Morphogenetic Movements Underlying Eye Field Formation Require Interactions Between the FGF and Ephrinb1 Signaling Pathways

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Developmental Cell, Vol. 6, 55–67, January, 2004, Copyright 2004 by Cell Press Morphogenetic Movements Underlying Eye Field Formation Require Interactions between the FGF and ephrinB1 Signaling Pathways

Kathryn B. Moore,1,4 Kathleen Mood,3 specification occurs (Hatakeyama et al., 2001; Livesey Ira O. Daar,3 and Sally A. Moody1,2,* and Cepko, 2001), the molecular mechanisms that con- 1Department of Anatomy and Cell Biology trol which embryonic cells become specified as the de- 2 Institute for Biomedical Sciences finitive retinal progenitors in the eye field remain The George Washington University Medical Center largely undefined. Washington, District of Columbia 20037 An accepted hypothesis of how the eye field forms is 3 Regulation of Cell Growth Laboratory that signals from surrounding anterior structures region- National Cancer Institute-Frederick alize the anterior neural plate (Perron and Harris, 1999). Frederick, Maryland 21702 The presumptive eye field then expresses several transcription factors that initiate the retina developmental program, e.g., rx1 (Mathers et al., 1997), pax6 (Chow et al., 1999), and six3 (Loosli et al., 1999). However, cellular Summary movements during gastrulation and neurulation, di- rected in part by eye field transcription factors, also The definitive retinal progenitors of the eye field are are critical (Chuang and Raymond, 2001; Kenyon et al., specified by transcription factors that both promote a 2001), and the signaling factors involved in these early retinal fate and control cell movements that are critical steps of eye field formation have not been identified. for eye field formation. However, the molecular signal- Several fibroblast growth factor (FGF) family members ing pathways that regulate these movements are have been implicated in affecting cell movements during largely undefined. We demonstrate that both the FGF gastrulation (Rossant et al., 1997; Wacker et al., 1998), and the anterior expression patterns of some FGFs and and ephrin pathways impact eye field formation. Acti- their receptors are consistent with a role in the morpho- vating the FGF pathway before gastrulation represses genetic movements of eye field cells (Friesel and Dawid, cellular movements in the presumptive anterior neural 1991; Friesel and Brown, 1992; Song and Slack, 1994; plate and prevents cells from expressing a retinal fate, Golub et al., 2000). Therefore, we investigated whether independent of mesoderm induction or anterior-pos- FGF signaling prior to gastrulation plays a role in de- terior patterning. Inhibiting the FGF pathway promotes termining which embryonic cells form the eye field. Us- cell dispersal and significantly increases eye field con- ing a constitutively active FGF receptor, we demonstrate tribution. ephrinB1 reverse signaling is required to pro- that enhanced FGF signaling prevents the normal retinal mote cellular movements into the eye field, and can progenitors from populating the presumptive eye field, rescue the FGF receptor-induced repression of retinal suggesting that low levels of FGF signaling are normally fate. These results indicate that FGF modulation of required for cells to adopt a retinal fate. This was con- ephrin signaling regulates the positioning of retinal firmed by demonstrating that reduced FGF signaling, progenitor cells within the definitive eye field. accomplished by expression of a dominant-negative receptor, enhanced the number of cells that become retinal progenitors. We further report that ephrinB1 signal- Introduction ing during gastrulation is required for retinal progenitors to move into the eye field, and that this movement can Retinal development consists of a series of steps that be modified by activating the FGF pathway. Our results progressively restrict the available cell fates. First, a demonstrate that FGF modulation of ephrin signaling is subset of embryonic cells are prevented from express- important for establishing the bona fide retinal progeni- ing a retinal fate by inherited maternal factors, whereas tors in the anterior neural plate. others become biased toward retinal fates due to their position within the neural inductive field of the animal hemisphere (Huang and Moody, 1993; Moore and Moody, Results 1999). As the CNS is regionalized, part of the anterior neural plate is specified as the eye field. Potential retinal Local Alterations in FGF Signaling Change Retinal progenitors need to be positioned within the eye field Fates of Dorsal Animal Lineages to receive the local environmental signals that will direct Blocking FGF signaling beginning at late cleavage their ultimate fates (Saha and Grainger, 1992; Li et al., stages does not affect the ability of retina-producing 1997). Only after these steps are accomplished do the blastomeres to populate the eye field and the retina steps of eye organogenesis, cellular lamination, and (Moore and Moody, 1999). However, enhanced FGF sig- phenotype specification occur. Although there has been naling prevented these blastomeres from adopting a great progress in understanding how retinal cell type retinal fate. Although overexpression of wild-type FGF receptor 1 (FGFR1) in the major retinal progenitor (D1.1.1; Figure 1A) did not alter that blastomere’s contri- *Correspondence: [email protected] bution to the retina (all embryos contained retinal clones 4Present address: Department of Neurobiology and Anatomy, Uni- and clone size was equivalent to that in GFP controls), versity of Utah, Salt Lake City, Utah 84132. overexpression of wild-type FGF receptor 2 (FGFR2) Developmental Cell 56

