Cornesse Y et al. (2005), Olfactory and lens placode formation is control...

XB-ART-2541

Dev Biol 2005 Jan 15;2772:296-315. doi: 10.1016/j.ydbio.2004.09.016.

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Olfactory and lens placode formation is controlled by the hedgehog-interacting protein (Xhip) in Xenopus.

Cornesse Y , Pieler T , Hollemann T .

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The integration of multiple signaling pathways is a key issue in several aspects of embryonic development. In this context, extracellular inhibitors of secreted growth factors play an important role, which is to antagonize specifically the activity of the corresponding signaling molecule. We provide evidence that the Hedgehog-interacting protein (Hip) from Xenopus, previously described as a Hedgehog-specific antagonist in the mouse, interferes with Wnt-8 and eFgf/Fgf-8 signaling pathways as well. To address the function of Hip during early embryonic development, we performed gain- and loss-of-function studies in the frog. Overexpression of Xhip or mHip1 resulted in a dramatic increase of retinal structures and larger olfactory placodes primarily at the expense of other brain tissues. Furthermore, loss of Xhip function resulted in a suppression of olfactory and lens placode formation. Therefore, the localized expression of Xhip may counteract certain overlapping signaling activities, which inhibit the induction of distinct sensory placodes.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 chrd dlx3 egr2 elavl1 emx2 en2 fgf4 fgf8 gal.2 grn hhip mapk1 nodal3.2 OPN4 otx2 pax2 pax6 pitx1 pitx3 ptch1 rax ret rho rpe sdhd shh sia1 six3 slc5a5 tbx2 tbxt vax1 wnt8a
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	Fig. 2. Temporal and spatial expression of Xhip. Whole mount in situ hybridization of Xhip in Xenopus embryos. Staging according to Nieuwkoop and Faber (1967). (a) Neurula stage (NF stage 18), anterior view. White arrowhead, preplacodal ectoderm; black arrowhead, expression of Xhip in paraxial stripes in the ventral neural tube. (b) Early tailbud (NF stage 20), frontal view. Arrowhead, presumptive placodal epithelium. (c) Dorsal view, anterior to the left. Arrow, lateral mesoderm; arrowhead, ventral neural tube. (c′) Transversal section. White arrowhead, chorda dorsalis; arrow, lateral mesoderm; black arrowheads, paraxial stripes in the ventral neural tube. (d) Mid-tailbud (NF stage 24), frontal view. Arrow, diencephalon. (e) Lateral view, anterior to the left. White arrowhead, pAD + pM, common anterodorsal and middle lateral line placode; white arrowhead, pP, posterior lateral line placode. (c′, e′, f) Transversal sections reveal Xhip expression in the chorda dorsalis at NF stages 18 and 24, but not at NF stage 28 (white arrowheads). (f) Expression of Xhip in the roof plate (white arrow), Xhip dorsal to the floor plate (black arrowhead). (g) In tailtip stage embryos (NF stage 34), frontal view, Xhip is expressed in the pineal gland (white arrow), in the anterodorsal lateral line placode (white arrowhead), in the olfactory placodes (black arrowhead) and in the anterior head mesenchyme (black arrow). (h) Lateral view, anterior to the left. A black arrowhead indicates Xhip expression in the olfactory placode. White arrow, pineal gland anlage. White arrowheads, anterodorsal, middle and posterior lateral line placode; black arrows from anterior to posterior, facial epibranchial placode fused with the anteroventral lateral line placode, glossopharyngeal placode, 1st, 2nd, and 3rd vagal epibranchial placode. (i) Ventral view of the head of the tadpole shown in j (NF stage 41); arrowhead, lower jaw; arrow, rim of the mouth opening. (j) Lateral view, anterior to the left. Black arrowhead, lower jaw; white arrowhead, rim-like staining for Xhip at the transition zone between the yolk mass and the ectoderm. (k) Ventral view of the proctodeum of the tadpole shown in j, white arrowhead as in j. Abbreviations: 1,2,3ep, 1st, 2nd, and 3rd vagal epibranchial placode; ahm, anterior head mesenchyme; cd, chorda dorsalis; de, diencephalon; fep, facial epibranchial placode fused with the anteroventral lateral line placode; gep, glossopharyngeal placode; mop, mouth opening; lj, lower jaw; lme, lateral mesoderm; pAD, anterodorsal lateral line placode; pgl, pineal gland; pM, middle lateral line placode; pOl, olfactory placode; pP, posterior lateral line placode; ppe, preplacodal ectoderm; ppl, presumptive placodal epithelium; pr, proctodeum; rp, roof plate; vnt, ventral neural tube.
