1. Biological relevance of cell-specific transcription regulation in retinoid signaling

2. Retinoid signaling during embryonic development

2.1 Xenopus laevis as a model system for early embryonic development

2.2 Model systems for early mouse development

3. Differentiation of P19-EC cells

3.1. Endoderm differentiation

3.2. Mesodermal differentiation

3.3. Neuronal differentiation

3.4. A speculative model for EC cell differentiation in relation to embryonic development

4. Role of RA during differentiation

4.1. Role for RA and RARs in early differentiation steps during development

5. A model for RA-induced EC cell differentiation: role for methylation and chromatin structure.

References
 

Discussion
 

In this thesis I describe various constraints on transcriptional responses to retinoids in different cell types. These experiments show that multiple factors, including receptor activity (Chapter 3, 4, 5, 7), cofactors (Chapter 5, 6, 8), and promoter architecture (Chapter 8) in concert to determine responsiveness to retinoids. These results were discussed in the previous chapters, but certain aspects deserve further attention.

I first evaluate results concerning the role of cell type specific activation by retinoid receptors. This leads to the question whether (cell-specific) retinoid signaling is required for embryonic development. In the remainder of the discussion I therefore focus on the role of retinoid signaling in embryonic development. I first briefly describe current models for Xenopus development with respect to formation and axial patterning of the three germ layers. This is followed by a detailed description of the processes involved in EC cell differentiation, which we use as a model-system for early embryonic development. Interestingly some mechanisms that are involved in Xenopus development may also be important for EC cell differentiation. Since retinoids are required to permit differentiation into endoderm, mesoderm and (neuro)ectoderm in EC cells, the roles of retinoids and their receptors in embryonic development is then discussed. Finally a model is proposed which may explain the different roles for retinoids in EC cell differentiation and in embryonic development.
 

1. Biological relevance of cell-specific transcription regulation in retinoid signaling

The availability of retinoic acid is modulated in vivo via biosynthesis and metabolism, catalyzed by various enzymes, probably acting in concert with CRBPs and CRABPs (94). Most of these enzymes are differentially regulated during development (see Chapter 1), and their expression is modulated by retinoids. The tissue-specific expression of particular metabolic enzymes also enables the synthesis of additional active retinoids such as 4-oxoRA, 9-cisRA, 13-cisRA, 3,4-didehydroRA, 14-HRR, which may fulfill specific functions. Evidence for distinct biological functions of different retinoid metabolites came from the identification of 4-oxoRA acting in early Xenopus embryos (101). This ligand was shown to be more potent than atRA in modulating positional information, although 4-oxoRA was slightly less potent than atRA in growth and differentiation assays in P19-EC cells. Furthermore 3,4didehydoROL can be formed from retinol in cultures of human skin keratinocytes, but not in other cell-types, indicating that the biosynthesis of vitamin A2 from ROL is regulated, further suggesting distinct functions for the two ligands in skin (108). Finally, the dual role 9-cis RA should be mentioned; it causes activation of RARs, and of RXRs as homodimers or as heterodimers with orphan receptors (74), while atRA can only activate the RAR pathway, arguing that biosynthesis of 9-cis RA can affect distinct processes. Interestingly, 9-cis RA binds with a much lower affinity than atRA to CRABPs (1,35), supporting the notion that the two retinoids are functionally different.

Expression patterns of the RARs during embryonic development have suggested specific functions for individual RARs. Disruption of individual receptors in mice (56) however argues strongly against a specific function for individual RARs and clearly indicates overlap of functions. Recent compound RAR^-/- mice give some insight to this issue. When expression of additional RAR isoforms was abolished, in a RAR^-/- background, severe abnormalities were found, which were at least to some extent dependent on the receptor (isoforms) deleted. Specific functions for individual RAR/RXR heterodimers became especially apparent in the RXR^-/- background (58). These experiments indicated that most functions of individual RARs are redundant, probably because cells express more than one RAR. When multiple RARs or RAR/RXRs are mutated, compensation by other RARs failed to occur either because particular cells failed to express additional RARs, or because the remaining receptors are less efficient in performing specific functions. This partial redundancy and receptor-specific functioning is nicely illustrated in the F9-EC cells in which individual receptors have been deleted (13,14,123,125). We have also presented evidence for receptor specific functions in P19-EC, where RAR is involved in regulating RA-dependent differentiation, while RAR controls growth and apoptosis (Chapter 7).