Figure 1. Activation of FGFR2 Signaling Prevents Cells from Adopting Retinal Fates (A) Nomenclature of animal 32-cell blastomeres (Jacobson and Hirose, 1981). Dorsal is to the top. (B) GFP-labeled D1.1.1 descendants in retina of control embryo. L, lens; di, diencephalon. (C) Wild-type FGFR2-expressing D1.1.1 cells in retina. (D) cFGFR2-expressing D1.1.1 cells are not in retina. (E) Retinal volumes after expression of gfp or test mRNAs. Asterisk indicates significant difference (p Ͻ 0.05) from controls. (F) Anterior view showing ␤-gal-injected D1.1.1 clone (red) in eye field (rx1, blue). (G) In cFGFR2-injected embryo, rx1 expression (blue) is missing on the injected side (arrow). (H) In XFD-injected embryo, rx1 expression (pink) is larger on the injected side (arrow).

reduced the numbers of D1.1.1 cells that populated the Activation of FGFR2 Signaling Represses retina from 50% in controls to 12%–25% (Figures 1B and Eye Field Formation 1C). To amplify the FGF signal, we utilized constitutively We tested whether cFGFR affects the formation of the active forms, cFGFR1 and cFGFR2, containing point eye field, which is the earliest specified retinal progenitor mutations that result in ligand-independent activation pool in the neural plate. We utilized only the cFGFR2 of FGF signaling (Neilson and Friesel, 1995, 1996). Over- construct because FGFR2 is more likely to be the endog- expression of either receptor completely blocked ac- enous signaling molecule through which eye field devel- cess of D1.1.1 descendants to the retina; no embryo opment is affected. It is expressed in bilateral patches contained labeled cells in the retina (cFGFR1, n ϭ 40; in the future dorso-anterior regions in the gastrula and cFGFR2, n ϭ 72, Figure 1D). The majority of embryos in the ectoderm surrounding the anterior neural plate, had smaller retinas on the injected side (Figures 1D and whereas FGFR1 is expressed more broadly in the gas- 1E), consistent with a subset of retinal progenitors being trula, predominately in paraxial mesoderm (Golub et al., absent on the injected side. Conversely, the average 2000). The expression of two eye field-specific genes retinal volume in embryos expressing a truncated FGF was repressed (Figure 1G; rx1 absent 69.2%, reduced receptor that inhibits FGF signaling (XFD; Amaya et al., 30.8%, n ϭ 26; pax6 absent 92.6%, reduced 7.4%, n ϭ 1991) was 11% larger than controls (p Ͻ 0.05; Figure 27). To determine whether retinal development was sim- 1E). A more dramatic effect was observed when XFD ply delayed, the expression of Xath5, a marker for differ- was expressed in a blastomere that gives rise to moder- entiating retinoblasts (Kanekar et al., 1997), was moni- ate amounts of the retina (D1.2.1). The percentage of tored in tadpoles; it was absent (66.7%) or reduced embryos that contained retinal progeny increased from (33.3%; n ϭ 15) in cFGFR2-injected embryos (data not 79% in controls (n ϭ 17) to 91% (n ϭ 55), and the size shown). Therefore, activation of the FGF pathway in the of the D1.2.1 contribution to the retina increased from major retina-producing lineage repressed retinal fate 12%–25% in controls to 40%–60% in XFD-expressing beginning at least at eye field formation. Conversely, embryos (p Ͻ 0.05). Thus, changes in FGF signaling repressing the FGF pathway via XFD expression moder- within dorsal animal lineages alter the size of the pool ately increased the size of the eye field (Figure 1H; of embryonic cells that contribute to the retina. 38.1%, n ϭ 42) and caused the XFD-expressing cells to Retinogenesis and FGFR-ephrinB1 Signaling 57

