternative pathways is associated with the risk of increased deposition of immune complexes and the resulting damage may outweigh the benefit. On the other hand, it seems that complement inhibition distal of the formation of the C3 convertases is safe and offers more promising therapeutic options for renal diseases for which no satisfying treatment has been established until now.
Independently of these promising therapeutic prospects, complement has become an invaluable tool in the diagnosis and monitoring of renal disease and results of complement studies have a strong impact on day-to-day decision making in the care of our patients with renal disease.

complement
glomerulonephritis
immune complexes
hemolytic uremic syndrome


Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.
Introduction
Most plants are not simply homogeneous resources to herbivorous insects but produce discrete organs with different functions. In addition to the vegetative leaf tissues, plants produce reproductive organs (flowers, seeds) that may exhibit significant differences in levels of both nutrients and defensive secondary metabolites. Specialized guilds of herbivores are known to exploit specific plant structures (Schoonhoven et al. 2005). Flowers and seeds may be attacked by insects that rarely feed on leaf tissues, and vice versa. Other herbivores exhibit more plasticity in food selection and will readily feed on all available parts of the plant. Even the latter herbivore types display some preference for a specific plant structure, presumably as a means of obtaining optimal nutrient intake, limiting competition, or obtaining enemy-free space.
The cabbage white butterflies Pieris brassicae L., Pieris rapae L., and Pieris napi L. are specialized on the plant family Brassicaceae. Interactions between these butterflies and their host plants have been explored in detail, and they have acquired the status of a model system in the field of insect–plant biology (Feltwell 1982; Chew and Renwick 1995; Renwick 2002). Glucosinolates, characteristic of the Brassicaceae, play a crucial role as chemical mediators of these interactions. Adult females of P. brassicae and P. rapae, as well as their larvae, exploit glucosinolates as token stimuli during selection of host plants for oviposition and feeding (Renwick et al. 1992; van Loon et al. 1992; Moyes et al. 2000; Schoonhoven and van Loon 2002). The majority of Pieris–Brassica interaction studies has focused on cultivated forms of Brassica oleracea L., which are biennials. Other wild crucifers, such as the annual black mustard, Brassica nigra L. (Koch), are reported to be host plants for P. brassicae in Europe as well (Harvey et al. 2003). We studied within-plant feeding site location in detail on three genotypes of B. nigra. We included the analysis of leaf and flower glucosinolates to assess whether a chemical basis for selection of either plant organ in terms of quality or quantity of these compounds could be determined. In addition, we investigated whether growth rates differed between caterpillars feeding on either leaves or flowers to assess whether the observed preference behavior would confer a nutritional benefit.
Methods and Materials
Plants Seeds of B. nigra var. abyssinica A. Braun from three early flowering accessions were obtained from the Centre for Genetic Resources (CGN, Wageningen, The Netherlands). The accessions were: A, CGN06619 (feral population, collected in 1975 from the Peloponesus, Greece); B, Junius CGN06618 (advanced cultivar, collected in 1977 in Germany); and C, Giebra CGN06620 (advanced cultivar, donated in 1965 by the Botanical Garden of the Justus Liebig University, Giessen, Germany). Sowing was done on April 7, 2004, in a greenhouse at 22 ± 2°C and 60 ± 5% RH. Two weeks later, plants were individually transplanted into 1.5-l pots and transferred to another greenhouse compartment on April 30, 2004. This compartment was kept at 22 ± 2°C, 60% RH, natural light supplemented by additional illumination by 500-W SON-T lamps (Philips, Eindhoven, The Netherlands) for 16 hr per day. Flower bud formation and flower opening were recorded daily to determine developmental stage (Harper and Berkenkamp 1975).
Insects Pieris brassicae caterpillars were obtained from a laboratory strain established in 2004 and reared on Brussels sprouts (B. oleracea var. gemmifera cv Cyrus) plants grown in a climatized greenhouse, under the same conditions as given above for B. nigra plants. The colony was maintained in a climatized room at 22 ± 1°C, RH 40 ± 5%, and a photoperiod of L16:D8.
Caterpillar Position on the Plant On the first day of an experiment, newly hatched P. brassicae larvae were collected from the laboratory culture and taken to the greenhouse. Three caterpillars were placed on the first true leaf below the lowest flowering branch of a plant in growth stage 4.1 (Harper and Berkenkamp 1975). Total numbers of plants used from each accession were A, 8; B, 7; and C, 9. Caterpillar position on the plants was scored each morning in four categories: on a true leaf, on a small leaf in the inflorescence, on a flower, and on the stem. Caterpillar instar stage was recorded daily until the fifth instar was reached. In the late fifth instar, caterpillars that had entered the wandering phase left the plants.
Caterpillar Growth on Leaves and Flowers Three neonate caterpillars were inoculated on each of 20 plants of accession A, on a mature leaf just below the inflorescence. The inoculation took place when plants had just started to flower (growth stage 4.1). Just after caterpillars had molted to the third instar, they were transferred to the lowest branches of the inflorescence on half of the plants. We applied a specially constructed water barrier to prevent caterpillars from migrating from leaves to the inflorescence or vice versa, while not influencing the microclimate around the plant (Fig. 1). Fresh body weights of larvae were determined to the nearest milligram on a Mettler electronic balance at three time points separated by 3-d intervals.
Fig. 1Schematic drawing (top and lateral views) and dimensions of water barrier applied around the stem of B. nigra plants to prevent movement of caterpillars from leaves to flowers and vice versa. A circular water container was manufactured of plastic. The middle circle consisted of a central platform, preventing drowning of caterpillars feeding on the inflorescence in the rare events of falling down or downward migration. In the center of the platform, a hole allowed the main stem to grow through. The platform was put in place around the main stem just below the branch carrying the first flower buds. The circular opening between the central hole and the stem was blocked with soft foam (not drawn)
Preparation of Plant Samples for Glucosinolate Analysis Ten seedlings of each of the three accessions were individually transplanted to 1 1/2-l pots. Plants were maintained at 22 ± 2°C, RH 60 ± 5%, during the 16-hr photophase (8 Philips SON-T Agro 400-W sodium lamps) and 8-hr scotophase.At day 47 after sowing, leaf and flower samples were taken from all 10 plants of each accession. After sampling, five plants of each accession were designated untreated (controls). Three groups of 10 newly hatched P. brassicae caterpillars were placed on three leaves of the five treated plants. As soon as the caterpillars had migrated to the flowers, which took place late in the second or early in the third instar (days 5–6), leaves damaged by the caterpillars were sampled. Leaves of a similar age were sampled from the control plants and intact flowers were sampled from both control and treated plants.The flower and leaf samples were kept in paper envelopes and stored in a −20°C freezer before and after freeze-drying. Samples were freeze-dried in the envelopes for 2 d (Labconco Freedry system). After freeze-drying, the envelopes were sealed in plastic and sent to the laboratory of NA in Denmark for chemical analysis.
