47
4. Discussion and Conclusions
In this research, using Xenopus as a convenient model system to rapidly test gene function we investigated the function of Noggin, an antagonist of BMP signaling. As described previously, Noggin, together with other growth factors, allows neural development by binding BMPs in the extracellular space in embryos, thus antagonizing BMP signaling. Our observations raise the possibility that in naive ectodermal explants (ACES cells), the high level of antagonism of BMP signaling by Noggin alone is able to trigger retinal gene expression and consequently drive these cells effectively toward retinal cell differentiation, presumably through several sequential induction steps. For directing retinal fate differentiation from embryonic stem cells, Noggin action in the extracellular space makes it as a convenient molecule, that could be simply administrated in culture medium, an easier approach than transfection of regulatory genes into cells. The data presented in this thesis highlight a central role for Noggin in retinal fate determination.
4.1 ACES cells expressing high doses of noggin are directed to retinal lineages
Animal caps from embryos injected with mRNAs transcribed from the genes of interest can be easily explanted, in vitro cultured and tested for the expression of specific markers. Using this animal cap assay, we investigated the effects of different doses of noggin mRNA on the expression of marker genes. As we expected, all noggin doses tested (2.5pg to 20pg) were able to activate the expression of Sox2, a general neuronal marker (Fig.3.1.1 A), since it has been known for a long time that Noggin can induce neuronal differentiation in Xenopus ectodermal explants by antagonizing BMP signaling (Lamb et al., 1993; Zimmerman et al., 1996). We also found that these cells expressed CycD1, Xotch, Xash1, NeuroD, that are neural precursor proliferation markers, as well as Rx1 and Pax6, undifferentiated retinal precursor markers (Fig.3.1.1 B; Fig.3.5.3; Fig.3.5.5). These findings demonstrate that all noggin doses tested induce ACES cells to undergo proliferation, neurogenesis and at least the initial steps of retinogenesis.
For induction of terminal retinal differentiation in ACES cells, we found that noggin has a dose-dependent activity, that is, only dosages of 15pg or more of noggin mRNA were able to elicit the expression of terminal differentiation markers of specific retinal cell types, such as Opsin (photoreceptors, Whitaker et al., 2004) and Brn3d (ganglion cells, Poggi et al., 2004) (Fig.3.1.1). However, low doses of noggin mRNA only induce the formation of retinal progenitors (Rx1- and Pax6- positive, Fig.3.5.3) but not differentiated retinal neurons (Opsin- and Brn3d- negative, Fig.3.5.3).
We also determined the cell identities of 20pg noggin-expressing ACES cells at early developmental stages (stage 15, at which the transplantation is performed). We found that 20pg of noggin mRNA induce the expression of the forebrain markers such as Otx2, BF1 and Eomes (Lupo et al., 2002), the expression of the presumptive neural crest marker Snail (Linker et al., 2000), and the expression of the eye field specific transcription factors such as Rx1 and Pax6, but not the expression of the hindbrain marker Krox-20 (Bradley et al., 1993) (Fig.3.1.4). These
48 observations suggest that high doses of noggin trigger the anterior neural induction, and at neurula stage high dose noggin-expressing ACES cells can be considered as a tissue with anterior neural tissue characteristics, which has the competence to develop into forebrain neurons, neural crest cells and retinal neurons.
We then characterized cell identities induced by different doses of noggin at stage 39, and we found that 2.5pg noggin-expressing ACES cells give rise to forebrain neurons, neural crests and cement gland-like structures, but not differentiated retinal neurons. By contrast, 20pg noggin-expressing ACES cells are almost entirely composed of differentiated retinal neurons and proliferating retinal progenitors when counting the cell numbers of different marker staining. It should be noted that, using in situ hybridization to detect the expression of neuronal forebrain markers we did not find any visible staining for Nkx2.4 and Emx1 on the sections of explants (Fig.3.1.3), while using RT-PCR analysis we found a weak expression of forebrain markers such as BF1 and Eomes (Fig.3.5.5) in the RNA extracts. These divergent results may be caused by the different sensitivities of the detection techniques. Moreover, there is a possibility that not all ACES cells received equal amounts of noggin mRNA following cell cleavage, leading to some cells expressing relatively lower doses of noggin, these cells will not undergo retinal differentiation but will differentiate into forebrain neurons.
