EGF receptor

Effects of Mutation or Deletion

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Egfr and neurogenesis (part 2/2)

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).

To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rho were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis. The effect of Egfr on neuroblast formation was monitored by the neuroblast marker Snail. Anti-Sna staining reveals three columns of SI neuroblasts in the control embryo: the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells. In Egfr and rho mutants, Sna-positive neuroblasts in the intermediate position are frequently missing, with a higher frequency of loss in Egfr embryos. In rho mutant embryos, the frequency of the loss of intermediate column neuroblasts is variable among embryos (Yagi, 1998).

To further examine the effect of the loss of Egfr signaling on the late events of neurogenesis, the progeny was traced for one of the intermediate neuroblasts, NB4-2. NB4-2 gives rise to the RP2 motor neurons, which can be identified by the expression of Even-skipped (Eve) and its unique position. Loss of RP2 neurons in stage 13 is observed (over half the cases examined) with the frequency of loss slightly higher in Egfr than in rho mutants, reflecting the earlier defect in neuroblast formation in stage 9. It is known that the Ras-MAPK signaling cascade is the major target of Egfr in many tissues. To understand whether Ras-MAPK signaling also mediates the Egfr signal in the neuroectoderm and to determine the relative contribution of each component of the pathway, the expression of esg and sc was examined in embryos lacking one of the Ras-MAPK signaling components. The phenotype of mutants lacking either Sos, Ras1, Draf or Dsor1 was examined. As in wild-type embryos, embryos mutant for any of the four genes examined express esg in three separate domains: procephalic neurogenic region, amnioserosa and neuroectoderm. In all cases, the anterior limit of the procephalic expression and the posterior limit of neuroectodermal expression are expanded to the terminus, consistent with the fact that Ras-MAPK is required for the terminal fate specification controlled by Torso receptor tyrosine kinase. All mutants exhibit specific defects within the neuroectoderm where esg expression is derepressed in the intermediate column. Essentially the same phenotype is also observed with sc expression, suggesting the loss of Ras-MAPK signaling has the same consequence as the loss of Egfr. All four Ras pathway mutants show, qualitatively, the same phenotype in the neuroectoderm. The neuroectoderm phenotype in Ras1 mutants is not rescued by a paternal copy of the wild-type gene, suggesting that a relatively high dose of the Ras signal is required for repression of esg and sc in the neuroectoderm (Yagi, 1998).

Rhomboid (rho) is initially expressed in the medial half of neuroectoderm, but repression of esg, ac and sc transcription by Egfr and Ras-MAPK occurs only in the intermediate column, posing a question as to whether or not the site of MAPK activation and the site of transcriptional repression exactly correspond. The spatial and temporal pattern of MAPK activation has been described by the use of an antibody that specifically reacts with the phosphorylated and activated form of MAPK (diphospho-MAPK=dpMAPK), which shows that dpMAPK is distributed in a broad domain in the neuroectoderm in stage 5-7 embryos. dpMAPK is distributed in an 8- to 10- cell-wide area in the neuroectoderm in stage-5 embryos and becomes restricted to the ventral region at the end of gastrulation. This rapidly evolving pattern of dpMAPK expression made it difficult to determine the exact correlation between distribution of dpMAPK and the DV subdomains in the neuroectoderm. A protocol was used to double label embryos with dpMAPK and antisense RNA probes to study the spatiotemporal relationship between expression of dpMAPK, its activator Rhomboid (Rho) and its downstream target, esg. Initial expression of dpMAPK overlaps with that of Rho in stage-5 embryos; dpMAPK expression remains in this broad domain when Rho expression became restricted to the medial column at gastrulation in stage 6, and finally narrows down to a 2- to 3-cell-wide stripe abutting the stripe of Rho at stage 7. Comparison with the mesodermal marker sna shows that the ventral border of dpMAPK expression abuts the neuroectoderm-mesoderm border. Examination of histochemically stained material reveals a sharp ventral border of dpMAPK expression, which gradually declines in the dorsal direction, resembling the pattern of Rho expression. In Egfr mutant embryos, dpMAPK staining is not detectable. These results demonstrate that MAPK activation in the neuroectoderm is dependent on Egfr and follows the spatial expression pattern of Rho, but persists for some time after termination of Rho transcription. The latter observation may reflect perdurance of Rho or its target protein, Spitz (Spi). Alternatively, a ligand other than Spi, such as Vein, might be activating Egfr. The dorsal limit of dpMAPK expression was determined relative to the three separate columns of neuroectoderm revealed by esg expression. In stage 5, the dorsal limit of dpMAPK reaches halfway within the intermediate column and subsequently retracts to the medial column in stage 6 and 7. These data indicate MAPK is activated at least in the ventral half of the intermediate column of the neuroectoderm when it is required to repress transcription of esg. It is concluded that transcription of esg is repressed by a marginal level of MAPK activation (Yagi, 1998).