Determination of Glucosinolate Content Freeze-dried samples were weighed accurately into 10-ml centrifuge tubes (excluding the petiole part of leaves). For samples below 300 mg, the entire sample [27–257 mg dry weight (DW)] was used to avoid errors associated with subsampling, but from a few samples above 300 mg, a representative subsample was taken. Each sample was extracted 3× in 70% boiling aq. methanol with benzylglucosinolate as internal standard. The combined extracts were adjusted to 10.00 ml, of which an amount was applied to an anion exchange column (Agerbirk et al. 2001a). To keep within the linear range of the additional sample preparation steps, the amount of crude extract applied depended on the weight of the sample extracted as follows (DW extracted/volume applied): 25–75 mg/8 ml, 75–150 mg/4 ml, and 150–300 mg/2 ml. Glucosinolates were converted to desulfoglucosinolates (Agerbirk et al. 2007), which were eluted and quantified by high-performance liquid chromatography (HPLC) (Agerbirk et al. 2001a) and identified by comparison of retention times and diode array UV spectra with those of authentic standards (Agerbirk et al. 2001b). A number of unidentified trace peaks with areas typically less than 0.2% of the desulfosinigrin area were ignored. Generally accepted relative response factors were used for calculation of glucosinolate levels based on the HPLC peak areas (at 229 nm, band width 8 nm) of the desulfoderivatives (Wathelet et al. 2004). The recoveries of internal standard in the three extraction series were [mean (SD)] 99% (6%), 97% (13%), and 98% (4%), as compared to HPLC peak areas obtained after application of the internal standard only to DEAE columns in parallel control experiments. Preliminary experiments without the addition of internal standard (comprising both leaf and flower samples from plants both with and without caterpillars feeding) confirmed the absence of benzylglucosinolate as an endogenous glucosinolate in the material, in accordance with previous reports (Feeny and Rosenberry 1982; Sang et al. 1984), allowing the use of this glucosinolate as internal standard. The identities of all desulfoglucosinolate peaks were confirmed by liquid chromatograph mass spectrometry (LC–MS) of representative samples with an Agilent 1100 Series LC (Agilent Technologies, Waldbronn, Germany) coupled to a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). An XTerra MS C18 column (Waters, Milford, MA, USA; 3.5 μM, 2.1 × 100 mm) was used at a flow rate of 0.2 ml min−1. The mobile phases were as follows: A, 0.1% (v/v) HCOOH and 50 μM NaCl; B, 0.1% (v/v) HCOOH and 80% (v/v) MeCN. The gradient program was 0 to 4 min, isocratic 2% (v/v) B; 4 to 10 min, linear gradient 2 to 8% B; 10 to 30 min, linear gradient 8% to 50% (v/v) B; 30 to 35 min, linear gradient 50 to 100% (v/v) B; 35 to 40 min, isocratic 100% B. The mass spectrometer was run in electrospray mode, observing positive ions. Mass spectral data were treated with the native DataAnalysis software.The extraction procedure was somewhat simplified compared to the standard protocol (Agerbirk et al. 2001a, originally optimized for seed extraction), as variable sample weights were accepted, and mechanical homogenization during extraction was avoided. These simplifications were justified by results of initial control experiments: A number of both leaf and flower samples of varying weights (up to 400 mg) were first subjected to the extraction described above. Then, the residue was further extracted for another three times, but this time with mechanical homogenization. In all cases, low relative amounts of glucosinolates (1–16% compared to the first extract) were detected in the extract obtained with mechanical homogenization (with a tendency for increasing percentage with increasing sample weight). Proportional amounts of internal standard were similarly recovered in all the extracts obtained with mechanical homogenization, showing that the internal standard had already been completely mixed with endogenous analytes during the extraction without homogenization. Hence, it was concluded that the ratio of internal standard to endogenous glucosinolates extracted without mechanical homogenization was representative of their initial concentrations.
Statistical Analyses Proportion of days caterpillars spent on different positions on the plant was treated as binomial data. Distributions over the four positions were compared by using the Kolmogorov–Smirnoff test (GenStat release 8.11; Anonymous 2005). Caterpillar weights were analyzed by analysis of variance. The amount (μmol/g DW) of each glucosinolate detected in the leaf and flower samples were analyzed with a generalized linear model after a logarithmic transformation. The effect of time (before and during caterpillar feeding), treatment (undamaged and damaged by caterpillars), plant tissue (leaf and flower), and genotype (accessions) and all possible two- and three-way interactions were investigated (GenStat release 8.11).
Results
Position on the Plant First and second instars fed primarily on leaves, whereas third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed (Fig. 2). The distribution of caterpillars over the four positions was statistically similar for the three genotypes (Kolmogorov–Smirnoff test, P > 0.05).
Fig. 2Frequency distribution of caterpillar position on B. nigra plants observed daily over a period of 12–13 d. On day 1, neonates were introduced on a true leaf just below the inflorescence. Four organ positions were distinguished: on a true leaf, on a small leaf in the inflorescence, on a flower, or on a stem in the inflorescence. Percentages are averaged based on three caterpillars per plant for eight, seven, and nine plants of accessions A, B, and C, respectivelyCaterpillars fed in discrete meals two to three times each hour, interspersed with phases without feeding. On a leaf, after termination of a meal, they moved away from the freshly damaged wound, after which they rested most of the time elsewhere on the leaf until they moved back to the feeding site. By contrast, in the inflorescence, during a single meal, caterpillars moved from flower to flower along flowering branches. A fifth-instar caterpillar consumed on average 135 ± 21 buds and flowers. When they were not feeding, P. brassicae larvae rested on stems immediately adjacent to the flowers.