Previous studies in Xenopus indicate that discrete BMP signaling levels induce three cell identities within the early ectoderm: low BMP signaling near the organizer specifies neural cell fate in the dorsal ectoderm, intermediate BMP levels induce neural crest in the marginal ectoderm, and high BMP activity results in epidermal fate in the ventral ectoderm (Wawersik, et al., 2005). According to other studies, the embryonic ectoderm can be subdivided into four domains: neural plate (demarcated by Sox2/Sox3 expression domain), neural crest (demarcated by FoxD3/Slug expression domain), pre-placodal ectoderm (demarcated by Six1 expression domain) and epidermis (Brugmann et al., 2004; Glavic et al., 2004; Schlosse and Ahrens, 2004). The expression levels of these fate specific genes in animal cap explants are responsive to the concentrations of Noggin. In particular, high concentrations of Noggin promote the expression of Sox2, intervening concentrations of Noggin promote the expression of FoxD3 and Six1, and low concentrations or an absence of Noggin promotes the expression of XK81 (epidermal marker) (Brugmann et al., 2004; Knecht and Harland, 1997; Marchant et al., 1998). In addition, it has been shown that Noggin, by antagonizing BMP signaling, can induce cement gland tissue at low concentrations and neural tissue at higher concentrations in Noggin-treated animal caps (Knecht and Harland, 1997). Another study conducted in Xenopus embryos shows that misexpression of pax6 in ventral animal blastomeres can convert these competent epidermal precursors to form ectopic retinal tissue, and coexpression of pax6 and noggin significantly enhances the frequency of ectopic retinal formation, suggesting that the ability of pax6 to elicit a retinal fate depends on neural inducing level (Kenyon et al., 2001). In our findings, it is very clear that both cement gland-like structures and neural crests are induced in low doses but not in high doses of noggin-expression ACES cells at late developmental stages. Collectively, these observations suggest that low doses of noggin induce neurogenesis, the formation of cement
49 gland-like structures and of neural crests, and high doses of noggin effectively direct ACES cells toward retinal differentiation following neurogenesis. It will be interesting to determine the dose ranges of noggin in the induction of different cell fates, which will be useful for in vitro expansion of specific cell types, such as neural crests.
It will be also interesting to determine the expression levels of noggin in different developmental stages in order to more precisely elucidate Noggin action during neural induction and retinal fate induction. Since neural induction and eye field specification occur sequentially in Xenopus embryos as described in Section 1.4, it remains a possibility that high doses of noggin mRNA in ACES cells have a longer life-span, resulting in several stepwise inductions ending with retinal differentiation, whilst the fact that low doses of noggin mRNA are insufficient to drive retinal differentiation might be due to their shorter life-span in ACES cells. To validate this idea, we intended to determine the levels of Noggin protein in ACES cells at different stages by Western blotting, using an antibody against Noggin protein. These experiments did not succeed, since this commercially available antibody gives a high background on blots of protein extracts from WT embryos, but no signal on blots of extracts from noggin-expressing ACES cells. We speculate this might be due to the secretion and diffusion of Noggin in the culture medium. Either further improvement of Western blotting or concentration of culture medium is worthy of trying to clarify this idea.
Animal caps can be induced to form neural tissue including eye structures upon treatment with secreted molecules, and thus have the potential to form retinal cells (Kessler and Melton, 1995; Bouwmeester et al., 1996). It is worthwhile to note the differences among the previously reported secreted molecules and Noggin. Animal cap explants treated with Vg1 protein, normally expressed in the vegetal site of Xenopus embryos, can form “embryoids” with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system (Kessler and Melton, 1995). It is now clear that Vg1 functions in the early blastula to induce the formation of dorsal mesoderm, and the formation of head structures is a consequential induction by dorsal mesoderm and probably by several BMP antagonists such as Chordin and Noggin induced by Vg1 (Birsoy et al., 2006). Cerberus is expressed in the Spemann organizer and its overexpression can induce the formation of secondary head structure so that it has been considered as a head inducer. Piccolo and co-workers found that Cerberus acts through multiple antagonisms, inhibiting BMP, Nodal and Wnt signaling pathways, and they suggest that head formation needs the triple inhibition of these pathways (Piccolo et al., 1999). Surprisingly, Noggin, as a specific antagonist of BMPs, induces retinal differentiation in the absence of mesodermal signals. This is intriguing, since no reports describe that Noggin by itself can elicit retinal fates. Furthermore, noggin overexpression experiments in Xenopus embryos carried out in our lab showed that noggin ectopic expression in the ventral side of embryos can induce with a high efficiency the formation of secondary axes with head, complete of cement gland and eyes (data not shown).
We cultured ACES cells from blastula stage in simple medium containing only saline, the only inductive cue is the noggin overexpressed in these cells by microinjection. It is easy to
50 conclude that neurogenesis and retinal differentiation can be attributed to the initial induction of Noggin. This work suggests that, at least in Xenopus, specific dosages of noggin can effectively drive pluripotent cells toward retinal cell fates.