Why does the high level of dpMAPK in the medial column fail to repress transcription of esg, ac and sc? One possibility is that a factor is present in the medial column that antagonizes or overcomes the events downstream of dpMAPK. A candidate for such a gene is vnd, which is expressed in the medial column in late stage 5 and is required for expression of ac. Expression of esg and sc was examined in vnd null mutant embryos: their expression in the medial column was found to be lost. To understand how vnd controls gene expression in the medial column, a target for vnd was sought in the cis-regulatory regions of an esg enhancer. Expression of esg is regulated by the neurogenic enhancer, which can be divided into two regions, the activator region, which mediates activation in the entire neuroectoderm, and the repressor region, which mediates Egfr-dependent repression. Expression of the esg-lacZ fusion genes was examined in the vnd mutant background. The construct esg-lacZ D1 containing the complete neurogenic enhancer reproduces neuroectodermal expression of esg and is regulated by vnd in the same manner as esg. In contrast, the construct esg-lacZ D5 lacks the repressor region for the Egfr-mediated regulation and is expressed in all three columns. Evidence is provided that vnd does not regulate esg-lacZ D5 and that the target site for vnd regulation is included in the repressor region. vnd is also shown not to be involved in activation of esg or Egfr; rather, it works to counteract the negative effect of Egfr (Yagi, 1998).

Given the results of the present work showing that vnd counteracts the negative regulatory effect of Egfr, a model is proposed for the DV structuring of the neuroectoderm. A gradient of nuclear localized Dorsal protein induces expression of dorsoventrally regulated genes such as dpp, sna, and twi, which determine the extent of the neuroectoderm, and the nested expression domains of rhomboid and vnd. rho determines the domain of MAPK activation, which covers the medial and intermediate columns. vnd is expressed in the medial column where it counteracts the Egfr signal to allow expression of esg. Thus the three columns in the stage 5-6 neuroectoderm are distinguished by unique combinations of activated MAPK and vnd expression. In the lateral column, neither of them are activated or expressed, and esg transcription is activated by default. In the intermediate column, MAPK is activated and represses esg transcription. In the medial column, vnd counteracts activated MAPK to allow the default pathway to activate esg transcription. It is possible that proneural genes are also regulated by the same mechanism. Loss of the Egfr signal leaves two domains, one with and the other without expression of vnd, the pattern likely to be reflected in the appearance of only two neuroblast columns in the later stage. Thus it is proposed that the primary role of Egfr signal in this stage is to define the intermediate domain to the neuroectoderm which is otherwise separated into two domains. It is possible that Egfr signal and vnd have later roles in promoting neuroblast formation in the intermediate and medial columns, respectively (Yagi, 1998).