Caterpillar Growth Caterpillars feeding on flowers of accession A since molting to the third instar had significantly higher fresh body weights on each of the three measuring points (mid-L4 and early and mid-L5 and late L5; Fig. 3) than those that were confined to leaves (P < 0.01, generalized linear model).
Fig. 3Fresh body mass of P. brassicae on either flowers (circles) or leaves (squares) of B. nigra in mid-fourth, mid-fifth, and late-fifth instar developing on B. nigra, accession A. Mean and SEM are plotted for 30 caterpillars
Glucosinolate Levels Five glucosinolates were identified: the aliphatic allylglucosinolate (sinigrin), which occurred in large amounts, and low levels of four aromatic glucosinolates including 2-phenylethylglucosinolate and the three indole glucosinolates indol-3-ylmethylglucosinolate (glucobrassicin), 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Despite the minor levels of the identified aromatic glucosinolates, they were quantified and included in the statistical analyses because of their well established chemosensory influences on Pieris species (Schoonhoven and van Loon 2002). No other significant peaks were detected. 2-Phenylethylglucosinolate was either below the detection limit in leaves (accession C) or present at very low levels (range 0.001–0.013 μmol/ g DW). We carefully distinguished the peak with the exact retention time of (desulfo) 2-phenylethylglucosinolate from an unidentified trace peak that was more intense in leaf chromatograms (estimated levels 0.03–0.35 μmol/g DW in leaves, 0.01–0.08 μmol/g DW in flowers), that had a retention time 0.7 min longer than that of (desulfo) 2-phenylethylglucosinolate, and that did not coelute with the authentic standard of 2-phenylethylglucosinolate after spiking.Genotype and tissue type were the most important factors affecting the levels of identified glucosinolates, and these factors showed a significant interaction (Table 1). No significant interactions were found between treatment and tissue (P > 0.05 for all glucosinolates). Glucosinolate concentrations have been graphically depicted for accession A in Fig. 4. Sinigrin was by far the most abundant compound in all three genotypes, with levels at least 44 times higher (range 44–305) in leaves and at least 87 times higher (range 87–447) in flowers of any of the three genotypes and treatments than the second most abundant compound, 4-hydroxyglucobrassicin. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. Sinigrin levels were ca. fivefold higher in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in leaves were higher than in undamaged plants (P < 0.05; Table 1). In flowers of accession A (Fig. 4), there was a similar tendency for induction of sinigrin by caterpillar feeding, but such induction was not seen with flowers from the other two genotypes (results not shown). Levels of other glucosinolates showed moderate variations that were not affected by the experimental variables (Table 1, Fig. 4 and results not shown). There was no systematic induction of any indole glucosinolate or of total indole glucosinolates in response to caterpillar feeding (results not shown).
Table 1Results from generalized linear model analysis on log-transformed data of glucosinolate concentrations in two tissue types (leaves and flowers) for three genotypes of B. nigraFactorSinigrinPEGB4OHGB4MeOGBTimensnsns***nsTissue******ns***nsTreatment*nsns*nsGenotype************nsTissue × genotype*****ns*nsTreatment means presence or absence of third-instar caterpillars feeding on flowers during 4 d. Effect of the factor “time” refers to changes over 4 d in intact plantsPE = phenyethylglucosinolate; GB = glucobrassicin; 4OHGB = 4-hydroxyglucobrassicin; 4MeOGB = 4-methoxyglucobrassicin*P < 0.05; **P < 0.01; ***P < 0.001Fig. 4Concentrations of five glucosinolates (mean + SEM) in leaves and flowers of 10 plants of accession A. For both leaves and flowers, the initial concentration just before treatment, the concentration in leaves or flowers of plants damaged by feeding caterpillars during 4 d, and the concentration determined in leaves or flowers of intact plants 4 d after caterpillars were introduced on the treated plants
Discussion
Our data show that P. brassicae third, fourth, and fifth instars preferentially feed on B. nigra flowers. Preference was absolute: although leaf material was available in surplus, caterpillars fed exclusively on flowers. Historically, P. brassicae has been commonly considered a folivore (Feltwell 1982), with its impact on plants examined primarily from this perspective. To the best of our knowledge, this is the first detailed study showing that P. brassicae larvae preferentially feed on the buds and flowers of its host plant. The status of P. brassicae as an agricultural pest has invariably resulted in a focus on cultivated, biennial forms of B. oleracea in studies of its host plant relationships. We also observed flower and silique feeding under field conditions (Smallegange et al., unpublished results).
The impact of folivores on plants has both direct and indirect effects. Directly, these herbivores reduce the leaf area available for photosynthesis, thereby decreasing the plants’ ability to garner resources for defense and reproduction (Marquis 1984; Mothershead and Marquis 2000; Schoonhoven et al. 2005). Indirectly, leaf herbivory can delay flowering (Meyer and Root 1993; Strauss et al. 1996) or can alter developing flowers both physically (Strauss et al. 1996; Lehtilä and Strauss 1999; Mothershead and Marquis 2000) and chemically (Lohman et al. 1996; Karban and Baldwin 1997). This may cause a reduction in plant attractiveness to pollinators or in the efficiency of pollen delivery, resulting in decreased pollination and, subsequently, reduction of seed set and overall fitness of the plant. Florivory, feeding on flowers, has received less attention than folivory or feeding on other vegetative plant parts despite its common occurrence and potentially high impact on plant fitness (recently reviewed by McCall and Irwin 2006). Feeding on flowers causes damage to structures essential for plant reproductive output (Juenger and Bergelson 1997; Adler et al. 2001). Indirectly, petal damage leads to a reduction in pollinator visitation that not only reduces reproductive success but also affects male fitness (via pollen removal) of the plant as well (Karban and Strauss 1993; Krupnick and Weis 1998; Adler et al. 2001). However, the compensatory abilities of plants can partially overcome the impact of floral herbivory (Hendrix 1988; Krupnick and Weis 1998). Removal of reproductive organs as a preferred mode of feeding has evolutionary consequences, as this type of attack is likely to exert a stronger selection pressure than leaf feeding, especially in annual species like B. nigra. Although many plant species accumulate higher concentrations of defensive secondary metabolites in reproductive organs than in vegetative organs (Schoonhoven et al. 2005), in B. nigra, these higher levels are not sufficient to deter the specialist feeder P. brassicae. A putative barrier to florivory by generalist herbivores lies in the higher quantities of secondary plant compounds that are typically found in these organs relative to the levels reported for leaves (Rask et al. 2000; Fahey et al. 2001; Brown et al. 2003; Strauss et al. 2004; this study). The location of high concentrations of defensive secondary plant compounds, including glucosinolates, in reproductive structures is consistent with the optimal distribution of chemical defenses predicted by plant defense theory (Zangerl and Bazzaz 1992; van Dam et al. 1996; Wallace and Eigenbrode 2002; Strauss et al. 2004). Myrosinases, β-thioglucosidase enzymes that convert glucosinolates into products such as isothiocyanates and nitriles, which are volatile in the case of, e.g., sinigrin and 2-phenylethylglucosinolate, occur in different forms in flowers and leaves (Rask et al. 2000). By using headspace techniques, isothiocyanates and nitriles have been reported from mechanically macerated bud samples of several Brassica species (Tollsten and Bergström 1988). Phenylacetonitrile was detected in headspace samples of intact B. rapa L. flowers (Omura et al. 1999). Benzylcyanide was released in significantly higher amounts from Brassica napus L. in midflowering than in bud stage (Jönsson et al. 2005). High levels of isothiocyanates are potentially toxic to both specialist and generalist herbivorous insects (e.g., Li et al. 2000; Agrawal and Kurashige 2003), while even low levels may be detected by specialized sensory receptors and assist specialized herbivores in host plant finding (Renwick et al. 2006).