4.2 ACES cells expressing high doses of noggin form eyes that are
morphologically and functionally identical to normal eyes
Xenopus embryos from which an eye field has been removed survive, develop normally, but lack the eye on the operated side. Using a transplantation assay, ACES cells expressing different doses of noggin and GFP or expressing GFP alone were implanted in host embryos from which one of the two eye fields had been removed. As control, we transplanted GFP-expressing ACES cells that never form an eye and only make epidermis, these cells exhibit extension in the host embryos, when grafted in the posterior neural tube, that undergoes consistent extension movements (Fig, 3.2.1). Similarly to cultured in vitro, the GFP-expressing ACES cells explanted from blastula stage lose competence to form neural tissue and only develop into epidermis. However, 2.5pg noggin-expressing ACES cells transplanted in host embryos, even if they showed no eye formation, stayed grouped and were neuralized (Fig, 3.2.3). By contrast, high doses of noggin-expressing ACES cells effectively form morphologically recognizable eyes complete with lens in the eye field transplants (73.54%, Fig. 3.2.1).
We reasoned, if ACES cells are overexpressed high doses of noggin and start to undergo retinogenesis in vitro, after transplantation to the region of eye field, they should form an eye under the inductive signals from underlying mesoderm and adjacent head mesenchyme. Therefore, we also implanted the high doses noggin-expressing ACES cells in posterior neural tube, an ectopic region far away from the eye forming inductive cues. Interestingly, the eyes form with even higher frequency in the posterior location than in the eye field region (93.33% vs. 73.54%), strongly suggesting that high doses of noggin itself is able to activate a developmental program that leads exclusively to retinal differentiation, resulting in the eye formation in vivo. Meanwhile, we found that the low doses noggin-expressing ACES cells were neuralized but failed to form eyes even if they received the inductive signals driven the eye formation in the head region, suggesting that neuralization is not a sufficient requirement for retinal fate determination. Therefore, retinal fate determination needs neuralization (induced by Noggin) and other secondary events activated only by high doses of noggin both in vivo and in vitro.
It has been shown that in Xenopus embryos the specification of the eye field occurs as early as gastrulation stages (around stage 12.5), and it is shown by the expression of a set of eye field transcription factors (Zuber et al., 2003). In our results, the data of RT-PCR molecular marker analysis on ACES cells showed that explants expressing high doses noggin in vitro cultured to stage 15 adopt cell fates including neural plate, presumptive retinal cells, and presumptive neural crests (Fig.3.1.4). This means that the retinal fate of high doses of noggin-expressing ACES cells has been defined before transplantation, and the only inductive cue in ACES cells is Noggin. Moreover, GFP expression demonstrates that the induced eyes derived from the noggin-injected ACES cells. Together with the elucidation above, we therefore speculate that the formation of eyes by high doses noggin-expressing ACES cells either in the eye field or in the
51 posterior neural tube can be attributed only to inductive cues from Noggin and its downstream secondary signalings.
In addition, GFP expression was never found in the lens of the induced eye, suggesting that noggin-treated explants have been specified to become retina. The lens is formed by lens-competent ectoderm only in the head region of host embryos (Servetnick and Grainger, 1991). When the high doses noggin-expressing ACES cells integrate into host embryos, the reciprocal interaction between the retina-forming ACES cells and lens-forming ectoderm (lateral to eye field) results in the formation of an induced eye having a histological structure equivalent to a normal eye (Fig.3.2.2). This also explains that in the posterior transplants, the eyes originating from high doses noggin-expressing ACES cells are composed of retinal pigment epithelium and neural retina, but not lens, since posterior ectoderm has no competence to form lens (Servetnick and Grainger, 1991). It has been shown that the vertebrate lens influences development of retina in addition to transmitting and focusing light on the retina (Strickler et al., 2007), therefore the eyes formed in the posterior location exhibit less organization.
Since eyes originating from eye field transplants with ACES cells expressing high doses of noggin have a morphology and histology indistinguishable from WT eyes and include all major retinal cell types, we therefore analyzed if they were also functional. First, electrophysiological recordings of single rod photoreceptors show noggin-induced photoreceptors respond identically to WT photoreceptors after a light stimulus. Second, the same expression pattern of c-fos gene expression in the noggin-induced retina and the WT retina indicates functional integrity of the different layers within the retina. Taken together, with these data we demonstrate that high doses noggin-expressing eye field implants are able to respond to light, and are thus functionally indistinguishable from normal eyes.