The receptor-specific functions observed by us and others suggest that RA-target gene activation is receptor specific. Direct proof for this receptor-specific activation of RA-target genes came from transfection experiments comparing the ability of isoforms of individual RARs or RXRs and AF-1 lacking mutants, or combinations of distinct RAR/RXR heterodimers to activate various RARE-containing promoters (67,92). The molecular basis underlying this receptor-specific activation is largely unknown, but might involve: 1) distinct affinities of the distinct RAR/RXR heterodimers for the specific RAREs present in RA-target genes. 2) differential activity of the activation domains of the individual RARs leading to activation by distinct mechanisms. Supported by the observation that all receptors contain an activation function within the AB-region, even though this region of the receptor is not conserved between the various receptor subtypes/isoforms (36,93). 3) different affinities of (distinct) ligand(s) for the various receptor subtypes might cause receptor specific activation. Allenby at al (2) have shown that atRA binds with higher affinity to RAR than 9-cis RA, while the binding affinities of atRA and 9-cis RA for RAR are comparable. In agreement with this, residues have been identified in RAR, RAR and RAR which can discriminate between RAR or RAR-specific synthetic retinoids. Mutation of these specific residues into those typical of a different subtype allows activation by the retinoid specific for the latter receptor (98). Interestingly, these critical residues reside in the ligand-binding pocket, suggesting that the ligand-binding pocket, and consequently the binding of specific ligands might contribute to receptor-specific activation (17).

In conclusion, retinoid signaling is regulated by the presence and levels of receptors and orphan receptors that compete for binding to the same response element, by the nature and concentrations of available ligands, and by the relative affinities of these ligands or isoforms of these ligands for the receptors present, the nature of the response element and the promoter-context within which this element is present, and finally by the activity of the activation functions of the RAR/RXR heterodimer bound to DNA. The activity of the AFs is determined by the nature of the AF (36,37), by the presence and levels of cofactors (Chapter 8), the phosphorylation status of the receptor (37; Chapter 4) and the ability to activate transcription synergistically in a promoter specific way (93,38; chap. 6). All parameters together regulate retinoid signaling and are likely to contribute to the cell-specific responses observed during embryonic development and in adult life.

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2. Retinoid signaling during embryonic development

To study the role of (cell-specific) retinoid signaling, we used embryonal carcinoma cells as a model system; these cells have been shown to require the presence of both retinoids and functional receptors (34,66,102) for RA-dependent differentiation. I discuss the processes taking place during early embryonic development (Xenopus laevis) and in the embryonal carcinoma cell system, with respect to the roles of retinoids and their receptors in the in vitro system compared with their roles in vivo.
 

2.1 Xenopus laevis as a model system for early embryonic development

Embryogenesis is a precisely controlled process involving the spatially and temporally defined differentiation of embryonic cells. The development of the three germ layers ectoderm, mesoderm and endoderm in the cell mass which arises from the fertilized egg is one of the important phases of embryogenesis. Furthermore, specification of the animal/ vegetal, dorsal/ventral, and anterior/posterior embryonic axes is important for development since these axes determine the future body plan. The animal/vegetal axis is already determined in the unfertilized oocyte, by unequal distribution of yolk and other cytoplasmic components. The animal part of the oocyte will give rise to ectodermal tissue, while the vegetal pole will form endodermal tissue. The sperm entry point determines the ventral side of the embryo, and thereby the dorsal/ventral axis. The anterior/posterior axis is determined during gastrulation, being regulated by signals from the organizer region, the dorsal mesoderm. Mesoderm formation occurs during the blastula stage, and is dependent on signals from the vegetal hemisphere (60,120). Ectodermal cells in the marginal part zone of the animal hemisphere respond to these signals and differentiate to mesodermal tissue. Based on experiments by Nieuwkoop and coworkers, it is thought that two discrete signals generated by the ventral and dorsal vegetal regions respectively, induce the specification of ventral mesoderm and dorsal mesoderm respectively (95,96). Use of the animal cap assay resulted in the identification of several polypeptide growth factors that can induce mesoderm from ectoderm. Factors identified so far include the TGF family members: activin, VG1, BMP-4 and nodal (60,120). Furthermore, several FGF family members, including eFGF and bFGF, have been implicated in mesoderm formation. Finally, Wnt family members have been reported to induce mesoderm. The mesoderm inducing factors described above can induce either dorsal mesoderm (activin, VG1, nodal), ventral mesoderm (BMP4, FGFs), or can induce the formation of an (additional) A/P axis (Wnt-8, Noggin). Subsequently, the induced mesoderm differentiates further in response to signals from the organizer region (dorsal mesoderm) (32,43) and additional signals are probably also required for the differentiation of more ventral mesoderm, which will result in the formation of blood, mesenchyme and muscle tissue (44). The so-called (Spemann's) organizer, which will later become head mesoderm and notochord, has the ability to induce a secondary axis when transplanted to the ventral side of a gastrula stage embryo. Several gene products which have been reported to be involved in A/P axis formation such as noggin, follistatin and chordin, are present in the organizer region (41,60). These molecules can promote neural induction of competent ectoderm. A recent model for neuralization proposes that formation of neuronal tissue from competent ectoderm is a default state (46), suggesting that animal cap cells can form neuronal tissue autonomously, and that repressive signals prevent neuralization at the ventral side of the embryo. The authors suggest that when BMP-4 signaling is prevented by the previously identified neural inducers such as noggin, follistatin and chordin, the competent ectoderm will from neuroectodermal tissue. This presumptive neural tissue is anterior in nature and is subsequently posteriorized by signals that pattern neural tissue. Anterior neuroectoderm will differentiate to forebrain, while posterior neuroectoderm will form midbrain, hindbrain and spinal cord. RA, FGFs and wnts are candidate signals that mediate posterisation (72,111,114).
 