be distributed more extensively through the eye field posterior neural fates (Munoz-Sanjuan and H-Brivanlou, (Figure 1H; 52%). 2001). However, cell fate analyses showed that cFGFR2 expression did not drive the D1.1.1 clone out of the Activated FGFR2 Signaling Causes Cells head, nor increase its size in posterior CNS. There also to Adopt Ventral Neural Fates were no observable changes in the expression of several Even though the eye field and subsequently the retinas genes with distinct anterior-to-posterior expression do- were smaller in embryos expressing cFGFR2, a com- mains (otx2, en2, krox20, hoxB9; supplemental data). plete fate map analysis showed that FGFR2 activation Therefore activating the FGF pathway in the major retinal did not reduce the overall size of the D1.1.1 clone in the progenitor transformed retinal fates to ventral neural head (data not shown), suggesting that the progeny fates via a pathway independent of hedgehog signaling, destined for the retina did not die but adopted other mesoderm induction, or posteriorization of CNS fates. fates. Consistent with this analysis, the neural tube was larger on the injected side in 85% of the embryos (n ϭ Gastrulation Movements Are Abnormal 72; Figures 2A and 2B), and the width of the neural plate in cFGFR2-Injected Embryos was increased by 36% (n ϭ 21; p Ͻ 0.001; Figure 2C). Since the cFGFR2 phenotype is notable at neural plate In many embryos, neural hypertrophy was more pro- stages and is not affected by signals that alter neural nounced ventrally, suggesting that retinal fates were plate patterning, we analyzed clones at gastrulation transformed to ventral neural fates. Several observations stages. At stage 10, all control D1.1.1 clones were confirm this. At neural plate stages, cFGFR2-expressing broadly dispersed from the animal pole to the blastopore ϭ cells were clustered at the midline, whereas in controls, lip, whereas in 27% of cFGFR2-injected animals (n cells were dispersed laterally (Figures 2D and 2E). At 11), the clones were more confined toward the midline ϭ tadpole stages, there were large coherent masses of (data not shown). At stage 12, all control clones (n medially located cFGFR2-expressing cells abutting the 18) were broadly dispersed across the dorsal animal affected retina (Figures 1D and 2F), the majority of which quadrant (Figure 3A), whereas those expressing cFGFR2 ϭ expressed the neural marker HNK-1 (Figure 2F). These were more tightly confined to the midline (91.7%, n cells were not displaced retinal progenitors, as none 24; Figure 3B). In contrast, those expressing XFD also ϭ expressed rx1 or Xath5 (data not shown). Instead, three were all fully dispersed (n 24; Figure 3C). At stage 14, ventral forebrain fates were expanded. The expression control D1.1.1 clones were broadly dispersed across ϭ domain of pax2, a marker for the optic stalk portion of the anterior neural plate (92.3%, n 26; Figure 3D), whereas those in cFGFR2-expressing embryos were the ventral forebrain, was expanded at neural plate and more closely confined near the midline (89.9%, n ϭ 27; tadpole stages (68%; n ϭ 22; Figures 2G and 2H). The Figure 3E), presaging the ventral neural tube hypertro- number of dopamine neurons in the ventral hypothala- phy and midline clusters of cFGFR2-expressing cells mus was increased (n ϭ 15; p Ͻ 0.001, Figure 2I), and (Figure 2). Having established that D1.1.1 clones ex- the expression domain of Nkx2.4, a ventral forebrain pressing XFD were dispersed (Figures 1H and 2C), we marker (Small et al., 2000), was significantly larger examined the clone of a blastomere that contributes (ventral/dorsal ratio ϭ 1.39 Ϯ 0.07 versus 0.89 Ϯ 0.02 only moderately to the retina (D1.2.1). Normally, D1.2.1 in controls; p Ͻ 0.0001; Figure 2J). Therefore, there progeny populate the lateral border of the eye field (Fig- is a compensatory increase in ventral neural fates ure 3F). Expression of XFD caused D1.2.1 progeny to when retinal fates are repressed in cFGFR2-expressing populate the eye field at higher frequencies (91%, n ϭ lineages. 72) than controls (72%, n ϭ 29), and greater numbers There are three known functions of FGF signaling that of cells were dispersed within the eye field (Figure 3G). might cause these fate changes. First, interactions be- Thus, enhanced FGF signaling repressed the dispersal tween the FGF and the Shh pathways are important in of cells in the anterior ectoderm, whereas reduced FGF forebrain and retinal patterning (Macdonald et al., 1995; signaling enhanced it. Ohkubo et al., 2002). However, Shh expression was not Since FGF signaling can affect cell movements and altered in cFGFR2-injected embryos, nor did increased morphogenesis (Ornitz and Itoh, 2001), cFGFR2 repres- hedgehog expression alter retinal clones (data not sion of cell dispersal during gastrulation suggests that shown). Second, early FGF signaling promotes meso- this pathway may inhibit the movements of retinal pro- derm formation (Slack et al., 1987; Amaya et al., 1991). genitors into the eye field. If true, then coexpression of Although the notochord histologically appeared en- transcription factors previously demonstrated to en- larged or duplicated in many embryos (Table 1), neither hance cell movements (Kenyon et al., 2001) should res- chordin nor Xbra1 expression was changed in either cue the cFGFR2 phenotype. Coexpression of otx2, pax6, whole embryos or animal cap explants in which cFGFR2 or rx1 with cFGFR2 in the D1.1.1 lineage each rescued was expressed (see supplemental data at http://www. the retinal effects of cFGFR2 expression (Figure 3H) developmentalcell.com/cgi/content/full/6/1/55/DC1), in proportions equivalent to their frequency to induce nor was somite size or number changed. The minimal retinal fates. fh4 did not rescue the phenotype, consis- effect on mesoderm formation is consistent with previ- tent with its inability to cause cell movements when ous reports that 32-cell injections minimize exogenous ectopically expressed (Kenyon et al., 2001). FGF effects on mesoderm induction (Moore and Moody, Although otx2, pax6, and rx1 each restored the D1.1.1 1999), and that cFGFR1 has more potent effects than clone to the retina, these genes were less successful at cFGFR2 on mesoderm induction (Neilson and Friesel, rescuing the other components of the cFGFR2 pheno- 1996). Third, FGF signaling is involved in establishing type (Table 1). cFGFR2/pax6-injected embryos showed Developmental Cell 58

Figure 2. Activation of FGFR2 Signaling Increases the Size of the Neural Tube and Changes Retinal Fates to Ventral Neural Fates (A and B) Neural tube is thicker on the cFGFR2-injected side (*). os, expanded optic stalk. (C) White lines indicate extent of sox3 expression on control (right) and cFGFR2-injected (*) sides of neural plate. (D) D1.1.1 clone (green) in stage 14 forebrain region of control. Arrow denotes midline. (E) D1.1.1 clone (green) in the same region of cFGFR2-injected embryo. (F) Expanded neural tube on cFGFR2-injected side (*). Brown stain indicates HNK-1 expression. (G) Expression of cFGFR2 in D1.1.1 clone (red) expands pax2 expression on the injected side (*) of neural plate. (H) Expression of cFGFR2 in D1.1.1 expands forebrain pax2 expression (white bracket) on the injected side (inj) of tail bud embryos. un, uninjected. (I) Number of dopamine cells in the hypothalamus of cFGFR2-injected embryos. *, p Ͻ 0.001. (J) The lengths of dorsal (black line) and ventral (Nkx2.4ϩ; white line) forebrain domains in cFGFR2-injected and wild-type (wt) embryos.

