Non-neural origin of pineal progenitors

During an unbiased neural plate fate-mapping study in zebrafish, we observed that the diencephalic territory expressing flh/noto – published as the pineal progenitor territory at neurula stage (Masai et al., 1997; Cau and Wilson, 2003) – is largely fated to contribute to the thalamus, raising the question of where pineal organ precursors can be found at this stage (Staudt and Houart, 2007). As classical fate-mapping experiments had placed pineal progenitors at the border of the anterior neural plate (Couly and Le Douarin, 1987; Eagleson and Harris, 1990), we decided to characterise gene expression in this region in zebrafish in more detail. The early neural plate is surrounded by a horseshoe-shaped domain of non-neural ectoderm, the pre-placodal region (PPR), that gives rise to the cranial placodes (Streit, 2007; Schlosser, 2014). The anterior PPR (which gives rise to the adenohypophysis, the anterior part of the pituitary gland) is marked by expression of the homeobox gene pitx3 (Fig. 1A,B), whereas the posterior PPR is marked by expression of the iroquois-related homeobox gene irx1b (Fig. 1A,C-F). The most anterior part of the irx1b-positive PPR that is known to give rise to the trigeminal placode also expresses neurogenin 1 (neurog1), which encodes a basic helix-loop-helix transcription factor involved in neurogenesis (Fig. 1B,E-H). irx1b is also expressed in the posterior neural plate, but the prospective neural crest that lies between the posterior neural plate and PPR, and is marked by expression of foxd3, is irx1b negative (Fig. 1F,H).

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Fig. 1.

The flh/noto/NOT1-expressing pineal progenitor region extends into the PPR. (A-H) Dual-colour in situ hybridisation for indicated genes on bud stage zebrafish embryos (anterior is leftwards). White asterisk in A marks pitx3-positive anterior PPR; black asterisks mark irx1b-positive posterior PPR. (D,F,H) Magnified views of the boxed areas in C,E,G, respectively; arrows indicate a putative pineal progenitor region; nc marks the neural crest in F,H. (I) In situ hybridisation for flh/noto (red) and immunohistochemical detection of DLX3 (green) in an early bud stage zebrafish embryo. (J) Magnification of boxed area in I showing partial overlap between flh/noto and DLX3 expression. (K) Transverse section of late bud stage embryo stained as in I and J at the level of the diencephalon. Arrow indicates the overlap between flh/noto and DLX3-expressing cells in the PPR (PrePlac.). All zebrafish wild-type double staining experiments were performed with at least 20 embryos, two independent experiments each. Images are representative of most or all embryos for each set of markers. (L) HH8.5 chick embryo stained using dual-colour in situ hybridisation for SOX2 (red) and NOT1 (blue; anterior is upwards). Arrowhead indicates level of section in M. (M) Transverse section through the neural tube of an embryo stained as in L at the level of the posterior forebrain (arrowhead in L). There is NOT1 staining in non-neural ectoderm overlying the SOX2-positive neural tube (n=3; two independent experiments). (N) Schematic colour-coded representation of gene expression patterns shown in A-K (Di, diencephalon; Hypoth, hypothalamus; Mes, mesencephalon; Tel, telencephalon). Scale bars: in A, 50 µm for A-C,E,G; in D,H, 15 µm for D,F,H,J; in I, 50 µm; 15 µm in K; 500 µm in L; 25 µm in M.