2.2 Model systems for early mouse development

The early developmental processes during Xenopus development described above are comparable to the processes occurring in other vertebrates such as zebrafish and mouse and thus appear to be conserved in evolution (5). Information on the signals mediating early development are available from Xenopus and many gene products which have been reported to be involved in mesoderm or neural induction in this modelsystem have been found to be are expressed during early mouse or zebrafish development (64,89), arguing that these processes are conserved throughout the evolution of vertebrates (112).

Since the study of mouse development is complicated by the poor availability of material and the importance of implantation during the blastocyst stage for embryonal development, in vitro modelsystems resembling early stages of mouse development have been widely used. Embryonic stem cells, derived from the inner cell mass of the blastocyst as well as embryonal carcinoma cells, malignant germ cells known as teratocarcinomas, which resembles inner cell mass cells are both used as model systems. Inner cell mass cells give rise to all embryonic tissues and some of the extra-embryonic tissues (77). ES cells differentiate spontaneously when grown in the absence of serum or LIF; however, fast and complete differentiation of these cells is obtained after RA treatment. Furthermore, the most studied EC cells such as F9-EC and P19-EC also require an inducer to differentiate. Because of the ease of keeping these cells undifferentiated in culture and the possibility of inducing differentiation in different directions via a process resembling early embryonic development, these cells are used widely as a model system for embryonic development (79,119). One of the most potent inducers of differentiation is RA: treatment of F9-EC cells results in differentiation to primitive, parietal or visceral endoderm-like cells, resembling extra-embryonic tissues, depending on the culture conditions (77). Treatment of P19-EC cells with different RA concentrations can lead to the formation of tissues derived from each of the three germ layers, including neural tissue (6,79). These characteristics make P19-EC cells an excellent model system to study the embryonic processes which occur normally during early development.

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3. Differentiation of P19-EC cells

In monolayer, P19-EC cells can differentiate to endoderm following RA treatment while RA treatment of aggregates followed by replating also results in formation of endodermal cells, but more importantly, also mesodermal tissue, including cardiac muscle and skeletal muscle (30,31,52). Furthermore, neuronal cells, such as neurons and astroglial cells are formed (51). As summarized in Table 1, formation of these distinct cell-types is dependent on the RA concentration present during the aggregation process (30). Aggregates from these cells can furthermore differentiate into distinct mesodermal cell types following treatment with DMSO (78) and sodium butyrate (30). Again, increasing concentrations of these chemicals modulate the differentiation of the outgrowing aggregates, resulting in more dorsal mesoderm like tissues such as skeletal muscle (Table 1).
 

3.1 Endoderm differentiation

For F9-EC and to a lesser extent for P19-EC, several inducers of differentiation in monolayer to the endodermal direction have been described and these are summarized in Table 2.
 

Table 2. Chemical inducer-dependent differentiation of EC cells in monolayer

inducer	cell		[inducer]	ref	possible mechanism
RA	F9		>10-8M	122	changes in gene expression
RA	P19		>10-9M	unp.	changes in gene expression
NaBu	F9		1-5 mM	63	c-jun; chromatin stucture
DFMO	F9		5 mM	39	repression of methylation
staurospo.	F9		40 nM	62	phosphorylation,c-jun activation
okadaic ac.		F9	10-20 nM	97	phosphatase inhibitor, c-jun activation

 