The growth rate we observed for caterpillars feeding on flowers was remarkable. The feeding site preference of L3–L5 caterpillars presents a case of within-plant selective foraging, the adaptive value of which might be the ingestion of higher nutritional content in flowers, known to act as nutrient sinks, compared to leaves. Nutrient acquisition during the larval stage can affect pupal survival, longevity, and fecundity of Lepidoptera (Feltwell 1982; Van der Meijden et al. 1984; Metspalu et al. 2003). For example, female pupae of Tyria jacobaeae were significantly larger when caterpillars were reared on leaves and inflorescences of Senecio jacobaea plants than when reared on leaves only. This is advantageous because large pupae have a higher eclosion success, and it may explain why T. jacobaeae females prefer flowering plants for oviposition (Vrieling and de Boer 1999).
Faster growth has often been interpreted as a mortality-reducing factor. It is assumed that by reducing the amount of time that larvae are early instars, when they are most vulnerable to attack by natural enemies, they will experience higher survival (the ‘slow-growth–high-mortality hypothesis’ sensu Clancy and Price 1987; Benrey and Denno 1997). This has been experimentally confirmed under field conditions for the interaction between P. rapae feeding on B. oleracea and its predators and parasitoids such as Cladophora glomerata (Loader and Damman 1991; Benrey and Denno 1997). However, for other tritrophic interactions, faster growth was associated with higher mortality due to natural enemies (e.g., Clancy and Price 1987). Biere et al. (2002) found that larvae of the moth Hadena bicruris exhibit a strong preference for seeds of their food plant, Silena latifolia, over leaf tissues. Herbivore development proceeded more rapidly on seeds, and the larvae were less susceptible to parasitism from their main endoparasitoid, Microplitis tristis.
Herbivorous insects specialized on glucosinolate-containing plants typically avoid the formation of toxic isothiocyanates by employing specialized detoxifying mechanisms. In the case of P. brassicae, this is accomplished by a nitrile specifier protein (NSP) in the gut that changes the products of the myrosinase-catalysed hydrolysis of glucosinolates from isothiocyanates to relatively harmless nitriles (Wittstock et al. 2004), which may be further metabolized before excretion depending on side chain structure (Agerbirk et al. 2006; 2007). Shortly before this mechanism was reported, it was discovered that allyl isothiocyanate, the volatile hydrolysis product of sinigrin, is toxic to the specialist feeder P. rapae when feeding on an artificial diet (Agrawal and Kurashige 2003). Assuming a 20% DW in the artificial diet as used by Agrawal and Kurashige (2003), the maximum concentration to which P. rapae caterpillars were exposed in their study was 8.5 μmol/g DW. This dose caused significant negative effects on growth rate and inhibited development. In contrast, sinigrin levels in B. nigra flowers were more than 10 times higher (Fig. 4) and flowers sustained higher growth rates of P. brassicae than leaves, which had average levels of sinigrin two times higher than the corresponding amount of allylisothiocyanate as applied in the artificial diet. Assuming that isothiocyanates were produced in significant amounts during ingestion of glucosinolate-containing foliage by P. rapae, an involvement of the glucosinolate–myrosinase system in defense against this specialist species was suggested. Brassica nigra plants that had been induced by previous P. rapae feeding damage had a 27% increase in sinigrin concentration, but also 43% higher trichome density (Traw and Dawson 2002). Our data confirm the reported induction of sinigrin in leaves upon feeding, but also demonstrate that the magnitude of the induction is minor relative to the difference between leaves and flowers. With the elucidation of the NSP-based mechanism for detoxification of the glucosinolate–myrosinase system, the suggested involvement of the glucosinolate–myrosinase system and defense to specialists now needs to be re-evaluated. Formation of isothiocyanates is to be expected only if the myrosinase activity in the plant is so high that available NSP cannot completely direct the reaction towards nitriles, combined with glucosinolate levels sufficiently high to produce toxic levels of isothiocyanates. The selective foraging on flowers by P. brassicae documented here demonstrates that the caterpillars are able to detoxify even high levels of sinigrin, such as those present in B. nigra flower tissue. Not only high glucosinolate levels but also high myrosinase activity would be needed to exert toxic effects on Pieris caterpillars, as has been experimentally demonstrated for another glucosinolate-adapted herbivorous insect, Plutella xylostella (Li et al. 2000). Glucosinolates occur in cells of all organs of Brassicaceous plants, whereas myrosinases can be found in scattered glucosinolate-free cells. It remains to be examined whether flower buds of different age, flowers, and siliques of B. nigra have cells that contain myrosinases. In the related B. napus L., expression of the MYR1-gene coding for particular myrosinase isoforms has been demonstrated to occur in all organs investigated, including petals (Falk et al. 1992), although myrosinase was absent in very young flower buds in this species (Andréasson et al. 2001). Myrosinase gene promoters fused to beta-glucuronidase as a reporter showed expression of myrosinase in idioblast myrosin cells in immature and mature seeds and phloem myrosin cells of B. napus (Thangstad et al. 2004). Data on headspace composition of inflorescences actually fed upon by Pieris spp. are not available. Follow-up studies on the types and quantities of volatile breakdown products emitted by herbivore-damaged flowers are required to quantify myrosinase activity levels in flowers. If these levels are low, the better performance associated with flower feeding might be reconciled with a role of the glucosinolate–myrosinase system in defense against the specialist P. brassicae. However, a likely and simple explanation of the better performance associated with flower-feeding may be that the caterpillars are able to efficiently detoxify the glucosinolate–myrosinase defense system in both organs, and that flowers, major nutrient sinks on a plant, are more nutritious than leaves. The fact that early instars did not migrate to flowers may be an adaptation to factors other than nutrient content: leaves are the site of oviposition, are present earlier in the season than flowers, and may provide better refuge from natural enemies for small instars. We have never observed P. brassicae or P. rapae ovipositing on flowers or flowering stems.