the greatest extent of rescue, but only rarely (12.5%) ephrinB1 Can Completely Rescue was the cFGFR2 phenotype completely rescued. Increas- the cFGFR Phenotype ing the pax6 mRNA dosage from 50 to 100 pg increased The regulation of retinal progenitor movements during the frequency of retinal clone rescue but still did not gastrulation and eye field formation likely involves the rescue the nonretinal phenotypes (Table 1). These data temporal and spatial coordination of cell adhesive indicate that another molecular component is involved events. FGFs are among several proteins that are impli- in the cFGFR2 inhibition of retinal fate acquisition. cated in regulating such events, and FGF receptors can Retinogenesis and FGFR-ephrinB1 Signaling 59

Table 1. Ability of Factors to Rescue cFGFR2 Phenotypes Percentage of Embryos Exhibiting Phenotype ephrinB1 ephrinB1 cFGFR2 otx2 rx1 pax6 (n ϭ 21) (n ϭ 8) Phenotype (n ϭ 48) (n ϭ 24) (n ϭ 29) (n ϭ 48) 100 pg 500 pg

No retinal clone 100 70 45 33 14 0 Smaller retina 100 100 75 72 11 0 Neural tube hypertrophy 85 86 81 56 36 0 Ectopic cell masses 100 100 88 72 22 0 Expanded notochord 80 72 75 75 5 0

interact with several cell adhesion molecules that regu- ephrinB1 (Jones et al., 1997), and a receptor for ephrinB1 late cell-to-cell interactions, including transmembrane (EphB2; Tanaka et al., 1998) are all expressed during (Type B) ephrins (Doherty et al., 1996; Holder and Klein, gastrulation and neural plate stages in the anterior neu- 1999; Palmer and Klein, 2003). The FGF receptor can ral plate region. A careful comparison of the expression associate with and induce the phosphorylation of domains of pax6, ephrinB1, and fgfr2 demonstrates that ephrinB1 within the same cell, resulting in effects on (1) strong ephrinB1 expression overlaps with that of cell adhesion when ectopically expressed in Xenopus pax6 (and rx1, not shown) along the anterior margin of (Chong et al., 2000). Furthermore, components of both the eye field, and lower levels of ephrinB1 are expressed the FGF and ephrin/Eph signaling pathways are ex- within the rest of the eye field; (2) ephrinB1 and fgfr2 pressed in coincident temporal and spatial patterns that expression overlap along an anterolateral crescent that would allow them to interact and regulate retinal progen- borders the eye field; and (3) fgfr2 expression does not itor movements. FGFR1, 2, and 4 (Golub et al., 2000), overlap with that of pax6 (or rx1, not shown) (Figure 4A).

Figure 3. Gastrulation Movements Are Al- tered in Clones with Enhanced or Repressed FGF Signaling (A) Animal pole at stage 12 showing control D1.1.1 clone (red). (B) Stage 12 embryos expressing cFGFR2 in D1.1.1 clone (red). (C) Stage 12 embryo expressing XFD in D1.1.1 clone (red). (D) Anterior view at stage 14 showing control D1.1.1 clone (red). (E) Stage 14 embryos expressing cFGFR2 in D1.1.1 clone (red). (F) Control stage 15 embryo showing control D1.2.1 clone (bright blue) at lateral edge of eye field (rx1 expression). (G) Embryos expressing XFD in D1.2.1 clones that extensively populate the eye field. (H) Percentage of embryos with D1.1.1 progeny in retina after coexpression of cFGFR2 and anterior neural plate transcription factors. *, p Ͻ 0.05. Developmental Cell 60

Figure 4. ephrinB1 Rescues the cFGFR Retinal Phenotype (A) Anterior views at stage 16 show control expression domains of pax6, ephrinB1, and fgfr2. Asterisks indicate the anterior tip of the neural groove, allowing an anatomical comparison of the domains. (B) Blastomeres that contribute large numbers (D1.1.1, top) or very small numbers (V1.2.1, bottom) of cells to the retina were injected with ␤-gal mRNA, and the location of anterior descendants (red) compared to the expression domains of pax6, ephrinB1, and fgfr2 (blue patches, arrows). Top, frontal views; bottom, pax6 is frontal; ephrinB1 and fgfr2 are side views. (C) Animal poles at stage 12 showing D1.1.1 clone (red) coexpressing cFGFR2 and ephrinB1. (D) Anterior view at stage 16 showing D1.1.1 clone (red) coexpressing cFGFR2 and ephrinB1. (E) Coexpression of ephrinB1 rescues the cFGFR2 repression of D1.1.1 contribution to the retina in a dose-dependent manner. ephrinB1- 60AA does not rescue. (F) Coexpression of ephrinB1 with cFGFR2 allows D1.1.1 progeny (green) to populate the retina.

Comparison of the location of blastomere clones that borders of the eye field may regulate the movements of have differential contributions to the retina further sug- cells into the eye field. gest that ephrinB1 and FGFR2 might regulate cell move- To test this, ephrinB1 and cFGFR2 mRNAs were co- ments in the vicinity of the eye field. Clones that contrib- injected into D1.1.1. At stage 12, the normal gastrulation ute to Ͼ50% of the retina (D1.1.1) are dispersed across pattern was restored; ephrinB1/cFGFR2 clones were the pax6-expressing eye field (100%, n ϭ 14) and are broadly dispersed anteriorly (Figure 4C; 67.9%, n ϭ 28), confined within anterior and lateral borders delineated and in 21.4% several cells were scattered long distances by high ephrinB1 (95.5%, n ϭ 22) and fgfr2 (94.4%, from the main clone, indicating increased cell move- n ϭ 18) expression (Figure 4B). In contrast, clones that ment. By stage 15, the normal phenotype of full anterior contribute to Ͻ1% of the retina (V1.2.1) populate the dispersal and population of a restored eye field was lateral boundary of the pax6 domain (100%, n ϭ 5), observed (Figure 4D; 100%, n ϭ 15). Coexpression of coinciding with the anterolateral patch that expresses ephrinB1 rescued the cFGFR2 repression of D1.1.1 pop- high levels of ephrinB1 (92.8%, n ϭ 28) and fgfr2 (90%, ulation of the retina in a dose-dependent manner (Fig- n ϭ 20) (Figure 4B). These patterns suggest that the ures 4E and 4F). The proportion of the retina derived expression of these two signaling molecules along the from the cFGFR2/ephrinB1-expressing clone increased Retinogenesis and FGFR-ephrinB1 Signaling 61