Most of these inducers have only been tested in the F9-EC cell system; experiments are currently underway to investigate the ability of P19-EC cells to differentiate in the endodermal direction in monolayer, as well as in the mesodermal and neuronal directions after aggregation. What might be the general mechanism underlying these differentiation events? Almost all of these chemicals induce c-jun expression (24,63,61,62,97). Interestingly, overexpression of c-jun both in F9-EC and in P19-EC leads to loss of EC-cell characteristics, accompanied by induction of differentiation (25,136,137), while overexpression of dominant negative c-jun prevents RA-induced differentiation of F9-EC cells (130). The adenoviral oncogene E1A can also influence the differentiation status of F9-EC cells presumably through regulation of c-jun expression (61). Furthermore, overexpression of E1A 13S in P19-EC cells results in loss of EC cell characteristics and an increase in TRE-binding activity and c-jun expression (23), supporting the involvement of c-jun in these differentiation events. It has been shown that E1A-dependent differentiation of P19-EC cells is dependent on p300 (117), suggesting an involvement for histone modification in this process. The c-jun promoter cannot be activated in undifferentiated EC cells, possibly due to transcriptionally inactive chromatin structure, methylation or the lack of signals required to activate critical transcription factors involved in c-jun promoter activation such as Sp-1, CAAT and AP-1 (24).

A different factor which can induce endoderm (and mesoderm) differentiation of EC cells is the so called END-2 factor secreted by END-2 cells, which can cause differentiation in aggregates of P19-EC cells (90). The observation that the differentiation in the endodermal direction can occur in a monolayer, and the results described above indicate that multiple factors not involving direct cell-cell contact can induce endodermal differentiation of EC cells, possibly depending on c-jun expression.
 

3.2. Mesodermal differentiation

Several of the mesoderm-inducing factors, identified in Xenopus animal cap assays, including activin have, been tested in the P19-EC cell system. Experiments involving the expression of polypeptide growth factors and their receptors and testing the effect of TGFs and activin on EC and ES cells, as summarized by Mummery et al (93) indicate that undifferentiated EC cells are not responsive to these factors. RA treatment in the presence of these growth factors did however influence differentiation, both stimulation and inhibition of differentiation has been reported for TGF (118) and for activin respectively (64,129). Treatment of RA treated aggregates with activin repressed both mesodermal and neuronal differentiation, while monolayer differentiation was not affected (90). A molecular basis for these effects has been suggested concerning the action of activin on P19-EC cells in monolayer, since addition of activin results in increased c-jun expression (84). Differential RA-dependent expression of various mesoderm inducing factors has been reported in EC and ES cells (42,64,91), arguing that these factors could play a role in these systems as well. Recently, direct evidence has been presented suggesting that the mesoderm-inducers activin A and BMP can induce mesodermal differentiation of ES cells in a chemically defined medium (50), but not in serum containing medium (see 50, 91). This could mean (1) that normal DCC-treated serum contains factors that repress the action of these mesoderm inducers (2) that growth in chemically defined medium allows signaling that is normally prevented by growth in serum (3) that ES cells differentiate spontaneously when treated with these growth factors (4) that EC cells and ES cells respond differently to activin. Interestingly, under these conditions, the concentration-dependent differentiation of ventral mesoderm and dorsal mesoderm in response to BMP-4 and activin A respectively, as found in animal cap assays in Xenopus, embryos can be mimicked in ES cells. Expression of mesoderm inducing polypeptide growth factors and their receptors during RA-induced differentiation, and the observed effect upon treatment of ES cells grown in chemically defined medium, suggest that these factors are also involved in mesoderm induction in this in vitro model system.

Based on both overexpression and gene disruption experiments, members of the myogenic family of basic helix-loop-helix (bHLH) transcription factors are proposed to promote myogenesis directly (132). Overexpression of MyoD in P19-EC cells resulted in aggregation-dependent but RA-independent differentiation of these transfectants into skeletal muscle (116). A different method to induce mesoderm is aggregation of EC cells and treatment with RA or DMSO (78), and the END-2 factor can also induce mesoderm (90). The mechanisms underlying these differentiation events are largely unknown but may involve induction of the expression of myogenic regulatory factors such as myf-5, MyoD and myogenin. The presence of undifferentiated EC cells that have not lost the ability to differentiate in aggregates upon treatment with an inducer is a requirement for mesoderm formation and this suggests a requirement for cell-cell interactions. Furthermore, inducer concentration-dependent effects observed for all of the chemical inducers suggest that a differentiation/transformation step could occur, (See Table 1) resulting in the same type of tissues after treatment with increasing concentrations of chemical inducer in the EC-cell system as are formed during differentiation of mesoderm in a ventral/dorsal direction in Xenopus. Interestingly it has been proposed that the organizer region, which has been reported to contain retinoids, can transform mesoderm. This suggests that both in Xenopus and in EC cells the effect of RA could involve differentiation of primitive (anterior) mesoderm through a RA-concentration gradient or a gradient of a signal transducer resulting from RA (emerging from the organizer region) into more dorsal mesoderm such as skeletal muscle and notochord.
 