When we analysed the expression of the bona fide pineal progenitor marker flh/noto in relation to these different domains, we found that its territory reaches further laterally than the expression of irx1b in the neural plate (Fig. 1C,D), suggesting that it might extend into the neural crest and/or PPR domain. In order to test whether flh/noto-positive cells reside in the PPR, we performed double staining for flh/noto and the pan-PPR marker DLX3, using a combination of in situ hybridisation and antibody staining, and observed that there is indeed an overlap of the expression of these two factors in the neural plate border region (Fig. 1I-K). The chick orthologue of flh/noto, NOT1, is not expressed in the ectoderm at early neural plate stages, but it appears in the non-neural ectoderm overlying the diencephalon at around Hamburger-Hamilton (HH) stage 8, when the neural tube is starting to close (Fig. 1L,M) (Hamburger and Hamilton, 1951). This comparatively ‘late’ onset of ectodermal NOT1 expression in the anterior neural folds of the chick embryo is consistent with what has previously been described (Stein et al., 1996). These findings show that the flh/noto-positive domain encompasses non-neural progenitors that are located within the PPR and that this potential non-neural contribution to pinealogenesis may be conserved in amniotes (chick). A diagram summarising the results from our gene expression mapping analysis in fish can be found in Fig. 1N.

In order to refine our mapping of the origin of pineal progenitors in fish, and to verify that the DLX3-positive region of the flh/noto domain is indeed contributing to the pineal organ, we uncaged fluorescein in the neural plate border at the level of the posterior diencephalon in transgenic Tg(her5:eGFP) embryos (Staudt and Houart, 2007). The accuracy of this approach was checked by fixing a few embryos minutes after uncaging and staining them for irx1b (Lecaudey et al., 2001). Although cells labelled within the posterior PPR and prospective neural crest domains (Fig. 2A) frequently contribute to the trigeminal placode and neural crest (Fig. 2D), and uncaging across both neural and non-neural territories in this region (Fig. 2B) leads to staining of both epithalamus and pineal cells (Fig. 2E), cells labelled in a small border domain immediately anterior to the irx1b-positive posterior PPR (Fig. 2C) most often ended up in the pineal organ proper, which can be labelled for the orthodenticle-like homeobox gene otx5 at 24 h post fertilisation (hpf) (Fig. 2F).

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Fig. 2.

Non-neural ectoderm contributes to the pineal organ. (A-C) Small patches of cells (white arrows) were labelled using uncaging of fluorescein (Fluo, red) at bud stage. Embryos were fixed immediately and subjected to in situ hybridisation for irx1b (blue) to demonstrate precision of labelling (anterior is leftwards). A, n=6; B, n=4; C, n=7. (D-F) Lateral views (anterior is leftwards) of the diencephalon of embryos labelled as in A-C, incubated for 24 h and labelled for expression of otx5 (red) marking the definitive pineal organ (red arrowheads). Cells labelled by uncaging (blue) in the otx5-positive pineal organ are labelled in E and F. D, n=11; E, n=11; E, n=19/23. (G-I) Sequential images of time-lapse recording of an embryo from late bud to the 26-somite stage in which Kaede-expressing cells have been converted to red fluorescence in the ectoderm flanking the diencephalon. The trajectories of three representative cells (blue, red and green arrowheads in G) are shown in red, green and blue in H,I. (J) Dorsal view showing converted cells in the pineal organ at 22 hpf. See also Movies 1 and 2. (K) Lateral view, anterior towards the left, showing converted cells in the ectoderm and pineal organ, but not in the brain proper, at 30 hpf. Four out of 12 embryos had no converted cells in the brain proper; all four of those showed converted cells in the pineal organ. E, eye; L, lens; T, telencephalon. Dotted lines in J and K outline the pineal organ. Scale bars: 50 µm; bar in A applies to A-C,G-I; bar in D applies to D-F,J,K.

To monitor how non-neural cells integrate into the pineal organ, we performed time-lapse light-sheet microscopy recordings of embryos injected with Kaede mRNA in which we photo-converted the non-neural ectoderm along the border of the anterior neural plate at the tail-bud stage (Fig. 2G-K; Movies 1 and 2) (Ando et al., 2002). In four out of 12 embryos treated in this manner, no photo-converted cells were found within the neural tube, indicating that only non-neural ectoderm had been labelled. In these four embryos, many labelled cells were found in the epidermis as expected and a few migrating neural crest cells were also labelled. Moreover, in all four of these embryos, a subset of labelled cells integrated into the dorsal-most neural keel between the six- and eight-somite stages (Fig. 2J), and contributed to the pineal organ that is morphologically distinguishable as a button-like structure on top of the diencephalon at 30 hpf (Fig. 2K). Retrospective tracing of three such cells revealed that they clearly originated within the non-neural ectoderm laterally flanking the diencephalic territory (colour-coded traces in Fig. 2H,I; Movie 2).