	Fig. 4. Hip as multifunctional antagonist of Shh, eFgf/Fgf-8 and Wnt-8 signaling pathways. (A) mHip1 inhibits Shh and eFgf pathways in animal cap explants. (a–e) Explants corresponding to NF stage 17. In situ hybridization with Ptc1 as a marker for active Shh signaling. (a, b, c) Control caps and caps either injected with Chordin (50 pg) or Shh (500 pg) mRNA do not express Ptc1. (d) Co-injection of Chordin and Shh mRNA (500 pg) induces Ptc1. (e) mHip1 mRNA (1 ng) suppresses Ptc1 induced by Chordin and Shh. (f–w) Explants stained for Xbra at NF stage 10.5 or stage 17, respectively. (f) Control caps and caps injected with mHip1 mRNA (k, p) do not express Xbra. (g, l) Xbra expression induced by BMP4 mRNA (1 ng) or by activin mRNA (5 pg; i, n) is not blocked by mHip1 in early explants (h, j) but in late explants (m, o). The induction of Xbra by either eFgf mRNA (10 pg; q, u) or Fgf-8 (150 pg; s) is inhibited by the co-injection of mHip1 (r, t, v), but Xbra expression is rescued by Elk mRNA (w, 300 pg). (B) (a–d′) Early gastrula stage embryos (NF stage 10), vegetal view. (a, b, b′) Xbra in situ hybridization and (c, d, d′) immunostaining of phosphorylated MAPK (MAPK-P). (a, c) Control embryos, Xbra and MAPK-P staining around the blastopore. Microinjection of mHip1 mRNA (750 pg) into one cell of a four cell stage embryo inhibits expression of Xbra (b, b′; 72%, n = 25) and phosphorylation of MAPK (d, d′; 67%, n = 15). LacZ mRNA was co-injected as a lineage tracer (light blue). (C) (a, b, e) Second axis formation induced by Wnt-3a (a; 0.5 pg; 84% 2° axis; n = 57) or Wnt-8 mRNA (b; 5 pg; 78% 2° axis, n = 49). Co-injection of mHip1 (600 pg) did not block Wnt-3a (c; 71% 2° axis; n = 32), but Wnt-8 activities (d; 10% 2° axis, n = 39). RT-PCR analysis of animal caps at NF stage 10.5. (f) Induction of Xnr3 and siamois by Wnt-8 (50 pg), but not by Wnt-3a (e; 0.5 pg), was inhibited by mHip1. (D) Comparison of Xhip, Shh, Fgf-8, and Wnt-8 expression. (a, b, c) Lateral views, anterior to the left. (a′, a′, b′, b′) Transversal sections. Shh expression in the ventricular layer of the ventral diencephalon (arrow), while Xhip expression was detected near the sulcus limitans (arrow) in early tadpoles. (a′) Shh expression was found in the chorda dorsalis (red arrowhead) and floorplate (white arrowhead) and (b′) Xhip transcripts only dorsally to the floorplate (black arrowhead). (a, b, c′) Horizontal sections, anterior up. (a) Shh transcripts were detected in the posterior endothelium of the endodermal visceral pouches (black arrow) and Fgf-8 in the anterior endothelium (c′, black arrow), whereas (b) Xhip expression was detected in the adjacent mesenchyme of the branchial arches (arrow). (d, e, f, g) Anterior view of neurula stage embryos. (d) Cleared embryo. Shh transcripts in the chorda dorsalis (white arrow) and prechordal plate (black arrow). (e, f) Xhip expression in the preplacodal region (white arrowhead) limited by Fgf-8 transcripts in the anterior neural ridge (black arrowhead) and in a line outside the neural plate (red arrowhead). (g) Xhip in the lateral placodes is juxtaposed toward Wnt-8 positive cells (arrow, arrowhead).