Neither P. rapae nor P. brassicae sequester glucosinolates for their own defense against natural enemies (Müller et al. 2003), in contrast to what had been published earlier by Aplin et al. (1975). The intensely green, clear regurgitant these caterpillars instantly, and typically, produce when attacked may contain highly concentrated glucosinolate hydrolysis products (nitriles) stored in the foregut. Presence of concentrated glucosinolate hydrolysis products may explain the obvious repellent effect of regurgitant to natural enemies such as Cotesia wasps and predators (Brodeur et al. 1996). Consequently, caterpillars may prefer flower tissues that contain high levels of these compounds, thus boosting their defense against both specialist and generalist natural enemies (Soler et al. 2005). Behavioral experiments with parasitoids and predators known to act as common natural enemies of P. brassicae (Feltwell 1982) are required to test this hypothesis.

brassica nigra
nutrition
herbivory
myrosinase
isothiocyanates
lepidoptera
pieridae


Cell identity in the preimplantation mammalian embryo: an epigenetic perspective from the mouse

The early preimplantation mouse embryo is a unique system where it is possible to explore the foundations of totipotency and differentiation. Following fertilization, a single cell, the zygote, will give rise to all tissues of the organism. The first signs of differentiation in the embryo are evident at the blastocyst stage with the formation of the trophectoderm, a differentiated tissue that envelopes the inner cell mass. The question of when and how the cells start to be different from each other in the embryo is central to developmental biology: as cell fate decisions are undertaken, loss of totipotency comes about. Although the blastomeres of the preimplantation embryo are totipotent, as the embryo develops some differences appear to develop between them which are, at least partially, related to the epigenetic information of each of these cells. The hypothesis of epigenetic asymmetries acting as driver for lineage allocation is presented. Although there are now some indications that epigenetic mechanisms are involved in cell fate determination, much work is needed to discover how such mechanisms are set in play upon fertilization and how they are transmitted through cell division. These considerations are further discussed in the context of preimplantation genetic diagnosis: does it matter to the embryo which cell is used for genetic diagnosis? The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future.
Embryonic development starts from a single cell, the zygote. In this cell, the two gametes convey and contribute information to start a new developmental programme. The formation of the newly fertilized zygote constitutes therefore the climax of totipotency because of the resulting zygote’s inherent ability to produce all cell types in a new organism.
In the mammalian embryo, the first differentiative event occurs as inner cells form upon cell division at the 8-cell stage. As a result of this division, an ‘inner’ and an ‘outer’ population of cells can be distinguished in the 16-cell stage embryo, which will respectively occupy different positions in the morula (Johnson and Ziomek, 1981). Much work has been done in trying to understand how cell polarity develops in the inner and outer cells, but I will not deal with this topic here and instead will refer the reader to an excellent review published elsewhere (Johnson and McConnell, 2004). The inner cells will develop into the inner cell mass (ICM) and the outer layer of cells will differentiate into the trophectoderm (Tarkowski and Wroblewska, 1967; Ziomek and Johnson, 1982). Morphologically, however, the first overt signs of differentiation are evident only at the blastocyst stage, with the formation of the trophectoderm, which is the first differentiated tissue to form as an epithelial layer that envelops the ICM (Fig. 1A). The latter, in contrast to the trophectoderm, retains its pluripotent character and the ability to self renew. While the ICM will give rise to the embryo proper, the trophectoderm will give rise to the extraembryonic tissues that will support development of the embryo during gestation. The ICM will also give rise to yet another extraembryonic tissue, the primitive endoderm, which is first visible as a cuboidal layer of cells lining the blastocoelic cavity on the fourth day of development. The trophectoderm and the ICM each display molecular identity, which is reflected in part by the expression of specific genes that are, for the ICM, involved in its specification and the maintenance of pluripotency (such as Nanog and Oct4) or, for the trophectoderm, that are required for its differentiation (such as Cdx2) (Palmieri et al., 1994; Nichols et al., 1998; Chambers et al., 2003; Mitsui et al., 2003; Strumpf et al., 2005). The bifurcation of these two lineages is complete at the late blastocyst stage. At this stage, cells from the ICM do no longer have the potential to form trophectoderm derivates in vivo upon transplantation (Rossant and Lis, 1979), indicating that these cells have lost their totipotency and that lineage allocation has definitely occurred.