Our gene expression and fate-mapping studies indicate that pineal precursors emerge from the PPR. Esterberg and Fritz previously demonstrated that PPR identity can be blocked in zebrafish by simultaneous morpholino (MO)-mediated knockdown of dlx3b and dlx4b, which encode the homeodomain transcription factors that are required for the specification of the neural plate border region (Esterberg and Fritz, 2009). Thus, we decided to use these dlx3b/4b MOs to functionally assess a requirement for the PPR in pineal organ formation. The pineal organ was reduced in size in embryos injected with MOs against flh/noto (Fig. 3A,B), and a similar reduction was found in dlx3b/4b double morphants (Fig. 3C). This result could indicate either that non-PPR tissues such as the neural crest or neural tissue proper also contribute to the pineal organ, resulting in partial pinealogenesis in the absence of the PPR, or they could simply be due to a hypomorphic effect of the MOs. However, the formation of the pineal organ is completely repressed by simultaneous knockdown of flh/noto and dlx3b/4b (or by injection of dlx3b/4b MOs into flh mutant embryos), leading to a complete absence of the gland in such triple loss-of-function embryos, suggesting a synergistic role of flh/noto and PPR identity in pinealogenesis (Fig. 3D).

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Fig. 3.

The PPR specifier genes dlx3b/dlx4b in conjunction with flh/noto are required and sufficient for pineal progenitor specification. (A-D) 24 hpf zebrafish embryos injected with the MOs indicated in B-D and stained for the pineal marker otx5 in red (lateral views, anterior is leftwards). There is reduced otx5 staining in B and C, and a complete absence in D. Scale bar: 50 µm. MO injection experiments were performed three times independently, injecting each MO or combination of MOs in parallel into 40-50 embryos from the same parents. Images are representative for each experimental condition. Fifty percent of the embryos injected with dlx3b/4b MOs looked as in C, the other 50% had a range of even weaker otx5 staining. (E,F) Injection of dlx3 and flh mRNAs at the one-cell stage results in ectopic induction of the pineal marker otx5 (blue) after 24 h of incubation (n=21/32; control embryo shown in E; two independent experiments). Inset in F is an image focusing on the ectopically induced otx5-expressing cells that are marked with the arrow in F. Scale bar: 50 µm. (G,H) Lateral views of embryos injected at the one-cell stage with hs:flh/noto and hs:dlx4b, heat-shocked at shield stage and stained by in situ hybridisation for otx5 (red) after 24 h of incubation. There is sparse ectopic induction of otx5 in superficial cells (arrowheads). Scale bars: 50 µm (bar in A applies to A-D; bar in E applies to E-H). (I) Quantification of otx5 induction in cells injected with hs:gfp alone, with hs:flh/noto and hs:dlx4b, or with hs:flh/noto alone (two independent experiments; six embryos were selected for statistical analysis; data are mean±s.d.). P<0.0001 (GFP versus flh+dlx), P<0.0001 (GFP versus flh), P=0.041 (flh+dlx versus flh).