	Fig. 5. The inhibition of Shh, Fgf and Wnt-8 signaling affects eye, brain, and placode development in tadpole stage embryos. (A) Overexpression of mHip1 and Shh. (a–g′) Microinjection of mHip1 (750 pg) into one cell of two-cell stage embryos. (h–n′) Overexpression of Shh (500 pg) into one cell of two-cell stage embryos. (b, e′, f, l′, m) Frontal view. (a, c, d, e, h, i, j, k, l) Lateral view. (g, n) Dorsal view. (a′–d′, f′, g′, h′–k′, m′, n′) Transversal sections. (a, a′) Formation of a giant eye. Note Xrx1-positive cells extend into the prospective midbrain region (white arrow in a′, 67%, n = 18). (b, b′) Repression of optic stalk marker Vax1 within the ventral fore- and midbrain (white arrows, 63%, n = 28). (c, c′) Displaced Rhodopsin positive cells in the enlarged eye (76%, n = 21). (d, d′) Abnormal lens formation revealed by Crystallin α expression (50%, n = 12). (e, e′) Enlarged olfactory placode as revealed by Emx2 expression, indicated by dashed open rectangles (65%, n = 17). (f, f′) Ventral shift of the dorsal limit of Nkx-2.1 expression (white arrows in f′, 55%, n = 11). (g, g′) Ventral expansion of Gsh-1 expression (38%, n = 8). (h–h′) Note the reduction of retinal tissue (Xrx1, dark brown) and lens tissue (Pitx3, red, 50%, n = 10). (i, i′) Expanded expression domain of the optic stalk marker Vax1 (white arrow in i′, 62%, n = 13). (j, j′) Loss of Rhodopsin positive cells (83%, n = 16). (k, k′) Absence of lens specific Crystallin α expression (57%, n = 14). (l, l′) Suppression of olfactory placode development as revealed by the loss of Emx2 expression, indicated by a red arrow (65%, n = 17). The dashed open rectangle marks the olfactory placode on the non-injected side (l′). (m, m′) Dorsal shift of ventral forebrain tissue as revealed by Nkx-2.1 expression (56%, n = 16), the eye structures are emphasized by dashed ellipses. (n, n′) Reduced expression of the dorsal neural tube marker Gsh-1 in the prospective midbrain (black arrow, 42%, n = 12). LacZ mRNA was co-injected as a lineage tracer (light blue in b′, c, c′, e, e′, f′, g, g′, l, l′, n′). (B) Influence of dnWnt-8, XFD and Fgf-8 overexpression on lens and olfactory placode development. (a′, b′, c′, d′, e′, f′) Frontal views, dashed open rectangles indicate the size of the olfactory and lens placodes, respectively. (a, a′, b, b′, c, c′, d, d′, e, e′, f, f′) Lateral views. (a–b′) Overexpression of dnWnt-8 (500 pg) resulted in enlarged olfactory (Emx2; 76%, n = 22) and lens placodes (Pitx3; 78%, n = 18). (c–d′) XFD (1 ng) injection did not alter the size of the olfactory placode but led to enlarged lens placodes (83%, n = 16). (e–f′) Additional Fgf-8 (10 pg) inhibits olfactory and lens placode induction (red arrows in e′ and f′; 76%, n = 19). (a–a′, c–c′, e–e′) Krox-20 and En-2 expression was followed to control hindbrain and midbrain–hindbrain formation, respectively. LacZ mRNA was co-injected as a lineage tracer (light blue in a′, a′, b′, b′, c′, c′, d′, d′, e′, e′, f′, f′). Abbreviations: ey, eye cup; mhb, midbrain–hindbrain boundary; olp, olfactory placode; r3, rhombomere 3; r5, rhombomere 5; tel, telencephalon.