Figure 1:
Cell lineages of the mammalian blastocyst and epigenetic marking
(A) Representation of the lineages in the mammalian blastocyst on the third day of gestation (E3.5). The blastocyst is composed of two distinct populations of cells: the trophectoderm (red) and the inner cell mass (ICM, green), which display molecular identity and epigenetic asymmetries. The embryonic-abembryonic regions (dotted line) of the blastocyst are determined by the position of the ICM, which lies within the embryonic region of the blastocyst. (B) Diagram illustrating some of the epigenetic marks. The DNA wrapped around the nucleosome (beige cylinders) is shown as light blue. The DNA is subject to DNA methylation, which constitutes one of the main epigenetic players. The core histones (beige) that form the nucleosome can be covalently modified (by acetylation, methylation, phosphorylation), particularly on their N-terminal tails. Each of these marks can have an effect on how the information contained in the DNA is read by modulating downstream events such as transcriptional activation or repression. For example, histone methylation (Me) can have a positive effect on transcription (green) or a repressive one (red). The marks can be present in different combinations and may change during the cell cycle. (C) Model for epigenetic marking and lineage allocation. In this model, an epigenetic mark would be laid down in a given cell during development. There could be other epigenetic event(s) that reinforce and/or are influenced by the first marking event. Cumulatively, this could result in determination of the fate of that cell towards a lineage in the blastocyst. The lineage specific marks could stabilize such cell identity and might be necessary for further differentiation. Alternatively, the acquisition of these epigenetic marks could be the result of cell fate determination. One should also consider that not only the nature of the mark would be important, but also the different regions of the chromatin that would be affected by such marks. Because the cells in the preimplantation embryo are totipotent and because the chromatin will still need to be dynamically remodelled during subsequent development, flexibility should be an important component of epigenetic mechanisms taking place during early development. As cell fate decisions are taken, a concomitant loss of totipotency takes place
Investigations during recent years have recreated an interest in whether the blastomeres of the mammalian embryo are truly alike throughout preimplantation development before the first differentiative division mentioned above. In other words, whether they acquire a ‘fate’ or whether they start to differ from each other prior to their spatial ‘inner/outer’ allocation within the embryo upon the formation of the morula. Of course, if this were to be the case, the big challenge would be to ascribe molecular mechanisms to these processes.
The question of when and how the cells start to be different from each other is not a trivial one. In particular, because as the first cell fate decisions are undertaken, a concomitant loss of totipotency occurs. The developmental time window when this first cell fate decision occurs comprises a number of epigenetic events (Morgan et al., 2005; Surani et al., 2007). These events include the reprogramming of the parental chromatin. Whether such epigenetic events are the cause or the consequence of reprogramming remains an exciting open question, but it is probably a combination of the two. Moreover, the two lineages of the blastocyst exhibit some epigenetic asymmetries.
This mini-review is divided in two parts, the first one will deal with the main epigenetic mechanisms that are known to occur during mammalian preimplantation development. The second one will give an overview on data obtained through experimental embryology manipulations and lineage tracing observations to study cell fate in the early mouse embryo.
Epigenetic mechanisms in the preimplantation embryo
In general terms, epigenetic mechanisms include DNA methylation, covalent modification of histones, chromatin remodelling and histone replacement through incorporation of the so-called histone variants (Fig. 1B). Histone marks have emerged as one of the main players involved in epigenetic mechanisms (Kouzarides, 2007). Histone modifications can be highly dynamic, or have a function in epigenetic memory. Although it is still unclear whether they are the actors of the epigenetic information or the epigenetic information itself, it is evident that covalent modifications of histones are essential components of the epigenome.
Histones can be modified by a number of enzymes that mediate methylation, acetylation, phosphorylation, ubiquitynation and ADP-ribosylation of specific amino acid residues (reviewed in Kouzarides, 2007). By and large, the highest density of modifications so far described occurs in histone H3, particularly on its tail. The effects of these modifications on the chromatin and on cellular processes are very diverse, and a modification of the same residue can even have opposite effects depending on the type of modification. For example, trimethylation of H3K9 is considered as a repressive mark, whereas acetylation of the same lysine has a positive effect on transcription (Bannister and Kouzarides, 2005). Likewise, methylation of arginine residues can have a positive effect on transcription (Chen et al., 1999) or a repressive effect (Pal et al., 2004), depending both on the targeted residue and on whether the methylation is symmetric or asymmetric. For some of the modified residues, there is a very clear view of the outcome of an eventual modification: H3K9me3 creates a specific docking site for the heterochromatin protein 1 (HP1), which subsequently recruits the H3K9 methyltransferase Su(var)3-9 and reinforces an autoregulatory loop for heterochromatin formation and maintenance (Bannister et al., 2001; Lachner et al., 2001; Nakayama et al., 2001).
The levels of regulation of epigenetic events in the preimplantation mouse embryo are multiple. They include the regulation of the subcellular localization of DNA methyltransferase activity, highlighted by the cytoplasmic retention of Dnmt1o (Carlson et al., 1992); the exclusion of a particular histone modification from the chromatin, which is exemplified by the lack of detection of H3K9me3 in the paternal pronucleus after fertilization resulting in an asymmetry of histone marks between the two pronuclei (Arney et al., 2002; Santos et al., 2005), the differential incorporation of chaperons and histone variants in the parental chromatin (van der Heijden et al., 2005; Torres-Padilla et al., 2006) and the acquisition of highly specific histone variants in the gametes (Clarke et al., 1992; Tanaka et al., 2001; Govin et al., 2007). Further, the maternal and paternal pronuclei exhibit different patterns of global DNA methylation: while the paternal pronucleus is rapidly demethylated—presumably through an active mechanism—right after fertilization, the maternal pronucleus is only passively demethylated through the subsequent rounds of replication and cell division that follow the first mitosis of the embryo (Mayer et al., 2000). Moreover, while the centromeric and pericentric paternal chromatin remain DNA methylated, the maternal DNA loses methylation in such regions (Rougier et al., 1998).
The changes in the levels of DNA methylation as development proceeds in the preimplantation embryo are dynamic. Global levels of DNA methylation have been analysed by immunofluorescence, bisulphate sequencing and restriction digestion (Rougier et al., 1998; Mayer et al., 2000; Santos et al., 2002; Aranyi and Paldi, 2006). Bisulphate sequencing and restriction digestion have also been used to analyse the methylation status of repeat sequences (such as L1 and IAP repeats) and some single-copy sequences (such as actin) (Howlett and Reik, 1991; Oswald et al., 2000). These studies have revealed that although global levels of DNA methylation decrease until the blastocyst stage, changes in DNA methylation do not occur to the same extent on all genes. Remarkably, imprinted genes (such as H19) and some repeat sequences (such as IAPs) do not undergo demethylation (Tremblay et al., 1997).