Conversely, combined ectopic expression by mRNA injection of dlx3b and flh/noto at the one-cell stage resulted in cells expressing the pineal organ marker otx5 in ectopic locations (n=21/32; Fig. 3E,F), whereas neither dlx3b nor flh/noto alone had this effect (n=34 for dlx3b and n=51 for flh/noto). Ectopic otx5-positive cells are only found anterior to the midbrain-hindbrain boundary, indicating a posterior restriction of competence for pineal precursor induction. The overall morphology of the neural tube in embryos injected with dlx3b and/or flh/noto tends to be highly abnormal, presumably owing to early effects of these factors on gastrulation and neurulation. Thus, we generated a conditional expression system by cloning dlx4b and flh/noto into heat shock-inducible plasmids that drive the expression of green fluorescent protein (GFP)-tagged versions of these two genes (Lewis et al., 2004). Embryos injected with these constructs at the one-cell stage and heat-shocked at 37°C at late gastrula/early neurula stage (75-90% epiboly) display sparse ectopic induction of otx5 in ectopic flh/noto-expressing cells and a higher rate of ectopic otx5 expression in double dlx4b/flh-expressing cells (Fig. 3G-I). Consistent with a non-neural origin of pineal progenitors, we observed that ectopic otx5-positive cells were almost exclusively found in non-neural ectodermal cells (and occasionally among migrating neural crest cells).

Two distinct cell populations, the PPR and the neural crest, form in close proximity in the neural plate border region of the posterior diencephalon, and the expression of the pineal progenitor marker flh/noto appears to overlap with both of these to some extent (Fig. 1; Patthey and Gunhaga, 2011, Groves and LaBonne, 2014). The overlap of flh/noto expression with dlx3 (Fig. 1I-K) and our MO knockdown experiments targeting dlx3b/dlx4b function (Fig. 3) have demonstrated that the PPR makes an essential contribution to pinealogenesis. To test whether the neural crest also contributes to the pineal organ, we blocked neural crest formation in zebrafish using MOs against the neural crest specifier genes foxd3 and sox10 (Whitlock et al., 2005), or injected the foxd3 MO into sox10 mutants. Neither individual nor combinatorial loss of foxd3 and sox10 function affected pineal organ formation, indicating that pineal precursors are not derived from the neural crest (Fig. S1).

Taken together, our experiments so far demonstrate that pineal precursors originate (at least in part) from the PPR, and that pre-placodal identity is required for pineal organ formation. Ablation experiments targeting precursors in late gastrula and neurula embryos indicated that pineal precursors are specified by neurula stage (Houart et al., 1998; Staudt and Houart, 2007). This evidence for early specification is supported by our dlx3b/4b loss-of-function experiments, as these two genes are only transiently expressed in the PPR at late gastrula and early neurula stages (Esterberg and Fritz, 2009), and by the conditional dlx4b/flh gain-of-function experiments in which ectopic pineal cell identity could specifically be induced during gastrulation.

Pineal organ formation requires Otx gene function cell non-autonomously

In previous studies, we observed that injection of MOs against otx1 and otx2 (otxH) resulted in embryos that lack the pineal organ (Foucher et al., 2006; Scholpp et al., 2007). Conditional ablation in the mouse had previously demonstrated a cell-autonomous requirement for Otx2 in pinealocyte development at later stages of development (Nishida et al., 2003). However, otxH morphant zebrafish display a more profound pineal defect, with a complete absence of a morphologically recognisable pineal organ (Fig. 4A,B) and of photoreceptor differentiation (assessed by expression of the photoreceptor marker α-opsin; Fig. 4C,D) at 48 hpf, as well as absence of expression of the pan-pineal marker otx5 at 24 hpf (Fig. 4E,F) and at the eight-somite stage (Fig. 4G,H). The complete absence of flh expression from the anterior neural plate of otxH morphants at late gastrula (bud) stage indicates that Otx gene function is already required for the earliest steps of pineal precursor specification (Fig. 4I,J).

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Fig. 4.