	Fig. 5. Rho expression in head of tadpole stage embryos
	hhip <hedgehog interacting protein> gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 34, anterior view, dorsal top. Image published in: Cornesse Y et al. (2005)
	hhip <hedgehog interacting protein> gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 18, anterior view, dorsal top. Image published in: Cornesse Y et al. (2005)

	Fig. 7. Suppression of Xhip function by antisense-morpholino injection resulted in loss of lens and olfactory placode formation. Whole mount in situ hybridization of Xhip-morpholino (a, f, j, n) and control-morpholino (e, i, m, q) injected embryos at NF stage 36. (a, f, j, n) Lateral anterior view of the non-injected side. (c, e) Frontal view, injected side to the right. (d, h, l) Horizontal vibratome sections of the corresponding embryos. In control-morpholino (5 pmol) injected embryos LacZ mRNA was co-injected as a lineage tracer (light blue). (a) Expression of Krox-20, En-2 and Emx2 was detected simultaneously. (b, c, d) Xhip-morpholino injection into one cell of a two cell-stage embryo (2.5 pmol/blastomere) did suppress the expression of Emx2 in the olfactory placode and branchial arches (74%, n = 31; red arrows), and that of Xpitx3 in the lens ectoderm (65%, n = 40; red arrow in g, h). (k, l) The expression level of Rx1 in the remaining eye cup was not altered, although the morphology of the eye cup was perturbed and the retinal pigment epithelium was not formed (68%, n = 38; d, h, l). (n) Xhip-morpholino injection did not suppress otic vesicle formation as shown by Pax2 expression, but RPE formation (100%, n = 47; red arrows in o, p). (r) Increasing amounts of mHip1 mRNA (50 or 100 pg) injected together with Xhip morpholino rescued the formation of lens and olfactory placodes. (s) Summary diagram of interactions of Hip, Hh, Wnt-8, Fgf-8 and activin. The arrows do not suggest direct interactions; for details, see Discussion. Abbreviations: ba, branchial arches; ey, eye; is, injected site; le, lens; mhb, midbrainindbrain boundary; nis, non-injected side; olp, olfactory placode; op, otic vesicle; os, optic stalk; r3/5, rhombomere 3/5; ret, retina; rpe, retinal pigment epithelium; tel, telencephalon.
	Fig. 6. Regulation of early eye field formation by Hip and Shh. (A) (a, g) Frontal views of embryos at NF stage 14, injected side to the right. (f, l) Lateral views of e and k. (a) Increased number of retinal progenitors upon mHip1 overexpression (750 pg; 71%, n = 46). (g) Reduction of Xrx1 expression after injection of Shh (500 pg; 50%, n = 32). (b, h) While Shh overexpression results in a slight reduction of Pax6 expression in the anterior neural plate and Pitx1 expression in the early lens field (45%, n = 11), both genes are up-regulated and expanded laterally after mHip1 injection (67%, n = 9). Microinjection of mHip1 mRNA results in strongly expanded domains of Six3 and Otx2 (c, 60%, n = 10; d, 100%, n = 8), but are almost normal upon Shh overexpression (i, 23%, n = 13; j, 67%, n = 12). (e, f) Overexpression of mHip1 results in reduction and lateral shift of Dlx3 (black arrows, 43%, n = 14). (k, l) Expression of Dlx3 in ectoderm surrounding the neural plate is increased after Shh injection (black arrows, 36%, n = 14). (B) (a) Frontal views of embryos at NF stage 13, injected side to the right. (a) Strong activation of Six3 upon injection of dnWnt-8 mRNA (63%, n = 11), while Dlx3 expression is reduced (b, 58%, n = 12). XFD overexpression did not alter Six3 (c, 95%, n = 9) or Dlx3 (d, 100%, n = 8) expression significantly. LacZ mRNA was co-injected and visualized by red- gal stain. (C) (a) BrdU-proliferation assay and (d) phospho-histone H3-proliferation assay. Frontal views of embryos at NF stages 14 and 15, respectively. mHip1 and Shh mRNA was injected into one cell of a two-cell stage embryo, injected side to the right. (b, c) Rx1 expression (red) was monitored to visualize mHip1 and Shh activity (indicated by dashed line). (b, e) The number of BrdU- and p-H3-positive cells is slightly enhanced by Shh and reduced upon mHip1 overexpression (c, f) versus control embryos (a, d). (g) Frontal views of tailbud embryos at NF stage 33 after TUNEL staining. (h) Shh overexpression reduces cell death, whereas the number of apoptotic cells is increased upon mHip1 injection (i). LacZ mRNA was co-injected as a lineage tracer (light blue). (jl) Mitotic arrest assay. (j) Control embryo, showing that normal Rx1 expression is maintained after HUA treatment. (i) mHip1 injection still expands the retinal territory (71%, n = 34), whereas in Shh injected embryos Rx1 expression is reduced (k).