During early stages of development, decisions involved in cell fate determination and pluripotency have to be assumed. These processes require the chromatin to be dynamically remodelled to ensure its plasticity. This implies that the mechanisms involved in regulation of chromatin structure need to ensure stability across generations and cell division, but they also need to be flexible (Reik, 2007). The double nature of a covalent modification either on histones and/or on the DNA as dynamic (because in principle it can be added and removed) and at the same time its potential ability to propagate a memory, fits well with these aforementioned needs. Moreover, in keeping with the importance of epigenetic mechanisms during early development, the possibility for an epigenetic mark(s) underlying these phenomena appears very attractive.
Concerning the epigenetic asymmetries of the ICM and the trophectoderm, the ICM displays, in global terms, higher levels of DNA methylation compared with the trophectoderm (Dean et al., 2001; Santos et al., 2002). Specific histone marks such as trimethylation of lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3, respectively) are enriched in the ICM compared with the trophectoderm (Erhardt et al., 2003). Likewise, the trophectoderm retains an imprinted form of X inactivation, where the paternal X chromosome is silenced (Heard and Disteche, 2006). This is in contrast to the ICM, where there is reactivation of the inactive X chromosome and a subsequent round of inactivation occurs at random in which either the maternal or the paternal chromosome is inactivated (Mak et al., 2004; Okamoto et al., 2004).
The aforementioned epigenetic asymmetries of the two lineages of the blastocyst are evident once lineage allocation has taken place and might reinforce their molecular identity. However, epigenetic asymmetry could also act as a driver for lineage allocation, in which case, the former would precede the latter (Fig. 1C). This constitutes a fascinating current working hypothesis.
Development of cell identity in the mouse embryo
From experimental embryology, we have learnt from pioneer experiments performed in the 50’s that after mechanical separation of the blastomeres of a 2-cell stage embryo and transfer into foster mothers, each of these two cells gives rise to an adult mouse (Tarkowski, 1959). This indicates that mouse embryos are very flexible in what people have referred to as developmental potential. Derivation of twins from mouse blastomeres at later stages of development (e.g. 4-cell stage or later) has not been possible. Although this has been linked to the low number of cells present in the resulting embryos and hence their inability to form an ICM with a normal cell number, rather than to their developmental potential or identity (Tarkowski and Wroblewska, 1967; Rossant, 1976). Indeed, when random single 4- and 8-cell stage blastomeres are aggregated with ‘carrier’ blastomeres, their progeny is able to contribute to all the tissues of the embryo (Kelly, 1977; Tarkowski et al., 2005), and in this sense, the blastomeres were considered to be totipotent. So, what are these carrier cells providing? Is it a simple matter of cell number? and/or of an appropriate environment? It is also possible however that some subtle intrinsic differences of these blastomeres might be masked by the limitations of the outcome of transplantation procedures, given that the results of these studies are very often far from 100% and the transplantation efficiency is never absolute. Whether this is solely related to technical difficulties linked to these challenging manipulations or to an intrinsic property of specific blastomeres of the embryo is impossible to ascertain.
Despite a controversial viewpoint on whether there is any polarity in the early mouse embryo or not, most reports coincide with the interpretation that a blastomere at the 2-cell stage contributes to both the ICM and the trophectoderm (reviewed in Edwards and Beard, 1997; Zernicka-Goetz, 2006). The suggestion of a given blastomere at the 2-cell stage having a ‘preferential’ fate towards either of the blastocyst lineage is not resolved and some researchers have suggested that a slight, but distinct difference in the fate of 2-cell stage blastomeres might be disturbed by experimental manipulations (Alarcon and Marikawa, 2005; Hiiragi et al., 2006). The role of extrinsic factors to the embryo, such as the shape of the zona pellucida, in axis specification of the blastocyst is also a matter of controversy (Gardner, 2007; Kurotaki et al., 2007). However, there are some indications that a bias for a blastomere to contribute to a given region of the embryo in the blastocyst could already exist at the 4-cell stage (Fujimori et al., 2003; Piotrowska-Nitsche and Zernicka-Goetz, 2005). These conclusions are mostly based on lineage tracing experiments of labelled blastomeres, and their degree of invasiveness is debatable.
Some groups have used the plane of division in relation to the animal–vegetal axis of the embryo as a sort of guideline to distinguish and characterize blastomeres according to their cleavage plane. By convention, the animal pole is demarcated by the position of the second polar body (which is extruded after resumption of meiosis II upon fertilization) and hence the vegetal pole lies on the opposite side (Fig. 2A). The division from the 2- to the 4-cell stage would segregate for the first time the ‘animal’ and the ‘vegetal’ components of the zygote if it occurs equatorially, that is, perpendicular to the animal–vegetal axis of the conceptus (Gardner, 2002). Thus, whereas a cell that derives from a meridional division (parallel to the animal–vegetal axis) inherits both components, an equatorial division gives rise to an ‘animal’ and a ‘vegetal’ blastomere (Fig. 2B). By looking into the plane of division and the order at which this division occurs from the 2- to the 4-cell stage and subsequent lineage tracing, a subgroup of embryos was identified where it is possible, with a relatively high probability, to predict the future position of the blastomeres in the blastocyst (Piotrowska-Nitsche et al., 2005). This group of embryos are referred to as ME embryos (for Meridional–Equatorial, reflecting the type of cleavage plane and order of division that generated them (Fig. 2B) (Piotrowska-Nitsche et al., 2005; Piotrowska-Nitsche and Zernicka-Goetz, 2005). Although the ME type embryos constitute only a small part (20%) of a complete litter, they provide a very nice system where it is possible to explore the foundations of differentiation in the embryo. Indeed, the ‘vegetal’ blastomere would most often populate the abembryonic region of the blastocyst, which contains mainly mural trophectoderm.