Otx function is required for pinealogenesis. Wild-type zebrafish embryos are shown in A,C,E,G,I and otxH MO-injected embryos in B,D,F,H,J (anterior is leftwards, lateral views are shown in A-H, dorsal views in I,J). Asterisks mark the location of the pineal organ in A-H. (A,B) The pineal organ is morphologically reduced in otxH embryos at 48 hpf. (C,D) Immunohistochemical staining for opsin at 48 hpf; staining is absent from the pineal area in D. (E,F) Double in situ hybridisation for otx5 and shh (both in blue) at 24 hpf. otx5-positive pineal progenitors are absent in F. Arrows indicate the zona limitans intrathalamica; this signalling centre is reduced in F. (G,H) In situ hybridisation for otx5 at the eight-somite stage, including dorsal views of the pineal region shown in the insets. (I,J) In situ hybridisation for flh/noto at bud stage; flh/noto-positive pineal progenitors are absent in J (arrow in J indicates persistent flh/noto staining in notochord). Each marker was tested in two independent experiments, comparing at least 30 control and otxH MO-injected embryos for each marker and stage. Images are representative of all analysed embryos. Scale bar: 50 µm.

To test whether pineal precursors require Otx function cell-autonomously, we transplanted otxH cells into wild-type embryos. Transplanted cells had a strong tendency to avoid the roof of the forebrain and the pineal organ proper (Fig. 5A,B). otx5-expressing cells are occasionally seen among otxH-injected cells, suggesting that the requirement for Otx gene function is not strictly cell autonomous (inset in Fig. 5B). Wild-type cells transplanted into otxH morphant embryos showed better integration and were more evenly spread throughout the embryo. Such mosaic embryos typically displayed formation of a slightly smaller and/or somewhat disorganised pineal organ in the correct location (Fig. 5C,C′). otxH cells contributed to the pineal organ in these embryos, indicating that wild-type cells rescue pineal precursor specification cell non-autonomously (Fig. 5C′).

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Fig. 5.

Wild-type cells can rescue pineal progenitor specification cell non-autonomously in otxH morphants. (A,B) Representative fluorescent images of wild-type zebrafish embryos containing cells grafted from an otxH embryo (green) and stained by in situ hybridisation for otx5 (red) at 24 hpf (lateral views, anterior is leftwards). Inset in B shows a dorsal view of the pineal region in more detail. otx5 is expressed in otxH cells that are adjacent to wild-type cells (arrow). Two independent experiments were performed grafting 13 and 19 embryos, respectively. (C) Bright-field image of an otxH embryo containing grafted wild-type cells and stained by in situ hybridisation for otx5 (red) at 24 hpf (lateral view, anterior is leftwards). (C′) Fluorescent images of the pineal region of the embryo shown in C in more detail; grafted wild-type cells are green. There is non-cell-autonomous rescue of otx5 expression (arrow). Inset in C′ shows a magnified dorsal view of the otx5-positive pineal organ in C′. Two independent experiments were performed grafting 17 and 14 embryos: 15/17 and 11/14 showed cell-non-autonomous pineal rescue. Scale bars: 50 µm in A,C′; 50 µm in insets (bar in the inset in C′ applies to the insets in both B and C′).

FGF signalling represses pineal organ specification

The pineal anlage is enlarged in mbl embryos, suggesting that WNT signalling promotes pineal precursor specification (Fig. S1) (Heisenberg et al., 1996; Masai et al., 1997). However, neither experimental up- nor downregulation of WNT signalling was able to rescue pinealogenesis in otxH morphants, suggesting that the pineal otxH phenotype is not related to WNT. Expression of erm, a bona fide target gene of the FGF signalling pathway, showed a significant increase in the neural plate border region of otxH morphants (Fig. 6A-B′), prompting us to test whether FGF inhibition can rescue pineal organ development in these morphants. We used the FGF inhibitor SU5402 at a concentration low enough not to result in severe morphological alterations of the brain and found that pinealogenesis is indeed rescued in otxH morphants treated with this pharmacological effector (Fig. 6C-F). Because SU5402 may interfere with signalling pathways other than the FGF pathway, we confirmed these results by blocking FGF signalling more directly using a zebrafish line carrying a heat shock-inducible dominant-negative FGF receptor transgene Tg(hsp70:dnfgfr1a-eGFP) (Lee et al., 2005). Heat-shock activation of this transgene at 30% epiboly (early gastrula) efficiently rescued the pineal organ in otxH morphants (Fig. 6G,H), whereas activation at a slightly later stage (70-75% epiboly, mid-gastrula) resulted in very few rescued cells in only 40% of the embryos. Similar to our SU5402 titration, we adjusted the duration of the heat shock in these experiments to avoid extensive brain abnormalities that would result from an ongoing strong inhibition of FGF signalling. Taken together, these results provide further evidence that pineal precursors are specified during gastrulation and demonstrate that excessive FGF signalling suppresses pinealogenesis in otxH morphant embryos.