Photoreceptor axons arriving in the Drosophila brain organize their postsynaptic target field into a precise array of five neuron "cartridge" ensembles. Hedgehog, an initial inductive signal transported along retinal axons from the developing eye, induces postsynaptic precursor cells to express the Drosophila homolog of the epidermal growth factor receptor (Egfr). HH alone is not sufficient for this cartridge assembly process, which depends on the presence of retinal axons. The Egfr ligand Spitz, a signal for ommatidial assembly in the compound eye, is transported to retinal axon termini in the brain where it acts as a local cue for the recruitment of five cells into a cartridge ensemble. Hedgehog and Spitz thus bring about the concerted assembly of ommatidial and synaptic cartridge units, imposing the "neurocrystalline" order of the compound eye on the postsynaptic target field (Huang, 1998).

In the mutant sine oculis, where only a few ommatidia may form in the eye disc, lamina development is restricted to the immediate vicinity of the small number of axons that grow into the lamina target field. The reduced number of arrays of retinal axons in these animals induces a proportionately reduced field of Dachshund-positive cells. A subset of these cells expresses the neuronal marker Elav. The onset of Dac expression is under the control of Hedgehog and neuronal differentiation (as indicated by Elav expression) involves a distinct signal. Is the putative signal for neuronal differentiation restricted to the immediate vicinity of a retinal axon fascicle? When axon fascicles enter a large field of postmitotic lamina precursor cells (LPCs) induced by hh+ somatic clones, Elav-positive cells are found only in the immediate vicinity of retinal axon fascicles. This local inductive effect is also observed for the expression of the gene argos (aos). aos is a direct transcriptional target of Egf receptor activation and encodes a secreted EGF-like product that can act as a negative regulator of Egfr signaling. In the lamina, Aos displays a punctate distribution surrounding the Elav-positive cartridge cells. As in the case of Elav, Hh is not sufficient to induce aos expression in the absence of retinal axons. Retinal axons thus appear to harbor a locally acting neuronal differentiation signal that is distinct from Hh. The local induction of Aos suggests that this signal may act via the Egf receptor (Huang, 1998).

The Drosophila homolog of the Egf receptor is strongly and specifically expressed by LPCs within the lamina target field. The onset of Egf receptor expression coincides with the terminal division of LPCs at the lamina furrow and the appearance of early markers such as Dac. Egf receptor immunoreactivity is found at higher levels among the older LPCs at the posterior of the lamina. The expression of the EGF receptor in the lamina depends on retinal innervation and is not detected in mutant animals, such as eyes absent (eya), that lack photoreceptor cells. Hh is sufficient for the onset of Egfr expression, as determined by ectopically expressing hh+ in the brain of an "eyeless" animal. R cell differentiation is blocked by the eya1 mutation or by maintaining hhts2 animals at the nonpermissive temperature from a time point in early larval development. Ectopic hh+ expression induces the expression of the EGF receptor in its normal anterior-to-posterior gradient. This and additional experiments show that Hh is both necessary and sufficient for the onset of Egf receptor expression in the lamina (Huang, 1998).

The notion that Egfr activity might play a role in cartridge neuron differentiation is suggested by the observation that elav expression in prospective L1-L5 neurons coincides with the expression of aos, a transcriptional target of Egfr activation in many tissues. To determine whether Egf receptor activity is required for cartridge neuron differentiation, a dominant-negative form of the Egf receptor (DN-Egfr) was used to block Egfr signal reception. In the developing eye, strong ectopic expression of DN-Egfr prevents the formation of ommatidial cell clusters. This effect of DN-Egfr is suppressed by a wild-type Egfr transgene, consistent with the notion that the truncated receptor acts by interfering with Egfr signal reception. Widespred induction of DN-Egfr results in a normal array of photoreceptor axons innervating the lamina target field and induces an apparently normal field of DAC-positive LPCs. However, Elav-positive cells are absent from the lamina in these animals. Large DN-Egfr-expressing somatic clones also lack Elav-positive cells when they include the lamina target field. These observations indicate that Egfr signal reception is required for cartridge neuron differentiation but not for the early steps of lamina development that are under Hh control (Huang, 1998).