3.3. Neuronal differentiation

Formation of neurons by P19-EC cells can only occur after aggregation in the presence of high concentration RA (>10^-7 M). It does not occur in response to other chemical inducers. The END2 factor, which can promote endodermal and mesodermal differentiation also only poorly induced neuronal differentiation in comparison with RA. Several gene products have been implicated in neuronal differentiation of P19-EC cells: WNT-1, Bcl2, Brn-2, BMP2 and BMP4, and EGFR in the presence of EGF as summarized in Table 3. In only one case (EGFR) could RA could be omitted for neuronal differentiation. Overexpression of RPTP overrides the need for the aggregation step although RA is still necessary for neuron formation.
 

Table 3. Gene products reported to influence neuronal differentiation of P19-EC cells. .

gene	RA	ref	experiments
Wnt1	+	121	overexpression results in disturbed neuroectoderm differentiation
Brn2	+	40	a.s.Brn-2 represses neuron or glia formation
BMP	+	41	recombinant BMP induces apoptosis thereby preventing neuron formation
EGFR	+	26	overexpresion of the EGF receptor in the presence of EGF leads to neuronal differentiation
EGFR	+	135	a.s.EGFR or truncated EGFR represses neuronal differentiation
RPTP	+	27	overexpression in the presence of RA causes neuronal differentiation in monolayer

 

As described above, neuroectoderm formation in Xenopus is thought to be a default status, caused by a block in BMP4 signaling by proteins present in the organizer (46). In P19-EC cells autonomous induction does not occur, probably because EC cells require expression of certain gene products to make them competent for neuralization. This could involve expression of EGFR, which is dependent on RA treatment of P19-EC aggregates (87,88). Furthermore, BMP4 expression is downregulated in F9-EC cells after RA treatment (107) and addition of recombinant BMP4 to P19-EC cells prevents RA-induced neuronal differentiation (41), making involvement of BMP-4 and the concept of the default state for neuralization of competent ectoderm a possible model for neuronal differentiation of P19-EC cells.

Members of the bHLH family could also be involved in neurogenesis, analogous to their role in myogenesis (132,49), Most of these bHLH transcription factors are expressed during later neuronal differentiation and can promote later differentiation events when injected into Xenopus embryos (68). However recently Ma et al (73) cloned neurogenin, a bHLH protein, that is expressed at early stages of neurogenesis, and promotes neurogenesis when injected in Xenopus embryos. Based on these experiments it was proposed that neurogenin is a neuronal determination factor. Interestingly McCormick et al (81) also cloned neurogenin from RA-treated P19-EC cell aggregates, and the expression of this gene product correlated with the onset of neuronal differentiation and preceded expression of other bHLH transcription factors such as NeuroD (81).

Other mechanisms could also account for neuronal differentiation. Experiments using P19-EC cells indicated that (dorsal) mesoderm/ectoderm interactions as found in Xenopus development cannot be excluded. Pruitt (103) used an in vitro cell-cell interaction method to follow neuronal induction. He introduced the LacZ gene with a gene trap vector into the PAX-3 gene which is specifically expressed in the neuroectodermal and mesodermal lineage. Using mixed aggregates of undifferentiated P19-EC PAX-3 reporter cells and P19-EC cells that were differentiated into mesoderm-like cells with RA or DMSO, he could show that the mesodermal cells transiently produce a PAX-3 and neuroectoderm-inducing activity, to which the undifferentiated ectodermal (reporter) cells respond, resulting in the formation of neurons. This neuroectoderm-inducing activity is reported to be more efficient than the RA-dependent neuronal differentiation in aggregates. The involvement of mesodermal tissue in neural-induction was further suggested by the observation that several primitive streak mesoderm-like P19-EC cell derivatives each expressing early mesodermal markers, can induce PAX-3 expression in the undifferentiated reporter cell (104). The transient ability of these RA or DMSO differentiated cells to induce both PAX-3 expression and neuroectoderm formation, suggests that neuroectoderm formation is dependent on an early mesoderm-like cell. The fact that the END-2 factor, DMSO or low [RA] all allow formation of mesoderm but not of neuroectoderm, while high [RA] permits formation of neurons suggest that RA is required for formation of a ("dorsal-like") mesoderm that can induce neuroectoderm.

It is presently unclear how these different neural induction pathways are connected. Knock out experiments both in mice and in EC cells of these putative neural determination factors, are necessary.
 