with larger ephrinB1 mRNA doses, such that all embryos dispersed (90.5%, n ϭ 21), and the majority of clones receiving 500 pg had retinal clones equivalent in size to spread across the entire animal hemisphere (76.2%, Fig- controls. Retinal size increased in a dose-dependent ure 5G). The ability of ectopic ephrinB1 to change the manner: 50 pg of ephrinB1 mRNA restored retinas to morphogenetic movements of ventral clones was de- wild-type (6.2 ϫ 10Ϫ6 ␮m3), and larger doses caused pendent upon reverse signaling since clones expressing significantly larger retinas (250 pg: 6.9 ϫ 10Ϫ6 ␮m3; 500 the ephrinB1(–60AA) mutant were mostly coherent (71.4%, pg: 7.5 ϫ 10Ϫ6 ␮m3;pϽ 0.05). Thus, increased ephrinB1 n ϭ 21) and positioned to one side of the animal pole signaling enabled more cells to adopt retinal fates. The (100%, Figure 5H). However, V1.1.1 clones expressing rescue was dependent upon the ability of ephrinB1 to XFD resembled the ephrinB1(Y305, Y310) phenotypes reverse signal since a mutant ephrinB1 that lacks its (51.2%, n ϭ 41; Figure 5I top); in the remainder, several intracellular signaling domain (ephrinB1–60AA; Chong cells were dispersed into the dorsal midline (Figure 5I, et al., 2000) did not rescue the cFGFR phenotype (Figure bottom). That these gastrulation movement changes re- 4E). Increased ephrinB1 signaling also rescued the non- sulted in an increased population of the eye field was retinal phenotypes of cFGFR2 (Table 1). A 100 pg confirmed by examining XFD-expressing V1.1.1 clones ephrinB1 dose significantly reduced the frequency of at neural plate stages. In controls, V1.1.1 clones lay neural tube hypertrophy, ectopic masses, and expanded ventral to the eye field (100%, n ϭ 45; Figure 5J), whereas notochords, and 500 pg eliminated all cFGFR2 pheno- in XFD-expressing embryos, these cells were often types. within the eye field (34%, n ϭ 97; Figure 5K). Thus, If an ephrinB1-FGFR interaction were responsible for both enhanced reverse ephrinB1 signaling and reduced controlling the cell movements that regulate which cells FGFR signaling caused greater cellular dispersal and gain access to the eye field, then ectopic ventral expres- movement into the retinal region of the animal hemi- sion of ephrinB1 should cause epidermal progenitors to sphere. move into the anterior neural plate and adopt a retinal fate. To test this, wild-type ephrinB1 was expressed in a ephrinB1 Is Necessary for Retinal Progenitors blastomere that normally never contributes to the retina to Move into the Eye Field (V1.1.1); 42% of embryos contained V1.1.1 progeny in A knockdown of ephrinB1 expression was performed ϭ the retina (Figure 5A; n 17). Since wild-type ephrinB1 using antisense morpholino oligonucleotides (MO) to may be subject to signaling modification by endogenous determine whether this protein is necessary for retinal FGFR signaling, we also expressed a mutant form progenitors to move into the eye field. To test the ability [ephrinB1(Y305, Y310)] in which the two tyrosines are of the ephrinB1-MO to block ephrinB1 protein accumu- altered to phenylalanines; this prevents ephrinB1 asso- lation, lysates from embryos injected with ephrinB1 ciation with and modulation by FGFR but does not alter mRNA alone or with MOs were analyzed on Western cell de-adhesion (Chong et al., 2000). The expression blots. ephrinB1-MO completely blocked the expression of ephrinB1(Y305, Y310) in blastomeres that normally of ephrinB1 protein (Figure 6A), whereas neither an in- never contribute to the retina (V1.1.1, V1.1.2; Figure 5C) verted ephrinB1-MO nor a standard control MO did. caused their progeny to populate the retina at significant The translation of a 5Ј truncated ephrinB1 (ephrin⌬UTR) frequencies (Figures 5A and 5D). In the case of V1.1.1, mRNA was not blocked by the ephrinB1-MO, and thus ephrinB1(Y305, Y310) caused a higher percentage of was used for rescue experiments. Translation of ephrinB3 embryos to contain retinal clones compared to wild- mRNA (Helbling et al., 1999), which encodes a related type ephrinB1. Reverse signaling is required for these ligand recognized by the same antibody, was not blocked effects, since the ephrinB1(–60AA) mutant did not cause by the ephrinB1-MO, indicating that ephrinB1-MO is changes in fate (Figure 5A). Consistent with these data, specific for this family member. reduction of FGF signaling via XFD expression also Injection of ephrinB1-MO into D1.1.1 prevented its caused the V1.1.1 clone to populate the retina (n ϭ 46, progeny from populating the retina in a dose-dependent Figures 5A and 5E). Expression of ephrinB1(Y305, Y310) manner, whereas the inverted ephrinB1-MO did not (Fig- in a blastomere that normally makes a minor contribution to the retina (V1.2.1) was sufficient to nearly double the ures 6B–6D). In the embryos that did contain D1.1.1 percentage of embryos containing retinal clones (Figure progeny in the retina, the retinal clones were always 5A), and increased the retinal cell contribution from Ͻ1% significantly smaller (1%–10%; Figure 6B). Higher doses in controls to 12%–50% (Figure 5B). Likewise, expres- phenocopied other aspects of the cFGFR2 phenotype sion of XFD increased the percentage of embryos con- including smaller retinas, larger neural tubes, and large taining V1.2.1-derived retinal clones (n ϭ 81, Figure 5A) cell masses adjacent to the retina (Figure 6C). Coexpres- ⌬ and the size of the V1.2.1 retinal clone (Ͻ1% in controls; sion of ephrinB1 UTR mRNA with the ephrinB1-MO fully 25%–50% in XFD). Together these results suggest that restored the D1.1.1 clone to the retina (Figure 6D). These an interaction with FGFR normally inhibits the function data demonstrate that the effects were specific to of ephrinB1 in promoting the recruitment of cells into ephrinB1 and that ephrinB1 signaling is necessary for the retina. appropriate movement of anterior ectodermal cells to To determine whether these transformations from epi- acquire a retinal fate. dermal to retinal fates correlated with cell movement changes, gastrulation movements were monitored. At Is the Rescue of Retinal Fate by Coexpression stage 12, control V1.1.1 clones were coherent (100%, of ephrinB1 and FGFR2 Mediated by n ϭ 21) and most had withdrawn from the dorsal side Transcriptional Regulation? of the animal pole (66.7%, Figure 5F). In contrast, V1.1.1 The results presented above suggest that either direct or clones expressing ephrinB1(Y305, Y310) were widely indirect signaling between FGFR2 and ephrinB1 affects Developmental Cell 62