In the developing eye, the Egfr ligand Spitz is required for the differentiation of all ommatidial cell types, with the exception of the founding R8 cell. Spi is expressed initially by the R8 cell and later by additional cells as they join the ommatidial unit. Spi antigen is found on retinal axons as they project into the lamina. Within the developing lamina, Spi is found on retinal axon fascicles immediately adjacent to Elav-positive cartridge neurons. Spi is thus present at the right time and place to be an Egfr-activating ligand required for cartridge neuron differentiation. Additional experiments show that spi is required for cartridge formation (Huang, 1998).

Spi is synthesized as a transmembrane molecule. An artificially truncated form of Spi (secreted Spi, or sSpi), containing most of the extracellular portion of the molecule, has been shown to activate the Egf receptor both in vivo and in cell culture. In the developing eye, ubiquitous expression of sSpi induces the differentiation of ommatidial precursors without their assembly into ommatidial cell clusters. sSpi expression likewise can trigger ectopic neuronal differentiation within the lamina. To determine whether these ectopic neurons included a bona fide L-neuron cell type, the specimens were stained with an antibody against the Brain-specific homeobox (Bsh) protein. Bsh expression is an early marker of L5 differentiation and coincides with the onset of elav expression in a single medial layer in the posterior two-thirds of the lamina. With the expression of sSpi, ectopic Bsh-positive neurons are found throughout the three medial cell layers of the lamina. These include cells at the anterior of the lamina, where Bsh-positive cells are not seen in the wild type. Thus, LPCs that are normally destined for elimination by apoptosis or that undergo neuronal differentiation precociously can assume a proper L-neuron identity. In sum, these data indicate that spi+ activity is sufficient for the onset of cartridge neuron differentiation in the lamina (Huang, 1998).

The role of an individual ommatidial fascicle as the "founder" of a cartridge ensemble, together with the precision of axon pathfinding in this system, serve to impose the "neurocrystalline" order of the compound eye on the developing postsynaptic field. This mechanism yields a precise numerical match of ommatidial and cartridge units. The component axons of an ommatidial fascicle might additionally make important individual contributions to the specification of the number and type of postsynaptic cells in a cartridge. For example, individual R axons may make important individual contributions to the spatial and temporal pattern of Spi expression. A dynamic interplay between the extracellular levels of SPI and its negative regulator, Aos, might provide the tight localization of spi+ activity necessary for this axon-cell signaling. Following cartridge neuron differentiation, a remarkable feat of "axon-shuffling" occurs as the six R1-R6 axons of an ommatidial fascicle separate and migrate laterally to form their synaptic connections in a set of six neighboring cartridges. In the adult lamina, a synaptic cartridge thus receives its complement of R1-R6 axons from six ommatidial units whose axons did not contribute to its induction. The assembly of this precise circuitry nonetheless relies on the order imposed on the lamina during its initial inductive phase. A test of this notion may provide a significant insight into the establishment of precise synaptic circuitry in this and other systems (Huang, 1998).

Cell adhesion molecules (CAMs) implement the process of axon guidance by promoting specific selection and attachment to substrates. In Drosophila, loss-of-function conditions of either the Neuroglian CAM, the FGF receptor coded by the gene heartless, or the EGF receptor coded by Egfr display a similar phenotype of abnormal substrate selection and axon guidance by peripheral sensory neurons. Moreover, neuroglian loss-of-function phenotype can be suppressed by the expression of gain-of-function conditions of heartless or Egfr. The results are consistent with a scenario where the activity of these receptor tyrosine kinases is controlled by Neuroglian at choice points where sensory axons select between alternative substrates for extension (Garcia-Alonso, 2000).