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Fig. 6.

Upregulation of FGF signalling activity in the neural plate border region is responsible for the absence of pineal progenitors in otxH morphant embryos. (A-B′) Representative images of dual colour in situ hybridisation for erm (blue) and flh (red) in wild-type (n=29; A,A′) and otxH (n=32; B,B′) bud stage zebrafish embryos; anterior is leftwards. (A′,B′) The boxed neural plate border region in A,B in more detail. (C-H) Wild-type (C,E,G) and otxH morphant (D,F,H) zebrafish embryos stained for fgf8 (red) and otx5 (blue) at 24 hpf (anterior is leftwards, dorsal towards the top; C,D, n=18). Embryos in E,F were treated with the FGF inhibitor SU5402 (n=25 and n=22, respectively). Embryos in G,H are transgenic for a heat shock-inducible dominant-negative FGF receptor and were heat-shocked at shield stage (n=19 and n=22, respectively). otx5-positive pineal progenitors are rescued by pharmacological inhibition of FGF signalling in F and by heat shock-induced receptor inhibition in H (compare with D). Inset in H shows a sibling from the Tg(hsp:dnfgfr1) cross that did not show rescue and is therefore likely to be from the 25% of offspring that are expected to be negative for the transgene. Results are based on two independent experiments. Scale bar: 50 µm.

Our findings suggest that the pineal precursor domain is restricted by FGF signalling. Indeed, transplants from fgf8-expressing (but not from gfp-expressing control) donor embryos into early gastrula stage (30% epiboly) wild-type host embryos inhibited flh/noto expression at the late gastrula stage, demonstrating that FGF signalling is sufficient to inhibit pineal specification in fish (Fig. 7A). In chick, the first faint expression of FGF8 in the midbrain-hindbrain boundary region is observed at HH8.5 (Fig. 7B), around the time of NOT1 induction in the anterior neural folds (Fig. 1L). Strong expression of FGF8 in the midbrain-hindbrain boundary and anterior neural folds is seen from around HH9 onwards (Fig. 7C). In order to test whether FGF8 also represses pineal identity in chick, we in ovo electroporated a FGF8-expressing plasmid into the prospective diencephalon of HH9/10 chick embryos. This treatment consistently resulted in downregulation or absence of NOT1 expression after 36 h of incubation (Fig. 7D,E). Many embryos electroporated with FGF8 showed morphological alterations that are characteristic of the diencephalon-to-midbrain transformation induced by FGF8 (Crossley et al. 1996), and only embryos that had retained a morphologically distinguishable diencephalic vesicle were included in this analysis. The competence for FGF signalling to repress pineal organ specification appears to be limited in time, as electroporation of FGF8 into the neural tube at HH13/14 did not result in a noticeable downregulation of NOT1 (n=7/7).

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Fig. 7.