Figure 5. ephrinB1 Causes Epidermal Pro- genitors to Move into the Eye Field and Popu- late the Retina (A) Percentage of embryos in which progeny from ventral blastomeres populated the retina. GFP indicates control embryos. (B) Expression of ephrinB1(Y305, Y310) in V1.2.1 progeny (green) in retina. (C) Progeny of V1.1.2 (green) do not normally populate the retina. (D) Expression of ephrinB1(Y305, Y310) in V1.1.2 progeny (green) in retina. (E) Expression of XFD in V1.1.1 progeny (green) in retina. (F) Stage 12 embryos showing control V1.1.1 lineage (red). Asterisk indicates the animal pole. Dorsal is to the top. (G) Stage 12 embryos expressing ephrinB1 (Y305, Y310) in the V1.1.1 lineage. (H) Stage 12 embryos expressing ephrinB1 (–60AA) in the V1.1.1 lineage. (I) Stage 12 embryos expressing XFD in the V1.1.1 lineage. (J) Anterior view of stage 15 control V1.1.1 lineage (bright blue) ventral to eye field (rx1). (K) Stage 15 embryos showing XFD-expressing V1.1.1 cells (bright blue) within eye field.

cell movement. Alternatively, FGFR signaling could lead ephrinB1-MO-injected embryos fully restored the D1.1.1 to alterations in transcription. It is possible that cFGFR2 clone to the retina (95.5%, n ϭ 22; Figure 6G). However, downregulates retinal fate by repression of pax6 and/or ephrinB1-injected embryos failed to display any ectopic rx1 transcription (Figure 1G) rather than of cell move- or expanded expression of pax6 (n ϭ 38; Figure 6H) or ment. However, in animal cap explants in which cell rx1 (n ϭ 40), indicating that ephrinB1 does not directly movement effects on cell fate are minimal, expression of regulate eye field genes but does effectively signal cFGFR2 in noggin-injected animal caps did not repress changes in cell movement. either pax6 or rx1 expression (supplemental data). Simi- larly, expression of cFGFR2 did not repress ephrinB1 Discussion expression (supplemental data), consistent with a previ- ous report (Jones et al., 1997). However, eye field tran- There are three major morphogenetic movements that scription factors likely act downstream of ephrinB1 to bring embryonic cells into the correct position to form promote a retinal fate. First, knockdown of ephrinB1 the retina. During gastrulation, epiboly movements posi- expression repressed both rx1 (88.6%, n ϭ 35) and pax6 tion dorsal ectoderm in register with signaling centers (84%, n ϭ 25; Figure 6E) expression, whereas neither that induce anterior neural ectoderm. During formation rx1 (1.9%, n ϭ 108) nor pax6 (0%, n ϭ 71; Figure 6F) of the neural plate, forebrain and eye field cells disperse mRNA injection caused ectopic or expanded expression and intermix with adjacent lineages (Wetts and Fraser, of ephrinB1. Second, coexpression of pax6 mRNA in 1988; Bauer et al., 1994). Finally, cells of the single eye Retinogenesis and FGFR-ephrinB1 Signaling 63