3.4. A speculative model for EC cell differentiation in relation to embryonic development

Combined together the experiments described above lead to a model. EC cells remain in an undifferentiated states, until treated with various chemicals. When these cells differentiate in the endodermal direction, they posses the ability to convert undifferentiated ectodermal cells after an aggregation step into an early/primitive mesoderm through an mesoderm-inducing activity such as the END-2 factor. This mesoderm can differentiate into more mature mesodermal tissue such as cardiac and skeletal muscle depending on the inducer concentration. Subsequent processes involving either mesodermal/ectodermal interactions, bHLH transcription factors or lack of BMP signaling will result in formation of neuroecotderm, which differentiates further into neurons. The model described above for early differentiation steps in Xenopus development is surprisingly similar to this in vitro differentiation model for the embryonal carcinoma cells.

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4. Role of RA during differentiation

Given the similarities between EC cell differentiation and early Xenopus development, it can be hypothesized that RA also fulfills an important function in the latter process. Just as RA is required to allow differentiation of EC cells, RA has also profound effects during vertebrate development and on pattern formation (21,82,84). The mechanism by which RA exerts its effect on this process is unknown, but based on the induction/transformation model for early development Durston has suggested that RA could be a likely candidate for the transformation signal. RA or 9-cis RA itself is unable to induce mesoderm or neuroectoderm but it can, when applied shortly before and during gastrulation, cause deletion of anterior structures in Xenopus, mouse and zebrafish. As already described in the introduction, direct HPLC measurements and indirect biochemical assays are indicative of the presence of biologically active retinoids in the organizer region, consistent with the idea that RA is a transformation signal.

In EC cells, RA is involved in the formation of mesoderm and neuroectoderm and their differentiation into more differentiated tissue, and in Xenopus RA is involved in pattern formation, during which differentiation of mesodermal and neuroectodermal tissue occurs. Although in the EC cell system RA can induce endoderm formation, no evidence for a role of retinoids in Xenopus endoderm differentiation has been presented so far. This leads to the question whether RA, modulates early differentiation steps in embryos in the same way as in the EC cell modelsystem.
 

4.1. Role for RA and RARs in early differentiation steps during development

Significant retinoid signaling was determined using a RARE coupled to a minimal hsp-LacZ reporter at E 6.5 to 7.5 in the mouse, in the posterior region of the embryo in all three germ layers, throughout the entire primitive streak, supporting a role for retinoids in A/P patterning along the body axis (109). The RARE-hspLacZ was also activated very rapidly during formation of the neural plate. However no evidence for the presence of a concentration gradient of RA was observed. Interestingly at the E 3.5 blastocyst small dots of RARE activity were observed both in the inner cell mass and the trophectoderm, which could mean that retinoid signaling might play a role at the early differentiation steps (endoderm formation), although the authors doubt its significance (109). Similarly also in zebrafish (53) and Xenopus (Hooyveld et al) retinoid signaling was determined using a GAL-RAR fusion construct, measuring luciferase or -galactosidase activity. In the zebrafish the first significant activity was found from the onset of gastrulation onwards at the dorsal side of the embryo in the embryonic shield, the equivalent of the Spemann organizer in Xenopus, and later at the end of gastrulation, most prominently at the posterior dorsal domain (53). Also during Xenopus gastrulation retinoid signaling is restricted to the dorsal side of the embryo. These data are in agreement with indirect measurements indicating the availability of higher concentrations of active retinoids in the Hensen's node in the early chicken embryo (18), and in the blastopore lip in the Xenopus gastrula (19). Furthermore indirect assays for the presence of active retinoids, analyzing the ability to synthesize RA from retinol, also indicated the presence of higher activities of RA synthesizing enzymes in the Hensen's node (46) and in the floor plate in the CNS (131) and the spinal cord (80) in the mouse. All these regions are believed to be involved in pattern formation. These experiments together suggest that the Speman organizer indeed contains higher concentrations of active retinoids which could be involved in A/P patterning. This idea is further supported by the retinoid regulation of HOX genes, which are thought to be involved in pattern formation (21,65,76), and by the identification of RAREs that are essential for HOX gene expression (29,76). Further evidence for a role of retinoids in early embryonic development came from retinoid deprivation of the yolk sac by injection of anti-sense oligo-nucleotides for retinol binding protein (7). The resulting embryos lack active retinoids as determined using the RAR2 promoter as reporter, and depending on the stage of injection, showed embryonic defects in vitelline vessels, the cranial neural tube and the eye. The downregulation of retinoid signaling is furthermore accompanied by a down-regulation of TGF-1 and Sonic hedgehog (Shh) expression, arguing that retinoids are present in the yolk material, and are required for important developmental processes.