The ocellar sensory system (OSS) offers a simple scenario for the study of axon guidance at the cellular level. During axon guidance, growth cones make decisions at choice points. In order to change trajectories at these choice points, it is assumed that signal transduction mechanisms should operate to transform specific extracellular information in the modulation of their actin cytoskeleton. In the OSS, the initial decision to attach or not to attach to the head epithelium appears to be a key choice (at the first choice point) for two types of sensory axons as they navigate to their respective targets in the brain. Due to the process of head eversion, ocellar pioneer (OP) axons must navigate in the extracellular matrix (ECM), free of adhesion to the underlying epidermis. Reciprocally, bristle mechanosensory (BM) axons should follow the epidermis before and after head eversion, since they do not reach the brain until this later stage. Should BM axons initially extend apart from the epithelium, they might possibly be unable to follow a physical substrate toward their brain targets after head eversion. Therefore, the process of head eversion establishes a constraint that prevents substrate redundancy between ECM and epithelium. OSS axons must make a second decision in order to leave the surface of the head and project to the brain (at the second choice point). OP axons leave the ECM surrounding the head capsule toward the brain before head eversion. In contrast, BM axons leave the head's internal epithelial surface after head eversion, when several BM axons have converged together. Each of these decision processes are abnormal in nrg, htl, and Egfr mutant individuals. OP axons can decide to attach to the epithelium, preventing them from reaching the brain. OP axons can fail to leave the internal surface of the head (even when extending free of epithelial attachment) and project to ectopic positions within the head after eversion. BM axons can be found extending, although they are abnormally separated from the epidermis after head eversion, suggesting a failure of attachment to the epithelium before head eversion. BM axons also can stall in the epidermis, suggesting that Nrg may also promote axon extension. Finally, BM axons can fail to leave the epidermis toward the brain, as they should, remaining instead within the epidermal layer, where they project in abnormal directions. In such cases, they can sometimes be observed to perforate the epidermis and project outside of the head, suggesting that BM axons navigate in the epithelial surface, using proteases to facilitate their movements. In contrast, when attached to the epithelium, OP axons seem to have difficulty extending properly. One possibility is that extension in the epithelium requires this perforating activity that BM axons have and that may be missing in OP axons that normally extend in the ECM (Garcia-Alonso, 2000).

Since nrg, htl, and Egfr mutants exhibit similar OSS axon phenotypes, it seems possible that they function in a common mechanism during OSS axon guidance. In order to test genetically if Nrg behaves as an upstream regulator of the Fgfr and the Egfr, different double mutant combinations were constructed between nrg and gain-of-function conditions for htl and Egfr (which should function independently of upstream regulation). Gain-of-function conditions of htl can partially suppress the nrg phenotype. Elp alleles behave as gain-of-function conditions of Egfr and behave as strong suppressors of nrg OSS axon phenotype. Therefore, although it is still possible that Nrg could also perform some role based on pure adhesion, the results are most consistent with the idea that both Htl and Egfr mediate Nrg function in OSS neurons (Garcia-Alonso, 2000).

Htl and Egfr exhibit some specificity in their effects on OSS axon guidance. Htl seems to be preferentially required by OP axons and BM axons to project to the brain, while Egfr seems to be more involved in BM axon attachment and extension in the epithelium. In contrast to in vitro studies in vertebrates, no evidence has been found that Htl promotes axon growth. Rather, in the Drosophila OSS, it seems that the Egfr is preferentially required for outgrowth. This discrepancy with the vertebrate in vitro studies could be explained if the Drosophila in vivo situation were more prone to the deployment of compensatory molecular interactions (which might mask a role of Htl in axon growth) than the in vitro situation. In such a case, a deficit of one RTK could be partially compensated by an increase in the activity of the other RTK. This could happen if, for example, different RTKs were regulated through a common negative feedback loop. This explanation would also help explain the presence of a mild phenotype in consititutively active lambda-Htl individuals and would account for those cases in which some nrg OSS alteration is enhanced by an increase or suppressed by a reduction in RTK activity. This explanation is also consistent with the lack of effect of the gain of function of one RTK over the loss of function of the other (since this condition would itself increment the activity of the former RTK) (Garcia-Alonso, 2000).