FGF signalling antagonises pinealogenesis. (A) Bud stage zebrafish embryo (anterior points to the top) containing transplanted fgf8-overexpressing cells in the right side of the neural plate (red), labelled using in situ hybridisation for flh (blue). flh-positive pineal progenitors are present on the right side (arrows; n=7/7). (B,C) Chick embryos (anterior towards the top) at HH8.5 (B) and HH9.5 (C) stained using in situ hybridisation for expression of FGF8 (n=6; two independent experiments). Expression is present in the midbrain-hindbrain boundary in B (arrowhead; the arrow marks expression in the tailbud region). Expression is present in the anterior neural folds (arrow) and midbrain-hindbrain boundary (arrowhead) in C. (D) HH18 chick embryo stained using in situ hybridisation for NOT1 (lateral view of diencephalon, anterior is rightwards). Inset shows same embryo at lower magnification. (E) Chick embryo electroporated at HH10 with FGF8 and eGFP, fixed after 36 h of incubation and stained by in situ hybridisation for expression of NOT1 (blue) and by immunohistochemistry for GFP (red, inset). Arrowheads mark the pineal organ; the arrow in the inset marks the location of electroporated cells at some distance from the pineal organ. NOT1 is downregulated in E compared with D (n=11/15; two independent experiments). (F) Zebrafish embryo (anterior is leftwards, dorsal towards the top) transplanted at 30% epiboly stage with heat shock-inducible dnfgfr-transgenic donor cells (green), heat-shocked at shield stage, fixed at 24 hpf and labelled by in situ hybridisation for otx5 (red). Ectopic pineal progenitors are induced following heat shock (n=9/12). Arrowhead highlights otx5-positive pineal organ; arrow indicates ectopic otx5-positive cells. (G) Lateral view (anterior towards the right) of a chick embryo electroporated at HH8 with dnFGFR and eGFP, and stained after 24 h of incubation by in situ hybridisation for NOT1 (blue) and by immunohistochemistry for GFP (red, inset). Arrowhead indicates NOT1-positive pineal organ; black and white arrows highlight ectopic NOT1-positive cells. (H) Oblique section of embryo along the line indicated in G. Arrowhead indicates pineal organ; arrows indicate clusters of NOT1-expressing cells in superficial ectoderm (n=6/13). (I) Lateral view of chick embryo electroporated at HH8 with dnFGFR and eGFP into the right anterior neural fold, and stained for NOT1 after 36 h of incubation (arrowhead indicates pineal organ). Inset shows GFP fluorescence before in situ hybridisation. There is ectopic induction of NOT1-positive pineal progenitor cells in electroporated neural tube (arrows; n=7/27). Scale bars: 50 µm in A for A-F; in B, 500 µm for B,C; in D, 200 µm for D,E,I; 100 µm in G for G,H.

FGF signalling is not only sufficient, but also necessary to delimit the pineal precursor domain, as transplantation of cells from Tg(hsp70:dnfgfr1a-eGFP) donors into wild-type zebrafish embryos at 30% epiboly followed by heat shock at 50% epiboly and fixed at 24 hpf resulted in ectopic patches of otx5 expression in the head ectoderm (Fig. 7F). Again, the competence for pineal induction appears to be restricted to the ectoderm anterior to the midbrain-hindbrain boundary, as posterior dnfgfr1a-expressing cells consistently failed to express otx5, which is reminiscent of the restriction observed in our dlx4b/flh overexpression experiments (Fig. 3F).

To test whether the requirement for FGF signalling in delimiting pineal progenitor induction is conserved in chick, we electroporated a dominant-negative FGF receptor 1 expression construct (dnFGFR) into the anterior neural folds at HH7/8 (one- to four-somite stage). Over half of the embryos electroporated at these comparably early stages die within 24 h of incubation or develop with severe neural tube defects that make the interpretation of their morphology impossible. However, in a subset of the surviving embryos, ectopic NOT1-expressing cells were observed in the superficial, non-neural ectoderm after 24 h of incubation (Fig. 7G,H). These non-neural clusters of ectopic NOT1 expression were hardly ever seen after 36-48 h of incubation, suggesting that ectopic NOT1-positive ectodermal cells may be eliminated or expelled from embryos. At these later stages, only a few cells that show weak ectopic expression of NOT1 were observed within the electroporated area of the neural tube of a subset of embryos (Fig. 7I). Taken together, FGF signalling antagonises pineal progenitor induction in both anamniotes (zebrafish) and amniotes (chick). In both systems, most ectopically induced pineal progenitors are found in the non-neural ectoderm, consistent with the idea that the non-neural ectoderm makes a significant contribution to the pineal organ.