Expression of RAR has been detected using RT-PCR at all stages during early mouse development, including oocytes. RAR was present in oocytes and in the morula and blastocyst stages. From the blastocyst stage onwards, all RARs could be detected using this technique and expression did not change until late gastrulation stage (134). Detailed in situ hybridization (ISH) studies of the expression patterns of receptors, binding proteins and an enzyme involved in RA synthesis during early implantation stage embryos from day 6.5 onward strongly indicate that retinoid signaling is important for these early stages of development (3,28,110,113), no expression was detected by ISH for the for retinoid receptors or binding proteins at blastocyst and early gastrula stage embryos (110). However, they are detectable by RT-PCR (134), and are therefore thought to be expressed at low levels at these early stages of development (110). RNase protection assays during early stages of Xenopus development have indicated that RAR and RXR are expressed until the beginning of gastrulation, while expression of RAR and RXR is detectable at all stages of development. RXR is expressed early pre-gastrula stages from maternal mRNA and is expressed at low levels after gastrulation, while RAR is specifically expressed at high levels during neurulation (11). ISH expression of XRAR further supports the involvement of RAR in neurulation and A/P axis formation, since RAR is expressed at both ends of the A/P axis in late gastrula stage embryos, and high expression is found during neurula stage embryos at both ends of the A/P axis (33). The observed expression of XRAR in the dorsal mesoderm around the organizer region supports a role of retinoids as transformation signal during neuronal differentiation (33). The presence of active retinoids and receptors, combined with the teratogenic effects found in mice and the developmental abnormalities found in zebrafish, mouse and Xenopus after exposure to retinoids during the gastrula stage strongly argue that retinoids are involved in A/P axis formation. Recently support for this model has been presented showing in vitro RA-dependent neural differentiation of neuroectoderm. Animal caps induced to form anterior neuroectoderm by injection of noggin, form more posteriorised neuroectoderm upon RA-treatment, strongly suggesting that RA may be the posteriorising signal for anterior neuroectoderm (99).

Targeted disruption of individual RAR isoforms did not however provide clear evidence for the importance of retinoid signaling for proper embryonic development (56). Mice lacking all RAR isoforms however, show homeotic transformations (70), while targeted disruption of two different RARs causes more severe skeletal abnormalities, indicating that retinoids are indeed required for pattern formation, and that functions of individual RARs are at least partially redundant (71,83). Furthermore also RXRs may fulfill an important role in the retinoid signaling since a phenotypic synergy was found by combined disruption of RXR with RAR or RAR (55,57,58). So far, no targeted disruption of any RAR/RXR combination caused inhibition of mesoderm or neuroectoderm formation, although differentiation of these germ layers is affected as demonstrated by homeotic transformations, heart defects, and neural crest abnormalities (56). Together these experiments support the role of RA as a transformation signal: differentiating or specifying the identity mesodermal or neuroectodermal tissue.

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5. A model for RA-induced EC cell differentiation: role for methylation and chromatin structure.

In the EC cell system too RA also contributes to the differentiation/transformation of mesodermal and neuroectodermal tissue as discussed above. However some aspects of RA-dependent formation and further differentiation of endoderm, mesoderm and neuroectoderm in EC cells seem distinct from these processes in early mouse or Xenopus development. It is clear that in EC cells, in contrast to early embryos, retinoid signaling is essential for formation of endoderm, mesoderm and neuroectoderm as shown by several lines of evidence. The RA-resistant P19-EC cell derivative, RAC65 (52) was shown to contain a mutated RAR that could prevent retinoid signaling by the normal receptors (66,102). Introduction of a dominant negative RAR in P19-EC cells further confirms that functional receptor signaling is required for differentiation of these cells in response to RA (van der Saag unpublished, 22). Furthermore, targeted disruption of RAR or RXR in F9-EC cells prevented, impaired or diminished RA-dependent differentiation in this cell line (13,14,20). Finally expression of a dominant negative RAR in F9-EC cells also prevented RA-dependent differentiation (34).

The mechanisms underlying endoderm formation in vivo are largely unknown but retinoids do not seem to be involved since no or hardly any retinoid signaling is detectable during this early differentiation step. Moreover gene disruption experiments do not show early lethality, while dominant negative RARs also fail to interfere with this early differentiation step (unpublished vand der Saag et al;12,115,128). Why are EC cells (and ES cells), in contrast with early embryos, dependent on the presence of retinoid signalling for (early) differentiation?

Induction of irreversible differentiation, as determined by endoderm-like morphology in monolayer and loss of growth in soft agar relies on the presence of retinoids for only a short period of time (10,86,87). Several other chemical substances can irreversibly induce differentiation of EC cells as discussed above. Furthermore it is thought that RA-dependent activation of RA target genes can trigger the differentiation process (42), as demonstrated by the phenotypes of EC cells that overexpress certain RA target genes such as c-jun(25,135) EGFR(26), NFB (48), MyoD (116). Finally, monolayer differentiation leads to endodermal differentiation while aggregation allows also differentiation in mesodermal and neuronal directions.