	Fig. 5. The inhibition of Shh, Fgf and Wnt-8 signaling affects eye, brain, and placode development in tadpole stage embryos. (A) Overexpression of mHip1 and Shh. (aV) Microinjection of mHip1 (750 pg) into one cell of two-cell stage embryos. (hV) Overexpression of Shh (500 pg) into one cell of two-cell stage embryos. (b, eV, f, lV, m) Frontal view. (a, c, d, e, h, i, j, k, l) Lateral view. (g, n) Dorsal view. (aVV, f V, gV, hVV, mV, nV) Transversal sections. (a, aV) Formation of a giant eye. Note Xrx1-positive cells extend into the prospective midbrain region (white arrow in aV, 67%, n = 18). (b, bV) Repression of optic stalk marker Vax1 within the ventral fore- and midbrain (white arrows, 63%, n = 28). (c, cV) Displaced Rhodopsin positive cells in the enlarged eye (76%, n = 21). (d, dV) Abnormal lens formation revealed by Crystallin a expression (50%, n = 12). (e, eV) Enlarged olfactory placode as revealed by Emx2 expression, indicated by dashed open rectangles (65%, n = 17). (f, f V) Ventral shift of the dorsal limit of Nkx-2.1 expression (white arrows in f V, 55%, n = 11). (g, gV) Ventral expansion of Gsh-1 expression (38%, n = 8). (hV) Note the reduction of retinal tissue (Xrx1, dark brown) and lens tissue (Pitx3, red, 50%, n = 10). (i, iV) Expanded expression domain of the optic stalk marker Vax1 (white arrow in iV, 62%, n = 13). (j, jV) Loss of Rhodopsin positive cells (83%, n = 16). (k, kV) Absence of lens specific Crystallin a expression (57%, n = 14). (l, lV) Suppression of olfactory placode development as revealed by the loss of Emx2 expression, indicated by a red arrow (65%, n = 17). The dashed open rectangle marks the olfactory placode on the non-injected side (lV). (m, mV) Dorsal shift of ventral forebrain tissue as revealed by Nkx-2.1 expression (56%, n = 16), the eye structures are emphasized by dashed ellipses. (n, nV) Reduced expression of the dorsal neural tube marker Gsh-1 in the prospective midbrain (black arrow, 42%, n = 12). LacZ mRNA was co-injected as a lineage tracer (light blue in bV, c, cV, e, eV, f V, g, gV, l, lV, nV). (B) Influence of dnWnt-8, XFD and Fgf-8 overexpression on lens and olfactory placode development. (aV, bV, cV, dV, eV, fV) Frontal views, dashed open rectangles indicate the size of the olfactory and lens placodes, respectively. (a, aW, b, bW, c, cW, d, dW, e, eW, f, fW) Lateral views. (aW) Overexpression of dnWnt-8 (500 pg) resulted in enlarged olfactory (Emx2; 76%, n = 22) and lens placodes (Pitx3; 78%, n = 18). (cW) XFD (1 ng) injection did not alter the size of the olfactory placode but led to enlarged lens placodes (83%, n = 16). (eW) Additional Fgf-8 (10 pg) inhibits olfactory and lens placode induction (red arrows in eVand fV; 76%, n = 19). (aW, cW, eW) Krox-20 and En-2 expression was followed to control hindbrain and midbrainindbrain formation, respectively. LacZ mRNA was co-injected as a lineage tracer (light blue in aV, aW, bV, bW, cV, cW, dV, dW, eV, eW, f V, f W). Abbreviations: ey, eye cup; mhb, midbrainhindbrain boundary; olp, olfactory placode; r3, rhombomere 3; r5, rhombomere 5; tel, telencephalon.