Figure 2:
Blastomere division planes according to the animal-vegetal axis in the embryo
(A) The animal–vegetal (A–V) axis of the preimplantation embryo is demarcated, by convention, by the position of the second polar body, which marks the animal pole. The opposite side to the site of extrusion of the polar body is, by default, the vegetal pole of the embryo. (B) The division pattern from 2-to- 4-cell stage of a typical embryo that undergoes one meridional (M) and one equatorial (E) division (ME embryo) is represented. The cleavage plane is depicted by a red dashed line (embryo on the left). The blastomere that divides earlier is represented on the left. A Meridional division has a cleavage plane that is parallel to the A–V axis of the embryo and hence gives rise to two cells containing both ‘animal’ and ‘vegetal’ components (two cells with pink and yellow motifs on the embryo depicted on the right). In contrast, when a 2-cell stage blastomere divides equatorially, a segregation of the ‘animal’ and ‘vegetal’ cytoplasm occurs and follows derivation of an ‘animal’ (pink) and a ‘vegetal’ (yellow) blastomere
In looking for epigenetic marks that could be involved in an eventual cell fate decision of the blastomeres of these ME embryos, it was found that the ‘vegetal’ blastomere displays the lowest levels of dimethylated arginine 26 of H3 (H3R26me2). If H3R26me2 participates in lineage allocation, one might predict that modulating the levels of histone arginine methylation, would have an effect over cell fate. Overexpression of the histone methyltransferase that methylates this residue on H3, PRMT4/CARM1, into individual blastomeres not only induced upregulation of Nanog and Sox2, but also resulted in an almost complete allocation of these blastomeres into the ICM compartment (Torres-Padilla et al., 2007).
It is interesting to note that in the mouse, the differences described in histone H3 arginine methylation appear at the 4-cell stage (Torres-Padilla et al., 2007), that is. as early as one cell cycle after the major wave of embryonic genome activation occurs (Schultz, 2002; Hamatani et al., 2004), which suggests that these events might be, at least in part, linked to the transcriptional programme of the embryo. The developmental stage at which genome activation occurs in other mammalian species varies considerably: it takes place at the 1-to- 2-cell stage in mice, the 4-to- 8-cell stage in cows and humans, and the 8-to- 16-cell stage in sheep and rabbits (Schultz and Heyner, 1992). Would this anticipate a different timing for an eventual ‘cell fate path’ for other species? Normal fertile adults can be derived from single blastomeres from 2-, 4- and 8-cell stage embryos in the rabbit, sheep and cattle (Moore et al., 1968; Willadsen, 1981; Willadsen and Polge, 1981). Thus, these species indeed support blastomere isolation and further development at later stages than the mouse does.
Transplantation of isolated 4-cell blastomeres into morula stage embryos has demonstrated that the blastomeres at the 4-cell stage are totipotent (Kelly, 1977). In this context, it is important to note that aggregating the ‘vegetal’ cell from ME embryos to form chimeric embryos, showed that this cell is able to contribute to all tissues in the embryo. However, aggregating the same blastomere with other ‘vegetal’ blastomeres from ME embryos exclusively, results in a failure to proceed through development (Piotrowska-Nitsche et al., 2005). Thus, the environment where the blastomeres develop is crucial for the success of the embryo throughout development, and in a ‘normal’ situation, where the embryo has not been perturbed and a given cell develops in its niche, some differences appear to develop, which are, at least partially, related to the epigenetic information of each of them (Torres-Padilla et al., 2007). If epigenetic asymmetries of the early embryo are related to lineage allocation, it is still uncertain whether they are a cause or a consequence for lineage choice. Also, it remains unknown whether such epigenetic asymmetries would affect only particular regions of the genome. For example, whether genes involved in specification of the ICM such as Sox2 and Nanog would all be targeted by the same epigenetic marks in the same blastomere or whether such marks would vary among genes and/or among blastomeres. Further, are ‘inner’ and ‘outer’ cells at the 16-cell stage distinguishable in terms of their chromatin landscapes?
The experiments showing that blastomeres are able to respond to the overexpression of a histone modifier and change their fate, indicate that these cells have not yet acquired a ‘fix’ destiny, but that they can still be responsive to some kind of signals. These experiments have an important impact on showing that manipulating the epigenetic information can affect cell fate in the preimplantation embryo, in line with the importance of epigenetic mechanisms being crucial for early development. Moreover, these results do illustrate that such cells can still be flexible and accommodate themselves after a perturbing event (in this case, overexpression of a histone methyltransferase and the downstream effects on the information that is. imparted through specific histone modifications).
These studies have originated some interest from the part of the medical community, particularly, in the context of preimplantation genetic diagnosis (PGD) (Goldman, 2007). Does it matter to the embryo which cell is used for genetic diagnosis? Might the death of one of these cells have an effect on subsequent development? This question is equally valid on the impact of cell loss upon cryopreservation (Cohen et al., 2007). Although these are very delicate questions with very likely no easy answer, from the perspective of the mouse embryo, at least four things are to be considered. A tendency for a blastomere of some 4-cell stage embryos to contribute to a given region of the embryo has been documented. Second, the blastomeres in the 4-cell stage show clear differences not only in the levels of histone methylation, but also in their transcriptional activiy when they develop without being perturbed. However (third), the cells undergo a redirection of cell fate when a histone methyltransferase is overexpressed, indicating that they can readapt. Finally, the environment in which cells develop seems to be crucial for completing development and somehow the remaining cells in the embryo could compensate provided they are somehow different from each other. It is also important to note that a 4-cell stage mouse embryo might correspond to a very different developmental stage than a 4-cell embryo in other mammalian species, as illustrated by the differences in the onset of genome activation between them. Indeed, PGD is most often performed at the 8-cell stage and some reports document a better rate of development when 6-to- 9-cell stage embryos are diagnosed, as opposed to 3-to- 4-cell stage embryos (Wang et al., 2007). Moreover, the effects of in vitro fertilization procedures and culture on embryonic development are also extremely important, as they have been shown to alter epigenetic information in the mouse (Li et al., 2005).
As a final consideration, I would like to leave the reader with an open perspective of some ongoing questions in the field. Although there are strong indications that epigenetic mechanisms are involved in cell fate determination, we are still far from establishing a direct link between an epigenetic mark(s) and the derivation of a particular cell lineage in the embryo. Much work is still to be done to determine how these mechanisms are set in play upon fertilization and how they are transmitted during subsequent cleavage stages. Also, what other epigenetic marks contribute to the inheritability of cell fate decisions? How do these marks relate to and influence each other? Are different lineage-specific genes marked by a different combination(s) of epigenetic marks? Do the marking of these genes occur at different stages of development? It is also tempting to expand these notions into the stem cell field and question whether these mechanisms would also underlie the intrinsic self renewal ability of adult stem cells and their potential to differentiate into other cell types. The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future.
Funding
The author acknowledges support from the PNRRE/INSERM.

epigenetics
mouse embryo
cell fate
pluripotency
histone methylation