Here I propose a model that could explain this apparent contradiction between the requirement of RA for EC cell differentiation into the three germ layers but not for formation of these tissues in Xenopus or mouse embryos. EC cells possess a methylation pattern, resembling normal tissue, and this remains relatively unchanged during culturing (4,59). RA-dependent differentiation of F9 and P19-EC causes an overall demethylation of DNA (105,127). Furthermore treatment of F9-EC with DFMO, which is a DNA methylation blocker, leads to RA-independent differentiation (39). Studies of the methylation patterns of early mouse embryos have indicated that the sperm genome in contrast with oocyte DNA is highly CpG methylated. After fertilization, demethylation occurs and the complete genome remains largely unmethylated until implantation. At a later stage, prior to gastrulation, when endoderm has already formed, massive de novo methylation occurs, leading to an adult-like methylation pattern (15,45). This could mean that ICM cells are largely unmethylated, while EC cells are methylated similarly as adult tissue. The fact that EC cells originate from tumors of embryos implanted in the testis, could explain why the genome of these cells is methylated.

It is widely accepted that methylation can influence transcription, and that different methylation patterns can thus influence gene expression. Possibly, the RA-dependent demethylation in EC cells allows activation of genes which are otherwise inaccessible for transcription. The importance of DNA methylation for embryonic development is demonstrated by the severe developmental abnormalities and embryonic lethality caused by targeted disruption of the DNA methyltransferase gene (69). Although P19-EC cells largely resemble ICM cells, there is a difference with respect to DNA methylation. Possibly an important role for RA could be to promote demethylation, thereby allowing genes that are normally required for mouse development to be expressed. Oct4 gene expression in undifferentiated EC cells is correlated with a non-methylated Oct4 promoter region resulting in down-regulating of the Oct4 promoter (9). The correlation between transcriptional activity, methylation and chromatin structure has been observed before (4,54). By comparing transcriptionally active chromatin found at unmethylated CpG islands and bulk chromatin from methylated DNA, three important differences were found. The first contains 1) naked DNA, 2) acetylated H3 and H4, 3) and low levels of H1 (126). This suggests that RA through DNA demethylation causes a change in chromatin structure, thereby allowing changes in gene expression. Furthermore both levels of histone acetylation and nucleosome subunit composition are changed during development (100), making gene-specific changes between transcriptionally active or inactive chromatin a likely additional explanation for the developmental regulation of gene expression. Most of the above described chemical inducers such as DFMO (39), butyric acid (63,106), HMBA (105), and DMSO (106) also cause changes in methylation pattern or chromatin structure, thereby allowing transcription of genes involved in the differentiation process that are transcriptionally inactive in the absence of these chemicals. On the basis of these observations the changes in DNA methylation patterns and chromatin structure could explain the RA induced differentiation of EC cells. Interestingly ornithine decarboxylase, which is required for de novo methylation, is down-regulated by RA, presumably at the transcriptional level (75), which might explain the observed RA-dependent demethylation. As a consequence, the promoters of the genes involved in differentiation are methylated in the absence of RA, thereby preventing transcription. RA-induced demethylation creates more open chromatin, which is accessible for transcription factors, leading to the RA-dependent change in gene expression. In agreement with this, cofactors for NRs have been shown to act by a destabilization of chromatin through acetylation of histone tails (133,139). The recent observation, linking repression by NR via NCoR, with deacetylation of histones, nicely explains how RAR/RXR can, through modification of histones, keep a RA-responsive promoter silent in the absence of RA, and activate transcription upon RA treatment (138,139). Interestingly, many gene products that have been reported to promote EC cell differentiation such as MyoD, c-jun, c-fos (as described above) are dependent for their transcriptional activity on the presence of p300/CBP. This suggests that these gene products also act in a response element-dependent fashion, by changing chromatin structure through the HAT-activity of p300/CBP, thereby enhancing transcription of promoters that contain binding sites for these transcription factors.

In conclusion, the recent identification of the mechanism underlying transcription repression and transcriptional activation strongly suggest that chromatin structure is an important aspect in transcription regulation. Since RAR can regulate the chromatin structure through the acetylation status of histones, I propose that RAR can through a RARE present in the promoter change the chromatin sturcture, allowing transcription of RA-target genes involved in the differentiation process. Methylation of a promoter of a RA target gene which contribute to the differentiation process, might be kept in a transcriptionally inactive state through methylation of CpG residues. Both metylation status and chromatin structure during differentiation of EC and ES cells, and a detailed analysis of the promoters of the methyltransferase gene(s) and of the genes involved in the acetylation/deacetylation in various cell types should be performed to test this hypothesis.

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