It is likely that the RTKs also mediate the function of signals other than Nrg during OSS axon guidance. This is suggested by the fact that the nrg phenotype is weaker than the RTK phenotype and by the fact that it can be enhanced by a reduction of 50% in the amount of Egfr. These signals could represent other CAMs or growth factor ligands diffusing from the brain. One suggestive possibility is that OP and BM axons depart from the head capsule in response to some as yet unidentified diffusible signal from their brain targets (acting in addition to Nrg signaling). Further studies will be necessary to evaluate this possibility (Garcia-Alonso, 2000).

What would be the signal that triggers Nrg-dependent RTK activation? Nrg, like its vertebrate homolog L1, can behave as a homophilic CAM. It has been proposed that the homophilic interaction of L1 would activate the function of the Fgfr. Therefore, it is proposed that the homophilic interaction between Nrg180 (the neural-specific form) molecules would give a positive input on RTK activity in both OP axons (from the beginning of axon extension) and BM axons (after several mechanosensory axons have converged), which would signal the axons to lift off from the epidermis and project to brain targets. However, BM axons initially extend as single processes and interact with the Nrg167 form in the epithelium, where this interaction would result in the activation of the RTKs, and RTK signaling would promote extension in the epidermis. This shift in the RTK's activity outcome might be caused by the involvement of some other molecule specifically interacting with Nrg167. In agreement with this idea, rescue experiments of nrg using Nrg180 reveal that BM axon extension on the epidermis cannot be implemented by this molecular form. Since homophilic binding between the different Nrg forms is possible, this high degree of specificity suggests the existence of additional molecules (specifically interacting with Nrg167) that mediate BM axon association with the epithelium. Some observations are consistent with this model. (1) OP axons fasciculate with one another (50 or so per ocellus) from the very beginning of axon extension. These axons fasciculate together due to the presence of Neurotactin and other CAMs in the membrane. Defasciculation of OP axons (caused by a lack of Neurotactin) increases the chances that OP axons extend abnormally in the epidermis. These results suggest that fasciculation helps generate a robust process of axon guidance. In this model, defasciculation would reduce the probability of Nrg180–Nrg180 interactions between OP axons, producing a deficit of RTK activity, therefore, making them more likely to extend attached to the epidermis. (2) BM axons initially follow the epidermis in isolation from other axons but begin to converge as they approach the dorsal antennal field. After several BM axons have converged together, the BM fascicle lifts off from the epidermis. Thus, the signal for BM nerves to leave the epidermis might be a given threshold of Nrg180–Nrg180 interactions between the different BM axons. If this model is correct, the specificity of the OP and BM growth cone interaction with the epidermis would reside in the way Nrg167 and Nrg180 differ in their interactions with other molecule(s). Nrg167 differs from Nrg180 in the cytoplasmic domain. It has been previously shown that the cytoplasmic domain can regulate the adhesive properties of the extracellular part of the protein. Therefore, it is possible that some CAM molecule might specifically interact with Nrg167 to help promote initial RTK activation and attachment to the epithelium in BM axons. This molecule could represent the Drosophila homolog of some of the known vertebrate heterophilic partners of L1 (Garcia-Alonso, 2000).

In summary, these results strongly support the idea that Neuroglian functions in axon guidance by regulating the activity of both the Fgfr and the Egfr and suggest a scenario where other CAMs and growth factor activities would act in concert with Neuroglian for RTK regulation. In addition, the possible existence of cross-regulatory circuits between RTKs would add another level of control that might help to understand the high degree of canalization displayed by the axon guidance process from Drosophila to mammals (Garcia-Alonso, 2000).

back to Egfr and neurogenesis part 1/2

Table of contents

EGF receptor : Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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