4.3 The possible mechanisms of action of Noggin in ACES cells
4.3.1 Correlation between Noggin-mediated retinal induction and antagonism of BMP signaling
As we know, Noggin serves as an antagonist to BMP ligands, binding them extracellularly thus preventing activation of BMP receptors, ultimately causing cells to adopt neural fates. The role of Noggin in neural induction has been studied for many years, but no direct correlation between Noggin and retinal fate specification had been previously shown. Only recently, the group of Reh reported that efficient generation of retinal progenitor cells from human embryonic stem cells with a combination of Noggin, Dickkopf-1 (dkk1, a secreted antagonist of the Wnt signaling pathway) and IGF-1 (insulin-like growth factor-1) (Lamba et al., 2006). However, the underlying mechanism has not been defined. In our findings, Noggin by itself initiates an entire developmental program driving pluripotent ACES cells to retinal fates without additional inductive factors. This is intriguing, since it raises the possibility that inhibition of BMP signaling by Noggin is sufficient to promote retinal differentiation. To test this hypothesis, we employed dominant negative type I BMP receptor (tBR) to inhibit BMP signaling and compared its effect with that of noggin on retinal fate induction.
52 There are multiple BMP ligands such as BMP2/4/7 involved in epidermal induction. It has been shown that inhibition of one of these BMP ligands does not induce neural tissue in ectoderm. However, expression of a single tBR is capable of inducing neural tissue in ectoderm (Suzuki et al., 1997). tBR is obtained by deleting the intracellular domain of the receptor and retains a transmembrane domain which is involved in receptor-receptor association in the plasma membrane (Graff et al., 1994). In Xenopus, it has been shown that tBR can antagonize multiple BMP ligands by disrupting functional hetero- or homo-oligomeric complexes of type I and type II receptors (Suzuki et al., 1997), but does not inhibit signaling of related TGF-β family ligands such as activin (Schmidt et al., 1995). tBR therefore is generally used as a factor to specifically antagonize the BMP signaling pathway completely in the early Xenopus embryos and animal cap explants (Suzuki et al., 1994; Sasaiet al., 1995; Suzuki et al., 1997; Massé et al., 2004).
However, we noticed that the activity of tBR in Xenopus embryos is different from that of noggin. When noggin is overexpressed in the ventral marginal zone of early embryos, secondary axes are induced, and most of the induced axes have head structures including eyes and cement gland. If noggin is overexpressed dorsally, the injected embryos display a hyperdorsalized phenotype. By contrast, when tBR is overexpressed in the ventral marginal zone of early embryos, secondary axes are induced, but in no case, they have anterior head structures and only possess the spinal cord (Fig.3.5.1 and Frisch and Wright, 1998). If tBR is overexpressed dorsally, the injected embryos are quite normal. We think that BMPs are normally blocked by Noggin and other BMP antagonist in the dorsal side of embryos so that ectopic overexpression of tBR dorsally exhibits no phenotypic effect. These observations are consistent with previously reported results (Suzuki et al., 1994). Therefore, although both Noggin and tBR can directly neuralize the animal cap ectodermal explants, we conclude that they have different activities in specification of anterior-posterior neural tissue in vivo.
Successively, we compared the activities of tBR and noggin in retinal cell fates induction. We found that tBR exhibits activities similar to those of low doses of noggin in ACES cells. First, both tBR and 2.5pg of noggin can properly neuralize the ACES cells and induce the formation of cement gland-like structures in ACES cells when cultured in vitro (Fig.3.5.2). Second, neither tBR- nor 2.5pg noggin-expressing ACES cells can rescue eye development in host embryos with one eye primordium removed (in majority cases 2.5pg noggin-expressing ACES cells did not form eyes in the transplants). Finally, molecular analysis by RT-PCR shows similar patterns of marker gene expression in ACES cells at stage 37 expressing either 800pg of tBR or 2.5pg of noggin (Fig.3.5.3). Since a dose-dependent activity of tBR is excluded, we thus conclude that ectopic overexpression of tBR can not completely block the BMP signaling, and behaves like low doses of noggin.