Figure 6. ephrinB1 Is Necessary for Retinal Progenitors to Move into the Eye Field (A) Embryos were injected with ephrinB1 mRNA alone, or plus an ephrinB1 morpholino (MO), inverted ephrinB1-MO (invert ctl), or standard control MO (std ctl), and analyzed by Western blot. The ephrinB1-MO does not block the translation of a 5Ј truncated ephrinB1 (ephrinB1⌬UTR) or of ephrinB3, which is recognized by the same antibody. (B and C) ephrinB1-MO injected into D1.1.1 (green) represses population of retina. (D) Percentage of embryos with D1.1.1 clones in retina. ephrinB1-MO caused a dose-dependent reduction (p Ͻ 0.05 at all doses). (E) ephrinB1-MO injection into D1.1.1 represses pax6 expression (arrow). (F) Ectopic expression of pax6 (red) does not induce ectopic ephrinB1 (blue). (G) Expression of pax6 in an ephrinB1-MO- injected embryo restores D1.1.1 clone (green) to retina. (H) Ectopic expression of ephrinB1 (red) does not induce ectopic pax6 expression (blue).

field are displaced or move laterally to form the left candidate for regulating retinogenic cell movements. and right eye primordia (Varga et al., 1999). Very little is Our data show that FGFR2 signaling represses cell dis- known about the molecular regulation of these critical persal at the midline of the neural plate, whereas early steps in retinogenesis. Although otx2, rx1, and blocking this function increases cellular dispersal into pax6 have been implicated in regulating retinogenic cell the eye field. Another candidate signaling pathway for movements (Chuang and Raymond, 2001; Kenyon et al., regulating retinogenic cell movements are ephrins and 2001), the upstream signaling factors have not been their tyrosine kinase receptors (Ephs), which have im- delineated. Since FGF signaling is involved in several portant roles in several developmental processes involv- morphogenetic movements (Rossant et al., 1997; Sun ing cell migration and morphogenetic movements et al., 1999; Wacker et al., 1998) and cell adhesive inter- (Jones et al., 1998; Holder and Klein, 1999; Xu et al., actions (Kinoshita et al., 1993), this pathway is an ideal 1999; Wilkinson, 2001; Williams et al., 2003). We found Developmental Cell 64

that ephrinB1 is normally necessary for retinogenic cells et al., 2000; PDGF, Bruckner et al., 1997), both of which to acquire a retinal fate, as evidenced by repression lead to the recruitment of signaling effectors to the cyto- of endogenous ephrinB1 translation by ephrinB1-MO. plasmic tail (Kullander and Klein, 2002). Reverse signal- Moreover, ephrinB1 signaling is sufficient to rescue the ing is important for many morphogenetic events (Adams inhibition of retinal fate caused by activated FGFR2, and et al., 2001; Xu et al., 1999; Huynh-Do et al., 2002), and that repression of FGFR signaling by XFD expression herein we demonstrate that the ability of ephrinB1 to phenocopies the ephrinB1 effects. alter the gastrulation movements of retinogenic cells, The downstream events of ephrin signaling are com- rescue the cFGFR2 effect, or cause ventral epidermal plex and not yet fully understood. However, it has been cells to populate the eye field is dependent upon reverse demonstrated that ephrinB1 can be modulated by FGFR signaling through its cytoplasmic domain. These results within the same cell. Chong et al. (2000) showed in Xeno- suggest that FGFR signaling is involved in regulating the pus that the FGF receptor associates with and induces ephrinB1 retinogenic effects because FGFR has been the phosphorylation of ephrinB1 both in vitro and in vivo. shown to interact with ephrinB1 within the same cell in Based on this known interaction, we propose that during a manner dependent upon specific tyrosine residues normal development cells are recruited to the eye field within the deleted domain (Chong et al., 2000). More- by activation of the ephrinB1 signaling pathway, which over, an ephrinB1 protein harboring substitutions for allows them to disperse. An activated FGFR is likely tyrosines 305 and 310 is able to effectively drive ventral to influence ephrinB1 function along the anterolateral blastomeres toward a retinal fate at higher percentages borders of the anterior neural plate, based on the endog- than does the wild-type ephrinB1 protein that is subject enous expression domains of fgfr2 and ephrinB1, sup- to FGFR interactions. These tyrosine residues are critical pressing this cell movement and thereby repressing a for FGFR-mediated rescue of ephrinB1-induced effects retinal fate. This hypothesis is supported by the observa- on cell adhesion (Chong et al., 2000) and are the in vivo tions that (1) retinogenic clones disperse across the phosphorylation sites in embryonic chick retina (Kalo eye field domains that express pax6, rx1, and moderate et al., 2001). Thus, although we do not know whether ephrinB1; (2) clones that barely contribute to the retina ephrinB1 forward signaling is involved in promoting reti- do not extend beyond a patch that coexpresses high nogenic cell movements, reverse signaling is critical to levels of both ephrinB1 and fgfr2; (3) ephrinB1 expres- promote this important step in retinal development. sion counteracts the retina-suppressing effects of activated FGFR2, restores the dispersal of FGFR2-expressing cells in the anterior neural plate, and causes ventral Experimental Procedures epidermal progenitors to disperse more than normal and enter the eye field. None of these functional effects Blastomere Injections Fertilized Xenopus laevis eggs were obtained by standard methods occurred with an ephrinB1(–60AA) construct lacking the (Moody, 1999) and injected with the following mRNAs: ␤-gal (100 intracellular domain that is modulated by FGFR; and (4) pg), gfp (200 pg), bhh (100 pg; Ekker et al., 1995), cFGFR1, cFGFR2 the repression of FGFR signaling by XFD expression (10–100 pg; Neilson and Friesel, 1995, 1996), otx2 (50 pg; Blitz and phenocopied the ephrinB1 effects. These results indi- Cho, 1995), pax6 (50 pg; Hirsch and Harris, 1997), rx1 (50 pg; Mathers cate that ephrinB1 is involved in promoting morphoge- et al., 1997), fh4 (50 pg; Dirksen and Jamrich, 1995), ephrinB1 (50– netic movements that are necessary to establish the eye 500 pg; Jones et al., 1997), ephrinB1Y305Y310 (100 pg), ephrinB1- 60AA (100–250 pg; Chong et al., 2000), ephrinB1(⌬UTR) (100–250 field and express a retinal fate. pg), and XFD (250 pg; Amaya et al., 1991). An ephrinB1 morpholino Type B ephrins can signal in two directions, forward (MO), designed to 12 bases of the 5Ј UTR and 13 bases of the ORF, and reverse (Wilkinson, 2001; Palmer and Klein, 2003). its inverted control, and a standard control were synthesized (Gene Forward signaling occurs when the ligand binds to an Tools). For rescue of MO effects, mRNA was synthesized from Eph tyrosine kinase receptor on an adjacent cell and ephrinB1⌬UTR, which lacks the 5Ј UTR and contains four point activates a signaling cascade within that cell (Gale et mutations in wobble codons. al., 1996). ephrinB1/Eph signaling is involved in several morphogenetic events, including dorsal axis formation Cell Fate Analysis (Tanaka et al., 1998), skeletogenesis (Compagni et al., Blastomeres were injected with a mixture of gfp and test mRNAs. 2003), and proper cell positioning in the intestinal epithe- Tissue sections of stage 37/38 embryos were analyzed for the pres- lium (Batlle et al., 2002). However, it is not known ence of GFP-labeled cells, and their distribution compared to estab- whether ephrinB1 binding to a cognate Eph receptor is lished lineage maps (Moody, 1987) and to gfp-injected controls. The required to promote a retinogenic fate. Targeted disrup- frequencies that embryos contained labeled cells in the retina in tion of three Eph receptors that can bind ephrinB1 have experimental and control groups were compared using the z-test of proportions. Retinal volumes were calculated from serial tissue not been reported to affect retinogenic cell fate deci- sections as described (Moore and Moody, 1999) and compared by sions (Henkemeyer et al., 1996; Orioli et al., 1996; Wil- the t test. liams et al., 2003). Nonetheless, our results strongly im- plicate signaling from the intracellular domain of ephrinB1 as critical to this process. Animal Cap Assays Ephrins can transduce signals via their cytoplasmic Both blastomeres of 2-cell embryos were injected with mRNA, and domain within their host cells, referred to as “reverse at stage 8/9 animal cap explants were isolated and cultured. At signaling.” The cytoplasmic tail of ephrinB1 becomes neural plate stages, explants were processed for either in situ hybridization (below) or PCR. RNA was isolated (Purescript, Gentra), tyrosine phosphorylated, either upon its binding to a reverse transcribed (Superscript First Strand, InVitrogen) and ampli- cognate receptor in an adjacent cell (Holland et al., 1996; fied by PCR, using primer sequences and conditions for Xbra and Bruckner et al., 1997) or by the activation of other signal- EF1␣ (Hemmati-Brivanlou and Melton, 1994), ephrinB1 (Jones et al., ing cascades within the same cell (FGF or FGFR, Chong 1997), and rx1 and pax6 (Onuma et al., 2002). Retinogenesis and FGFR-ephrinB1 Signaling 65