	Fig. 2. Temporal and spatial expression of Xhip. Whole mount in situ hybridization of Xhip in Xenopus embryos. Staging according to Nieuwkoop and Faber (1967). (a) Neurula stage (NF stage 18), anterior view. White arrowhead, preplacodal ectoderm; black arrowhead, expression of Xhip in paraxial stripes in the ventral neural tube. (b) Early tailbud (NF stage 20), frontal view. Arrowhead, presumptive placodal epithelium. (c) Dorsal view, anterior to the left. Arrow, lateral mesoderm; arrowhead, ventral neural tube. (cV) Transversal section. White arrowhead, chorda dorsalis; arrow, lateral mesoderm; black arrowheads, paraxial stripes in the ventral neural tube. (d) Mid-tailbud (NF stage 24), frontal view. Arrow, diencephalon. (e) Lateral view, anterior to the left. White arrowhead, pAD + pM, common anterodorsal and middle lateral line placode; white arrowhead, pP, posterior lateral line placode. (cV, eV, f) Transversal sections reveal Xhip expression in the chorda dorsalis at NF stages 18 and 24, but not at NF stage 28 (white arrowheads). (f) Expression of Xhip in the roof plate (white arrow), Xhip dorsal to the floor plate (black arrowhead). (g) In tailtip stage embryos (NF stage 34), frontal view, Xhip is expressed in the pineal gland (white arrow), in the anterodorsal lateral line placode (white arrowhead), in the olfactory placodes (black arrowhead) and in the anterior head mesenchyme (black arrow). (h) Lateral view, anterior to the left. A black arrowhead indicates Xhip expression in the olfactory placode. White arrow, pineal gland anlage. White arrowheads, anterodorsal, middle and posterior lateral line placode; black arrows from anterior to posterior, facial epibranchial placode fused with the anteroventral lateral line placode, glossopharyngeal placode, 1st, 2nd, and 3rd vagal epibranchial placode. (i) Ventral view of the head of the tadpole shown in j (NF stage 41); arrowhead, lower jaw; arrow, rim of the mouth opening. (j) Lateral view, anterior to the left. Black arrowhead, lower jaw; white arrowhead, rim- like staining for Xhip at the transition zone between the yolk mass and the ectoderm. (k) Ventral view of the proctodeum of the tadpole shown in j, white arrowhead as in j. Abbreviations: 1,2,3ep, 1st, 2nd, and 3rd vagal epibranchial placode; ahm, anterior head mesenchyme; cd, chorda dorsalis; de, diencephalon; fep, facial epibranchial placode fused with the anteroventral lateral line placode; gep, glossopharyngeal placode; mop, mouth opening; lj, lower jaw; lme, lateral mesoderm; pAD, anterodorsal lateral line placode; pgl, pineal gland; pM, middle lateral line placode; pOl, olfactory placode; pP, posterior lateral line placode; ppe, preplacodal ectoderm; ppl, presumptive placodal epithelium; pr, proctodeum; rp, roof plate; vnt, ventral neural tube.
	rax (retina and anterior neural fold homeobox) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior right, dorsal up. Key: ret= retina; le= lens.
	hhip ( hedgehog interacting protein) expression in retina of Xenopus laevis embryo, assayed via in situ hybridizartion, NF stage 36, lateral view,anterior left, dorsal up. Key: ret= retina; le= lens; nis = non-injected side.