Overexpression of tBR disrupts functional complexes of type I and type II receptors on the membrane and causes the failure of the transmission of BMP signals since it lacks the intracellular domain, responsible of the phosphorylation of downstream BMP effectors, thereby resulting in blocking BMP signaling. However, why tBR even at very high doses can not induce
53 retinal fates in ACES cells while high doses of noggin can? We could think of two possibilities: tBR can not block BMP signaling completely or at sufficiently high levels, or Noggin does not behave only as a BMP antagonist. For the first assumption, it is not so surprising since there is a possibility that unknown BMP type I receptors exist in Xenopus but have not been cloned yet, and another possibility is that induction of retinal cell fates by high doses of noggin might employ other types of BMP receptors. Therefore, further investigation by using combination of both dominant negative type I and type II BMP receptors or using more potent BMP inhibitors, such as Smad5-sbn (a dominant negative form of Smad5, Marchal et al., 2009), might provide more complete blockade of BMP signaling, thereby helping to establish a correlation between Noggin-mediated retinal induction and antagonism of BMP signaling. As to the second assumption, our research presented here can not support it, but future studies by immunoprecipitation experiments to search for Noggin interactors might give some evidence. 4.3.2 The correlation of Noggin and downstream secondary signalings
Recently, we performed microarray analysis to search for downstream effectors of Noggin. The samples we tested include: GFP-expressing ACES cells, 2.5pg noggin-expressing ACES cells and 20pg noggin-expressing ACES cells, which were all harvested at stage 15, RNAs were extracted and probed on Agilent Xenopus oligo-Microarray Kit 4 x 44K multiplex format (Agilent Technologies, Santa Clara, California). The transcriptional profile of each condition was pairwisely compared. Although the detailed description of microarray analysis is not presented in the thesis, we present preliminary functional validation of some candidate genes. Interestingly, many of the differentially expressed genes can be classified into several important signaling pathways such as Shh, Wnt and BMP. It is likely that investigation of these signaling pathways will give a clear repertoire of the factors besides Noggin required for successful retinal specification.
Since the data of microarray analyses show that some components of Shh signaling pathway are transcriptionally upregulated in ACES cells expressing 20pg of noggin mRNA, we then proposed that Shh signaling is activated as a downstream event of the action of high dose noggin in ACES cells and plays a synergistic role with Noggin in the induction of retinal cell fates in ACES cells. To test this hypothesis, we used Cyclopamine (an inhibitor of Shh signaling, Decembrini et al., 2009) to block the Shh signaling in ACES cells expressing 20pg of noggin. The efficiency of eye formation by 20pg noggin-expressing ACES cells treated with Cyclopamine is decreased when compared with untreated noggin-expressing ACES cells. We also found that in the treated-ACES cells the expression of terminal differentiation markers such as Brn3d and Opsin are downregulated, but the expression of Snail is activated (Fig.3.5.5). As we discussed in Section 4.1, the neural crest fate (Snail-positive) is induced with intervening levels of Noggin-mediated BMP antagonism. The activation of Snail strongly suggests that blockade of Shh signaling counteracts the inhibition of BMP signaling by high doses noggin in the ACES cells, and consequently, the expression of EFTFs and of the terminal differentiation markers are downregulated. We therefore propose that Shh signaling pathway plays a synergistic role with high doses of noggin in promoting retinal cell fates in ACES cells.
54 BMP and Shh signalings have been known to collaboratively regulate developmental events and gene expression in several aspects of embryonic development. For example, in the D-V patterning of neural tube, dorsally produced BMPs and ventrally produced Shh antagonize each other, and through this mutual antagonism, they set up opposing gradients that control the distinct combination of genes, thereby establishing the polarity of cell differentiation (Wilson and Maden, 2005). In notochord, BMP antagonists (such as noggin) are normally coexpressed with Shh and might generate a pemissive environment for Shh-mediated induction of floor plate (Patten and Placzek, 2002). Moreover, the coordinated actions of BMP and Shh also appears in the somite patterning (Marcelle et al., 1997), in the development of vertebrate limb (Drossopoulou et al., 2000), and in the specification of the polarity of the optic vesicle (Kobayashi et al., 2010). Our findings provide more information about cross-talk between BMP and Shh signaling. This study represents an initial step in understanding the signaling interactions involved in generating retinal cell types in ACES cells.
Further work is needed to better study the correlation between Noggin and Shh signaling as well as other signaling pathways, by using treatments with agonists and antagonists. Some attempts have been done to investigate whether activation of Shh signaling is sufficient to induce retinal fate in ACES cells by using its agonist SAG (Smoothened-agonist, Frank-Kamenetsky et al., 2002) to activate Shh signaling in GFP-expressing and 2.5pg noggin-expressing ACES cells. We have some preliminary results which positively support our hypothesis but they need to be carefully replicated and verified. If we can identify some key factors (genes and/or signaling molecules) acting synergistically with Noggin to induce retinal fates, this knowledge not only sheds light on the molecular mechanisms essential for stem cell differentiation but also is useful in driving embryonic stem cells toward retinal fates in combination with Noggin. If these key factors are agonists or antagonists of a certain signaling pathway, they can be easily synthesized in large quantities and applied in cell culture.