Whole-Mount In Situ Hybridization Bruckner, K., Pasquale, E.B., and Klein, R. (1997). Tyrosine phos- Embryos were injected with a mixture of ␤-gal and test mRNA, phorylation of transmembrane ligands for Eph receptors. Science assayed for ␤-Galactosidase activity, and processed for whole- 275, 1640–1643. mount in situ hybridization using standard methods (Sive et al., Cho, K.W., Morita, E.A., Wright, C.V., and De Robertis, E.M. (1991). 2000) and the following probes: Xbra (Smith et al., 1991), chordin Overexpression of a homeodomain protein confers axis-forming ac- (Sasai et al., 1994), engrailed 2 (Hemmati-Brivanlou et al., 1991), tivity to uncommitted Xenopus embryonic cells. Cell 65, 55–64. ephrinB1 (Jones et al., 1997), fgfr2 (Friesel and Brown, 1992; Golub Chong, L.D., Park, E.K., Latimer, E., Friesel, R., and Daar, I.O. (2000). et al., 2000), hoxB9 (Cho et al., 1991), Krox20 (Hemmati-Brivanlou Fibroblast growth factor receptor-mediated rescue of x-Ephrin B1- et al., 1991), Nkx2.4 (Small et al., 2000), pax2 (Heller and Brandli, induced cell dissociation in Xenopus embryos. Mol. Cell. Biol. 20, 1999), pax6 (Hirsch and Harris, 1997), rx1 (Mathers et al., 1997), shh 724–734. (gift of R. Moon), sox3 (gift of R. Grainger), and Xath5 (Kanekar et al., 1997). To determine whether the neural plate was expanded, the Chow, R.L., Altmann, C.R., Lang, R.A., and Hemmati-Brivanlou, A. width of sox3 expression on both cFGFR2-injected and uninjected (1999). Pax6 induces ectopic eyes in a vertebrate. Development sides were measured with an eyepiece micrometer. The mean percent 126, 4213–4222. difference between sides was compared to similar measurements from Compagni, A., Logan, M., Klein, R., and Adams, R.H. (2003). Control control embryos by the t test. 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